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https://openalex.org/W2605434096	https://pure.aber.ac.uk/portal/files/11251302/ncomms14914.pdf	English	null	Past penguin colony responses to explosive volcanism on the Antarctic Peninsula	Nature communications	2,017	cc-by	22,734	Citation for published version (APA): Roberts, S. J., Monien, P., Foster, L. C., Loftfield, J., Schnetger, B., Pearson, E., Juggins, S., Hocking, E., Fretwell, P., Ochyra, R., Haworth, A., Allen, C., Moreton, S., Davies, S., Brumsack, H.-J., Bentley, M. J., Hodgson, D. A., & Ireland, L. (2017). Past penguin colony responses to explosive volcanism on the Antarctic Peninsula. Nature Communications, 8, Article 14914. https://doi.org/10.1038/ncomms14914 Past penguin colony responses to explosive volcanism on the Antarctic P i l Past penguin colony responses to explosive volcanism on the Antarctic Peninsula Roberts, Stephen J.; Monien, Patrick; Foster, Louise C.; Loftfield, Julia; Schnetger, Bernard; Pearson, E.; Juggins, S.; Hocking, Emma; Fretwell, Peter; Ochyra, Ryszard; Haworth, Anna; Allen, Clare; Moreton, Steven; Davies, Sarah; Brumsack, Hans-Jurgen; Bentley, Michael J.; Hodgson, Dominic A.; Ireland, Louise Published in: Nature Communications DOI: 10.1038/ncomms14914 Publication date: 2017 Published in: Nature Communications DOI: 10.1038/ncomms14914 Citation for published version (APA): Roberts, S. J., Monien, P., Foster, L. C., Loftfield, J., Schnetger, B., Pearson, E., Juggins, S., Hocking, E., Fretwell, P., Ochyra, R., Haworth, A., Allen, C., Moreton, S., Davies, S., Brumsack, H.-J., Bentley, M. J., Hodgson, D. A., & Ireland, L. (2017). Past penguin colony responses to explosive volcanism on the Antarctic Peninsula. Nature Communications, 8, Article 14914. https://doi.org/10.1038/ncomms14914 Document License CC BY General rights i h d General rights Copyright and moral rights for the publications made accessible in the Aberystwyth Research Portal (the Institutional Repository) 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. • Users may download and print one copy of any publication from the Aberystwyth Research Portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain Y f l di t ib t th URL id tif i th bli ti i th Ab t th R h P t l • Users may download and print one copy of any publication from the Aberystwyth Research Portal for the purpose of private study or research. • Users may download and print one copy of any publication from the Aberystwyth Research Portal for the purp research esearch. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the Aberystwyth Research Portal arch. • You may not further distribute the material or use it for any profit-making activity or commercial ga Y f l di t ib t th URL id tif i th bli ti i th Ab t th R h P t l • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the Aberystwyth Research Portal You may not further distribute the material or use it for any profit making activity or commercial gain • You may freely distribute the URL identifying the publication in the Aberystwyth Research Portal 1 British Antarctic Survey (BAS), Natural Environmental Research Council (NERC), High Cross, Madingley Road, Cambridge CB3 0ET, UK. 2 Institute for Chemistry and Biology of the Marine Environment (ICBM), Carl-von-Ossietzky-Str. 9–11, 26133 Oldenburg, Germany. 3 Department of Geosciences, Bremen University, Klagenfurter Str. 2–4, 28359 Bremen, Germany. 4 School of Geography, Politics and Sociology, Newcastle University, Newcastle-upon-Tyne NE1 7RU, UK. 5 Department of Geography, Northumbria University, Ellison Building, Newcastle-upon-Tyne NE1 8ST, UK. 6 Institute of Botany, Polish Academy of Sciences, ul. Lubicz 46, 31–512 Krako´w, Poland. 7 School of Earth and Ocean Sciences, Cardiff University, Main Building, Park Place, Cardiff CF10 3AT, UK. 8 NERC Radiocarbon Facility (Environment), Scottish Enterprise Technology Park, Rankine Avenue, East Kilbride, Scotland G75 OQF, UK. 9 Department of Geography and Earth Sciences, Aberystwyth University, Aberystwyth SY23 3DB, UK. 10 Department of Geography, Science Laboratories, Durham University, South Road, Durham DH1 3LE, UK. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to S.J.R. (email: sjro@bas.ac.uk) or to P.M. (email: monien@icbm.de). Take down policy 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. tel: +44 1970 62 2400 email: is@aber.ac.uk tel: +44 1970 62 2400 email: is@aber.ac.uk tel: +44 1970 62 2400 email: is@aber.ac.uk Download date: 24. Oct. 2024 Past penguin colony responses to explosive volcanism on the Antarctic Peninsula Stephen J. Roberts1,, Patrick Monien2,3,, Louise C. Foster1,4,, Julia Loftﬁeld2, Emma P. Hocking5, Bernhard Schnetger2, Emma J. Pearson4, Steve Juggins4, Peter Fretwell1, Louise Ireland1, Ryszard Ochyra6, Anna R. Haworth1,7, Claire S. Allen1, Steven G. Moreton8, Sarah J. Davies9, Hans-Ju¨rgen Brumsack2, Michael J. Bentley10 & Dominic A. Hodgson1,10 Stephen J. Roberts1,, Patrick Monien2,3,, Louise C. Foster1,4,, Julia Loftﬁeld2, Emma P. Hocking5, Bernhard Schnetger2, Emma J. Pearson4, Steve Juggins4, Peter Fretwell1, Louise Ireland1, Ryszard Ochyra6, Anna R. Haworth1,7, Claire S. Allen1, Steven G. Moreton8, Sarah J. Davies9, Hans-Ju¨rgen Brumsack2, Michael J. Bentley10 & Dominic A. Hodgson1,10 Changes in penguin populations on the Antarctic Peninsula have been linked to several environmental factors, but the potentially devastating impact of volcanic activity has not been considered. Here we use detailed biogeochemical analyses to track past penguin colony change over the last 8,500 years on Ardley Island, home to one of the Antarctic Peninsula’s largest breeding populations of gentoo penguins. The ﬁrst sustained penguin colony was established on Ardley Island c. 6,700 years ago, pre-dating sub-fossil evidence of Peninsula- wide occupation by c. 1,000 years. The colony experienced ﬁve population maxima during the Holocene. Overall, we ﬁnd no consistent relationships with local-regional atmospheric and ocean temperatures or sea-ice conditions, although the colony population maximum, c. 4,000–3,000 years ago, corresponds with regionally elevated temperatures. Instead, at least three of the ﬁve phases of penguin colony expansion were abruptly ended by large eruptions from the Deception Island volcano, resulting in near-complete local extinction of the colony, with, on average, 400–800 years required for sustainable recovery. 1 British Antarctic Survey (BAS), Natural Environmental Research Council (NERC), High Cross, Madingley Road, Cambridge CB3 0ET, UK. 2 Institute for Chemistry and Biology of the Marine Environment (ICBM), Carl-von-Ossietzky-Str. 9–11, 26133 Oldenburg, Germany. 3 Department of Geosciences, Bremen University, Klagenfurter Str. 2–4, 28359 Bremen, Germany. 4 School of Geography, Politics and Sociology, Newcastle University, Newcastle-upon-Tyne NE1 7RU, UK. 5 Department of Geography, Northumbria University, Ellison Building, Newcastle-upon-Tyne NE1 8ST, UK. 6 Institute of Botany, Polish Academy of Sciences, ul. Lubicz 46, 31–512 Krako´w, Poland. 7 School of Earth and Ocean Sciences, Cardiff University, Main Building, Park Place, Cardiff CF10 3AT, UK. 8 NERC Radiocarbon Facility (Environment), Scottish Enterprise Technology Park, Rankine Avenue, East Kilbride, Scotland G75 OQF, UK. 9 Department of Geography and Earth Sciences, Aberystwyth University, Aberystwyth SY23 3DB, UK. NATURE COMMUNICATIONS \| 8:14914 \| DOI: 10.1038/ncomms14914 \| www.nature.com/naturecommunications ARTICLE Received 16 Nov 2016 \| Accepted 14 Feb 2017 \| Published 11 Apr 2017 Received 16 Nov 2016 \| Accepted 14 Feb 2017 \| Published 11 Apr 2017 ARTICLE ) GC 47 Anvers Trough PD BI James Ross Island HB Weddell Sea Bransfie ld St ra it GC114 GC57 1/2/4/6/7 11/12 Penguin Record type Temperature Sea ice Tephra 0 250 500 km b © DigitalGlobe, Inc., All Rights Reserved 15 10 20 25 30 40 55 45 5 60 50 35 40 25 30 5 10 5 20 40 60 45 20 50 15 35 20 15 35 45 15 10 25 55 45 35 35 20 10 5 60 30 30 10 10 15 30 35 50 30 25 15 15 25 15 25 20 40 5 30 30 20 20 25 30 40 20 25 40 25 30 5 10 5 5 15 5 4 0 30 45 30 62°12'30"S 62°13'S Ardley Island Yanou Lake (YAN) Ardley Lake (ARD) Current penguin nesting area AD3 AD4 58°57'W 58°56'W Raised beaches 13.4–15 m 10.5–11.1 m 4.5–6.6 m d Y5 Y2 Y4 G Sinks Meltwater streams Lakes/ponds Ardley Lake Lake catchments 0 0.5 km 1 14 8 9 5 3 60°S 65°S 65°S 70°S 60°S 3 60°W PC460/1 a South Shetland Islands Deception Island Po la r F r ont Sept. (max.) Sept. Nov. Dec. Oct. Nov. Oct. Dec. F e b . ( m i n . ) GC 47 Anvers Trough PD BI James Ross Island HB Weddell Sea Bransfie ld St ra it GC114 GC57 1/2/4/6/7 11/12 Penguin Record type Temperature Sea ice Tephra 0 250 500 km b © DigitalGlobe, Inc., All Rights Reserved 58°56'W 59°0'W Fil de s P en insula 62°12'S 62°14'S c Maxwell Bay Fild es S t r ait d ARD YAN 0.15–0.4 0.4–1 0.1–0.15 NDVI Index 2 km 0 1 c King George Island Ardley Island b 12 13 © DigitalGlobe, Inc., All Rights Reserved 58°56'W 59°0'W Fil de s P en insula 62°12'S 62°14'S c Maxwell Bay Fild es S t r ait d ARD YAN 0.15–0.4 0.4–1 0.1–0.15 NDVI Index 2 km 0 1 c King George Island Ardley Island b . 12 13 © DigitalGlobe, Inc., All Rights Reserved c King George Island b 12 13 b a Maxwell Bay Figure 1 \| Study sites and the location of different types of records examined. ARTICLE ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14914 explosive (Volcanic Explosivity Index (VEI)25 of 6–7), producing an estimated 30–60 km3 of volcanic ash24,26, but the 20 þ eruptions of the ‘historical’ era (last c. 200 years) and the 30 þ eruptions identiﬁed in Holocene (11.75–0 ka) records have been smaller and less explosive. Most small eruptions from Deception Island present an immediate hazard to penguins nesting within its crater and in the immediate vicinity from ash-fall and pyroclastic ﬂows. However, ﬁne volcanic ash is often widely dispersed across the AP by strong Southern Westerly winds20,22,27,28. For example, following the December 1967–1970 CE eruptions (c. 0.1 km3, VEI ¼ 3)20, a ﬁne layer of tephra o2 mm thick was deposited on Ardley Island and the Fildes Peninsula, King George Island and South Shetland Islands (SSI), c. 120 km north-west of Deception Island (Fig. 1a)21, and in the James Ross Island (JRI) ice core, c. 200 km away on the north-eastern AP29. Even relatively minor E E vidence of different penguin species’ responses to changes in climate and other environmental variables are based on short-term observational records (typically the last 30–60 years1–8), studies of the age and provenance of sub-fossils found in abandoned penguin colonies9–16, and genetic and genomic studies of their evolutionary history17–19. Most of these studies have focussed on how climatic, oceanographic and anthropogenic factors affect penguin populations through access to food and nesting sites and predator–prey dynamics. As far as we are aware, no studies have investigated the long-term impact of large explosive volcanic eruptions on penguin colony size and distribution. Deception Island off the north-western Antarctic Peninsula (AP) (Fig. 1a) is a highly active volcano20–24. ARTICLE Its Late Pleistocene- early Holocene, caldera-forming eruption(s) were exceptionally 15 10 20 25 30 40 55 45 5 60 50 35 40 25 30 5 10 5 20 40 60 45 20 50 15 35 20 15 35 45 15 10 25 55 45 35 35 20 10 5 60 30 30 10 10 15 30 35 50 30 25 15 15 25 15 25 20 40 5 30 30 20 20 25 30 40 20 25 40 25 30 5 10 5 5 15 5 4 0 30 45 30 62°12'30"S 62°13'S Ardley Island Yanou Lake (YAN) Ardley Lake (ARD) Current penguin nesting area AD3 AD4 58°57'W 58°56'W Raised beaches 13.4–15 m 10.5–11.1 m 4.5–6.6 m d Y5 Y2 Y4 G Sinks Meltwater streams Lakes/ponds Ardley Lake Lake catchments 0 0.5 km 58°56'W 59°0'W Fil de s P en insula 62°12'S 62°14'S c Maxwell Bay Fild es S t r ait d ARD YAN 0.15–0.4 0.4–1 0.1–0.15 NDVI Index 2 km 0 1 c King George Island Ardley Island 1 14 8 9 5 3 60°S 65°S 65°S 70°S 60°S 3 60°W PC460/1 a b South Shetland Islands Deception Island Po la r F r ont Sept. (max.) Sept. Nov. Dec. Oct. Nov. Oct. Dec. F e b . ( m i n . ) GC 47 Anvers Trough PD BI James Ross Island HB Weddell Sea Bransfie ld St ra it GC114 GC57 1/2/4/6/7 11/12 12 13 Penguin Record type Temperature Sea ice Tephra 0 250 500 km b © DigitalGlobe, Inc., All Rights Reserved © DigitalGlobe, Inc., All Rights Reserved Figure 1 \| Study sites and the location of different types of records examined. (a) Antarctica, the northern Antarctic Peninsula (AP), the Anvers Trough marine core GC047 sampling site and the South Shetland Islands (SSI), located between the mean modern-day (NSIDC 1981–2010 CE; 20% sea-ice coverage) Austral summer minimum (February or Feb.) and winter maximum (September or Sept.) sea-ice limits (solid white lines) in the Bransﬁeld Strait area. The minimum (Feb.) sea-ice extent on the western side of the AP is located off-image further south of the December (Dec.) sea-ice extent (sea-ice data are from https://nsidc.org/data/seaice_index/). This ﬁgure illustrates how King George Island becomes sea-ice-free before other parts of the SSI and AP, enabling earlier nearshore sea-ice-edge foraging during the Austral spring/summer; locations 1–13 refer to penguin sub-fossil data shown in Fig. NATURE COMMUNICATIONS \| 8:14914 \| DOI: 10.1038/ncomms14914 \| www.nature.com/naturecommunications ARTICLE 5c and listed in Supplementary Data 5; other marine core locations used in tephra analysis are shown in Supplementary Fig. 20; BI ¼ Beak Island, HB ¼ Hope Bay, PD ¼ Palmer Deep; (b) King George Island (KGI) showing the location of records examined in this study, with legend as in a; (c,d) Fildes Peninsula, Ardley Island and the study sampling sites at Yanou Lake (YAN, on KGI), Ardley Lake (ARD) and previous study sites (Y2, Y4, G, AD3, AD4) (refs 42–44) on Ardley Island where bio-geochemical evidence of penguin occupation exists, and the location of meltwater pond Y5. The catchment area of Ardley Lake is c. 66,250 m2 and has an elevation range of c. 16–60 m above present sea level (m a.p.s.l.; mean elevation: 31±10 m a.p.s.l.) deﬁned from the King George Island Digital Elevation Model (KGI-DEM). Published raised beach elevations are in m a.p.s.l. as deﬁned in ref. 85. Vegetated areas in c were mapped from a Normalized Difference Vegetation Index (NDVI) analysis of satellite data acquired on 4 November 2013. See Supplementary Note 3. This ﬁgure includes material copyright of DigitalGlobe, Inc., All Rights Reserved, used with permission under a NERC-BAS educational license and not included in the Creative Commons license for the article. 15 10 20 25 30 40 55 45 5 60 50 35 40 25 30 5 10 5 20 40 60 45 20 50 15 35 20 15 35 45 15 10 25 55 45 35 35 20 10 5 60 30 30 10 10 15 30 35 50 30 25 15 15 25 15 25 20 40 5 30 30 20 20 25 30 40 20 25 40 25 30 5 10 5 5 15 5 4 0 30 45 30 62°12'30"S 62°13'S Ardley Island Yanou Lake (YAN) Ardley Lake (ARD) Current penguin nesting area AD3 AD4 58°57'W 58°56'W Raised beaches 13.4–15 m 10.5–11.1 m 4.5–6.6 m d Y5 Y2 Y4 G Sinks Meltwater streams Lakes/ponds Ardley Lake Lake catchments 0 0.5 km 1 14 8 9 5 3 60°S 65°S 65°S 70°S 60°S 3 60°W PC460/1 a South Shetland Islands Deception Island Po la r F r ont Sept. (max.) Sept. Nov. Dec. Oct. Nov. Oct. Dec. F e b . ( m i n . Past penguin colony responses to explosive volcanism on the Antarctic Peninsula 10 Department of Geography, Science Laboratories, Durham University, South Road, Durham DH1 3LE, UK. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to S.J.R. (email: sjro@bas.ac.uk) or to P.M. (email: monien@icbm.de). 1 NATURE COMMUNICATIONS \| 8:14914 \| DOI: 10.1038/ncomms14914 \| www.nature.com/naturecommunications TURE COMMUNICATIONS \| 8:14914 \| DOI: 10.1038/ncomms14914 \| w ARTICLE ARTICLE Ardley Island to provide a long-term and continuous record of changes in penguin occupation, because, unlike other shallow ponds on the island (Lake Y1, G, AD3 and AD4, Supplementary Notes 1–3), it has remained above sea-level for the last c. 9 ka (refs 45,46) (Fig. 1d). volcanic eruptions can be potentially devastating to the ecology at locations far from the eruption source30. In terms of penguin mortality, the ﬁne basaltic-andesitic tephra, most commonly produced by Deception Island eruptions, contains large volumes of ﬁne and physically abrasive glass, poisonous aerosols (for example, SO2, F), as well as trace elements and toxic metals (for example, Cd, As, Pb)31 that can adversely affect their physiological functions. ( ) ( g ) Our control site, Yanou Lake (62 13.243 S, 58 57.591 W; c. 11 m a.p.s.l.), located opposite Ardley Lake on Fildes Peninsula, is a former shallow marine basin, submerged below sea level until c. 6.5 ka, with basal sediments deposited in a glaciomarine environment between c. 12 and 7.8 ka (ref. 45). The marine embayment evolved into a shallow near-shore lagoonal basin c. 6.5–6.0 ka, and, when sea-level fell below the retaining sill at c. 14.5.–11 m a.p.s.l. (max.–min. elevation; ref. 45). Yanou Lake was formed and then developed into a predominantly freshwater lake until the present day. As there is no evidence of past or present penguin occupation in the Yanou Lake catchment, this record enabled us to separate the bio-geochemical inputs associated with penguin guano in the Ardley Lake record from those associated with the underlying geology, soils, tephra deposition and natural lake development. Gentoo penguin (Pygoscelis papua) populations across the AP and elsewhere have remained stable or increased over the last 30 years5,32,33, while more ‘sea-ice dependent’ species such as Ade´lie penguins (Pygoscelis adeliae) have declined32,34. These changes have been linked to ‘recent’ regional atmospheric and oceanographic warming on the AP7,8. Conversely, Ade´lie colony populations are increasing in parts of Antarctica (for example, the Ross Sea) where sea-ice is currently expanding35, or have increased during previous colder periods36. Recent AP-wide reductions in land and sea-ice extent/seasonality have altered access routes to breeding sites and have also caused changes in prey availability by shifting the location and distribution of northern-AP shelf-edge krill spawning grounds1,3,8,9,35,37. ARTICLE Since AP krill biomass has not changed signiﬁcantly over the same period35,38, recent changes could potentially relate to the early– mid twentieth century ‘krill-surplus’, which resulted from a reduction in predation pressure on krill due to the exploitation of large numbers of seals and whales in the late nineteenth century to mid twentieth century16,38,39. Gentoo penguins prefer to breed in ice-free areas, and are generalist, inshore predators (foraging within c. 20–30 km of their breeding sites), compared to the more krill-dependent, wider-ranging Ade´lie penguins, while reduced competition from other species on the AP has increased the availability of gentoo prey species across all trophic levels4,16,40. Intriguingly, genetic studies have shown that recent AP species- speciﬁc population trends are different from those of previous warm-periods, but that the response of gentoo penguins to warming is consistently positive16,17. In this study, we undertook detailed geochemical and multi- proxy analyses on an 8,500-year lake sediment record from Ardley Lake (ARD) and compared the results with a lake sediment record from Yanou Lake (YAN). Using variations in inorganic bio-element geochemistry, we established a novel method to determine the proportion of guano present in the ARD and YAN lake sediments—the ornithogenic sediment fraction or Fo.sed.. By converting the percentage Fo.sed. into guano-inﬂuenced sediment dry mass accumulation rates, we modelled estimates of penguin population change for the Ardley Lake colony (see Methods for details). We then determined local- regional drivers of past colony change, by comparing results from the ARD and YAN records with Peninsula-wide records of: (1) lacustrine and sub-fossil evidence of past penguin colony presence11,43; (2) palaeoclimate29, palaeoceanographic47,48 and relative sea level (RSL)45 change, including the ﬁrst AP lake sediment biomarker-based quantitative temperature reconstruction (Yanou Lake), and a new shelf-edge marine sediment record (Anvers Trough) (Fig. 1a; see Methods for details); (3) explosive volcanism from Deception Island, which we conclude had a signiﬁcant impact on colony population at least three times during the Holocene. Ardley Island in the SSI (Fig. 1c,d) contains one of the largest breeding gentoo penguin colonies in the Antarctic41 with c. 5,000 pairs (c. 1.3% of the current global gentoo population), alongside c. 200 breeding pairs of Ade´lie penguins and o50 breeding pairs of chinstrap penguins (P. antarctica) (Supplementary Fig. 1). ARTICLE Between 1950 and 1997 CE, the north-western AP was one of the most rapidly warming regions in the Southern Hemisphere7, and between 1980 and 2005 CE the number of breeding pairs of gentoo penguins on Ardley Island increased by c. þ 310% (minimum–maximum breeding pair count data), whereas between 1980 and 2014, Ade´lie ( 12%) and Chinstrap ( 3.7%) penguin populations have both declined (Supplementary Fig. 1). As each penguin produces c. 84.5 g of guano per day42, every breeding season c. 139 tonnes (dry mass) of penguin guano is discharged onto Ardley Island42, either accumulating in soils and shallow meltwater ponds and lakes (Fig. 1d), or discharging into the sea. Of these, lakes are protected by a permanent water column and can accumulate sediments without signiﬁcant disturbance. Geochemical analyses of guano signatures in lake sediments can thereby provide long-term records of past penguin presence in their catchments43,44 (Supplementary Note 1). NATURE COMMUNICATIONS \| 8:14914 \| DOI: 10.1038/ncomms14914 \| www.nature.com/naturecommunications ARTICLE (a) Antarctica, the northern Antarctic Peninsula (AP), the Anvers Trough marine core GC047 sampling site and the South Shetland Islands (SSI), located between the mean modern-day (NSIDC 1981–2010 CE; 20% sea-ice coverage) Austral summer minimum (February or Feb.) and winter maximum (September or Sept.) sea-ice limits (solid white lines) in the Bransﬁeld Strait area. The minimum (Feb.) sea-ice extent on the western side of the AP is located off-image further south of the December (Dec.) sea-ice extent (sea-ice data are from https://nsidc.org/data/seaice_index/). This ﬁgure illustrates how King George Island becomes sea-ice-free before other parts of the SSI and AP, enabling earlier nearshore sea-ice-edge foraging during the Austral spring/summer; locations 1–13 refer to penguin sub-fossil data shown in Fig. 5c and listed in Supplementary Data 5; other marine core locations used in tephra analysis are shown in Supplementary Fig. 20; BI ¼ Beak Island, HB ¼ Hope Bay, PD ¼ Palmer Deep; (b) King George Island (KGI) showing the location of records examined in this study, with legend as in a; (c,d) Fildes Peninsula, Ardley Island and the study sampling sites at Yanou Lake (YAN, on KGI), Ardley Lake (ARD) and previous study sites (Y2, Y4, G, AD3, AD4) (refs 42–44) on Ardley Island where bio-geochemical evidence of penguin occupation exists, and the location of meltwater pond Y5. The catchment area of Ardley Lake is c. 66,250 m2 and has an elevation range of c. 16–60 m above present sea level (m a.p.s.l.; mean elevation: 31±10 m a.p.s.l.) deﬁned from the King George Island Digital Elevation Model (KGI-DEM). Published raised beach elevations are in m a.p.s.l. as deﬁned in ref. 85. Vegetated areas in c were mapped from a Normalized Difference Vegetation Index (NDVI) analysis of satellite data acquired on 4 November 2013. See Supplementary Note 3. This ﬁgure includes material copyright of DigitalGlobe, Inc., All Rights Reserved, used with permission under a NERC-BAS educational license and not included in the Creative Commons license for the article. NATURE COMMUNICATIONS \| 8:14914 \| DOI: 10.1038/ncomms14914 \| www.nature.com/naturecommunications 2 NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14914 Results G Guano geochemistry. The geochemistry of Ardley Lake (ARD, study site) and Yanou Lake (YAN, control site) sediments showed signiﬁcant ﬂuctuations with sediment depth and modelled age (Figs 2–4, Supplementary Notes 4–7, Supplementary Figs 3–13, 16, Supplementary Tables 1–7, Supplementary Data 1). R-mode cluster analysis performed on the inter-correlation coefﬁcients of major and trace element concentrations in the ARD record clearly separated guano-derived elements (Sr, Cu, Zn, Se, Ca, Se, P, C, N, S) and guano-associated elements (Cd, As, Hg) from lithogenic elements (for example, Al, Mg, Si, Sc, Ti, Zr, Y and REE) derived from weathering of local volcanic bedrock (Fig. 2, Supplementary Fig. 8b, Supplementary Table 4). No similarly clear separation of elements was evident in the YAN control record (Supplementary Fig. 8c; Supplementary Table 5). The positive correlation between Ca and P (r ¼ 0.63, Po0.001; Supplementary Table 4) suggests that hydroxylapatite is the main phosphate phase in the ARD record (Supplementary Notes 1,8). Selected element-aluminium cross-plots (Fig. 3a) show that Ardley Lake sediments clearly represent a mixture between two end members: eroded Ardley Island bedrock and ornithogenically derived (bird-formed) soil and sediment from the lake’s catch- ment, whereas the geochemistry of Yanou Lake sediments is Ardley Lake (62 12.774 S, 58 56.398 W; c. 16 metres above present mean sea level, m a.p.s.l., as deﬁned in ref. 45) is the only large (c. 7,270 m2), permanent closed-basin water body on Ardley Island (c. 268,510 m2), and its only permanent depositional ‘sink’ (Fig. 1d, Supplementary Fig. 2). With two prominent meltwater inﬂows, and an overspill outﬂow bounded by its retaining sill at c. 18–16 m a.p.s.l. (max.-min. elevation45), the Ardley Lake catchment area (c. Results G 66,250 m2) is well-situated in the centre of 3 NATURE COMMUNICATIONS \| 8:14914 \| DOI: 10.1038/ncomms14914 \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14914 0 4 8 Visible tephra deposits Airfall tephra Redeposited tephra Munsell colour 2.5Y 2.5/1 Black 2.5Y 3/3 Dark olive brown 2.5Y 3/1 Very dark grey 2.5Y 3/1 Dark grey 2.5Y 3/2 Very dark greyish brown 2.5Y 4/3 Olive brown 2.5Y 4/2 Dark greyish brown 2.5Y 4/4 Olive brown Sedimentology Silt Clay Sand Tephra-rich (<50% tephra) Tephra bed (>50% tephra) Nodules / peloids Matrix-supported diamicton Clast-supported diamicton Moss bed (>50% moss) Moss fragments (<50% moss) 1 2 4a 5 Penguin bones (B1–B6) and bone fragments 1 3 0.1 0.3 0.5 0.1 0.25 0.4 0 0.4 0.8 Sum of squares CONISS XRF Zones (13) Guano phases 50 150 1 3 5 50 150 M7 M8A M7A Munsell colour Visible tephra Sedimentology Munsell colour Visible tephra Sedimentology Clay Silt Sand Grain size Fine Med. Coarse 0 50 100 150 200 250 300 350 Depth (cm) 0 50 100 150 200 250 300 350 Depth (cm) TC/Al TN/Al TS/Al P/Al Zn/Al (x 10–4) As/Al (x 10–4) Cu/Al (x 10–4) Guano and guano-related elements 4b 3 Diatom zones Lake environment T4 T1 T3 B1 B2 B3/4 B5 B6 B1 B2 B3/4 B5 B6 b c ab T6 T7 T8? T5b T5a T4 T1 T3b c ab T6 T7 T8? T5b T5a Fresh– brackish D1 D2 D3 D4 D5 D6 Fresh– brackish to brackish F.-Brack. Brackish– fresh Fresh– brackish and fresh Fresh and fresh– brackish B6 B1 B2 B3/4 2.6 3.0 0.08 0.12 100 200 300 0.5 1.5 0 0.01 0.02 0 4 8 10 20 30 Depth (cm) Guano and guano-related elements Lithogenic elements Si/Al Ti/Al Sr/Al (x 10–4) Ca/Al Hg/Al (x 10–4) Se/Al (x 10–4) Zr/Al (x 10–4) 0 50 100 150 200 250 300 350 1 3 5 4b 4a 2 Moss Bulk organic (fresh) Lacustrine algae SH13-Terrestrial Guano-influenced sed. MARINE13 (ΔR=700±50) Bulk organic (brackish) Penguin bone–collagen Age (cal a BP) 0 2,000 4,000 6,000 8,000 10,000 M7 M8A M7A B1 B2 B2.1 B2.2 B4.1 B6.1 B6.2 B6.3 B6.4 B3 B4 B5 B6 Figure 2 \| Whole core geochemistry of Ardley Lake sediments. Downcore proﬁles of selected aluminium normalized guano-elements (P, Zn, As, Cu, TN, TS, TC, Ca, Sr, Hg, Se), lithogenic elements (Si, Ti, Zr) and age-depth model (ARD-M5) of the ARD record (Supplementary Data 1,2). Results G MARINE13 (ΔR=700±50) Bulk organic (brackish) Penguin bone–collagen Age (cal a BP) 0 2,000 4,000 6,000 8,000 10,000 M7 M8A M7A B1 B2 B2.1 B2.2 B4.1 B6.1 B6.2 B6.3 B6.4 B3 B4 B5 B6 Figure 2 \| Whole core geochemistry of Ardley Lake sediments. Downcore proﬁles of selected aluminium normalized guano-elements (P, Zn, As, Cu, TN, TS TC Ca Sr Hg Se) lithogenic elements (Si Ti Zr) and age depth model (ARD M5) of the ARD record (Supplementary Data 1 2) Intact juvenile Depth (cm) Figure 2 \| Whole core geochemistry of Ardley Lake sediments. Downcore proﬁles of selected aluminium normalized guano-elements (P, Zn, As, Cu, TN, TS, TC, Ca, Sr, Hg, Se), lithogenic elements (Si, Ti, Zr) and age-depth model (ARD-M5) of the ARD record (Supplementary Data 1,2). Intact juvenile Pygoscelis Papua bones found at core depths are labelled B1–B6 (scale bars are 1 cm): B1 is a cnemial crest bone of the tibiotarsus (left leg); at B2, two base occipital condyle bones from a poorly fused skull(s); B3 is a heavily weathered metatarsal (foot) bone; at B4, two weathered carpometacarpus (wing) bones; B5 is a talon; B6 are mid-upper neck cervical vertebrae bones, found intact vertically (4 of 5 bones shown). Bone-collagen was extracted and radiocarbon dated from bones B2.1, B4.1, B6.2 (see Supplementary Fig. 4 and Supplementary Table 2 for details). strongly associated with local bedrock and tephra deposition. The inﬂuence of penguin guano on the sediment samples from Ardley Lake is further supported by their C/N and C/P ratios (C/N ¼ av. 6.6, C/P ¼ av. 3.3), which are close to those previously reported in ornithogenic soils (C/N ¼ 6.3, C/P ¼ 1.2–3.7) (ref. 49) (Fig. 3b). In contrast, they differ signiﬁcantly from ratios found in phyto- plankton and local plants, for example, carpet mosses, lichens (Usnea antarctica), liverworts and vascular plants, such as Deschampsia antarctica (Fig. 3b, C/N ¼ 21–114)50 present in the catchment area of both lakes. Guano phases. Sustained high concentrations of bio-elements (weighted mean Fo.sed.410%) and stratigraphically constrained incremental sum of squares (CONISS) cluster analysis were used to deﬁne ﬁve phases of elevated guano ﬂux (GP-1 to GP-5) (shown as 1–5 in circles in Figs 2, 4 and 5; see Methods for details). Periods of exceptionally elevated guano within each guano phase (darker green shading in Fig. Results G Intact juvenile Pygoscelis Papua bones found at core depths are labelled B1–B6 (scale bars are 1 cm): B1 is a cnemial crest bone of the tibiotarsus (left leg); at B2, two base occipital condyle bones from a poorly fused skull(s); B3 is a heavily weathered metatarsal (foot) bone; at B4, two weathered carpometacarpus (wing) bones; B5 is a talon; B6 are mid-upper neck cervical vertebrae bones, found intact vertically (4 of 5 bones shown). Bone-collagen was extracted and radiocarbon dated from bones B2.1, B4.1, B6.2 (see Supplementary Fig. 4 and Supplementary Table 2 for details). 0 4 8 Visible tephra deposits Airfall tephra Redeposited tephra Munsell colour 2.5Y 2.5/1 Black 2.5Y 3/3 Dark olive brown 2.5Y 3/1 Very dark grey 2.5Y 3/1 Dark grey 2.5Y 3/2 Very dark greyish brown 2.5Y 4/3 Olive brown 2.5Y 4/2 Dark greyish brown 2.5Y 4/4 Olive brown Sedimentology Silt Clay Sand Tephra-rich (<50% tephra) Tephra bed (>50% tephra) Nodules / peloids Matrix-supported diamicton Clast-supported diamicton Moss bed (>50% moss) Moss fragments (<50% moss) 1 2 4a 5 Penguin bones (B1–B6) and bone fragments 1 3 0.1 0.3 0.5 0.1 0.25 0.4 0 0.4 0.8 Sum of squares CONISS XRF Zones (13) Guano phases 50 150 1 3 5 50 150 M7 M8A M7A Munsell colour Visible tephra Sedimentology Munsell colour Visible tephra Sedimentology Clay Silt Sand Grain size Fine Med. Coarse 0 50 100 150 200 250 300 350 Depth (cm) 0 50 100 150 200 250 300 350 Depth (cm) TC/Al TN/Al TS/Al P/Al Zn/Al (x 10–4) As/Al (x 10–4) Cu/Al (x 10–4) Guano and guano-related elements 4b 3 Diatom zones Lake environment T4 T1 T3 B1 B2 B3/4 B5 B6 B1 B2 B3/4 B5 B6 b c ab T6 T7 T8? T5b T5a T4 T1 T3b c ab T6 T7 T8? T5b T5a Fresh– brackish D1 D2 D3 D4 D5 D6 Fresh– brackish to brackish F.-Brack. Brackish– fresh Fresh– brackish and fresh Fresh and fresh– brackish B6 B1 B2 B3/4 2.6 3.0 0.08 0.12 100 200 300 0.5 1.5 0 0.01 0.02 0 4 8 10 20 30 Depth (cm) Guano and guano-related elements Lithogenic elements Si/Al Ti/Al Sr/Al (x 10–4) Ca/Al Hg/Al (x 10–4) Se/Al (x 10–4) Zr/Al (x 10–4) 0 50 100 150 200 250 300 350 1 3 5 4b 4a 2 Moss Bulk organic (fresh) Lacustrine algae SH13-Terrestrial Guano-influenced sed. NATURE COMMUNICATIONS \| 8:14914 \| DOI: 10.1038/ncomms14914 \| www.nature.com/naturecommunications Results G 4b) were deﬁned as 4 the upper bound (95% conﬁdence level) of the whole-record weighted mean Fo.sed. value of 27.93% (green dotted line in Fig. 4b). g g The ﬁrst occupation of the Ardley Lake catchment by penguins occurred between 6.7 and 6.3 cal ka BP (GP-1), with further Fo.sed. maxima between 5.8–5.3 (GP-2), 4.5–4.3 (GP-3), 4.0–3.0 (GP-4) and 2.7–1.3 (GP-5) cal ka BP (Figs 4b and 5, Table 1). No similar patterns were found in the control site YAN (Fig. 4a, Supplementary Figs 10,11c). Intact juvenile gentoo penguin bones and bone fragments were found in guano phases GP-2 and GP-4 and in sediments deposited between GP-2 and GP-3 (Fig. 2, Supplementary Fig. 4). Guano dry mass accumulation rates (Fo.sed. DMAR in g cm 2 a 1, thick black line in Fig. 4b) account for variations in the sedimentation rate that reﬂect changes in erosional input into Ardley Lake, and, therefore, do not always correspond to higher guano-inﬂuenced sediment Fo.sed. percentages. Since Fo.sed. DMAR values underpin In the ARD record, in-phase changes in several aluminium- normalized bio (associated) elements, such as P, Zn, As, Cu, Ca, Sr, Hg, and Se, together with TN, TC (equivalent to TOC) and TS were recorded (Fig. 2, Table 1, Supplementary Figs 6–8,11b,16). This enabled us to derive the mean relative proportion of guano or ornithogenically derived sediment (% Fo.sed.) in the Ardley Lake sediment matrix using a mixing equation (equation (1, Methods) based on the element/aluminium (Al) ratio of selected bio-elements (Cu, P, Sr, Zn) in the Ardley Lake sediments, the mean Al-normalized Ardley Island bedrock51 composition and Al-normalized mean composition of guano-bearing ornithogenic sediment and soils49 from King George Island as end members (see Methods for details). NATURE COMMUNICATIONS \| 8:14914 \| DOI: 10.1038/ncomms14914 \| www.nature.com/naturecommunications 4 4 NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14914 NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14914 \| 0 1 2 3 2 4 6 8 10 Al (mass %) a Mg (mass %) 0 0.2 0.4 0.6 0.8 2 4 6 8 10 Al (mass %) Cu (mg g–1) 0 0.2 0.4 0.6 0.8 1.0 2 4 6 8 10 Al (mass %) Zn (mg g–1) 0 5 10 15 20 2 4 6 8 10 Al (mass %) Ca (mass %) 0.5 1.0 1.5 2.0 2.5 2 4 6 8 10 Al (mass %) Sr (mg g–1) 0 2 4 6 8 10 2 4 6 8 10 Al (mass %) P (mass %) r = 0.89 P<0.0001 r = –0.63 P<0.0001 r = –0.90 P<0.0001 r = –0.94 P<0.0001 r = –0.88 P<0.0001 r = –0.90 P<0.0001 0 1 2 3 2 4 6 8 10 Al (mass %) a Mg (mass %) r = 0.89 P<0.0001 0 0.2 0.4 0.6 0.8 2 4 6 8 10 Al (mass %) Cu (mg g–1) r = –0.88 P<0.0001 0 0.2 0.4 0.6 0.8 1.0 2 4 6 8 10 Al (mass %) Zn (mg g–1) r = –0.90 P<0.0001 a Al (mass %) 0 5 10 15 20 2 4 6 8 10 Al (mass %) Ca (mass %) r = –0.63 P<0.0001 0.5 1.0 1.5 2.0 2.5 2 4 6 8 10 Al (mass %) Sr (mg g–1) r = –0.90 P<0.0001 0 2 4 6 8 10 2 4 6 8 10 Al (mass %) P (mass %) r = –0.94 P<0.0001 P (mass %) Ardley Island bedrock Fildes Peninsula bedrock Ardley Lake (ARD) sediments Yanou Lake (YAN) sediments ARD samples Liverworts Moss Lichens Ornithogenic soils (g Ornithogenic soils (h Ornithogenic soils (p Vascular plants 0 10 20 30 40 50 0 1 2 3 4 5 TN (mass %) TC (mass %) r = 0.98 P<0.0001 b Yanou Fildes Peninsula bedrock \| Scatterplots of element/aluminium ratios and comparison of C ake and Yanou Lake sediments (this study) ornithogenic soils49 a ARD samples Liverworts Moss Lichens Ornithogenic soils (guano) Ornithogenic soils (humus) Ornithogenic soils (phosphatic clay) Vascular plants 0 10 20 30 40 50 0 TC (mass %) 0 10 20 30 40 50 0 1 2 3 4 5 TN (mass %) TC (mass %) r = 0.98 P<0.0001 b Yanou Lake (YAN) sediments Fildes Peninsula bedrock gure 3 \| Scatterplots of element/aluminium ratios and comparison of C/N and C/P ratios. NATURE COMMUNICATIONS \| 8:14914 \| DOI: 10.1038/ncomms14914 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14914 ARTICLE ation models, guano phases GP-2, GP-3 and GP-4 corres- to a higher number of penguins present in the Ardley Lake h i Fi b ( M h d S l N details). A good correspondence between the Ardley Lake fossil record and the sub-fossil record of former penguin c f h AP i d i h h d il ARD samples Liverworts Moss Lichens Ornithogenic soils (guano) Ornithogenic soils (humus) Ornithogenic soils (phosphatic clay) Vascular plants 0 10 20 30 40 50 0 2 4 6 8 P (mass %) TC (mass %) 0 10 20 30 40 50 0 1 2 3 4 5 TN (mass %) TC (mass %) r = 0.98 ARD data: linear regression summary statistics Redfield ratio P<0.0001 r = 0.89 P<0.0001 Ardley Lake (ARD) sediments Ornithogenic soils Ardley Island bedrock 0 1 2 3 2 4 6 8 10 Al (mass %) a b Mg (mass %) 0 0.2 0.4 0.6 0.8 2 4 6 8 10 Al (mass %) Cu (mg g–1) 0 0.2 0.4 0.6 0.8 1.0 2 4 6 8 10 Al (mass %) Zn (mg g–1) 0 5 10 15 20 2 4 6 8 10 Al (mass %) Ca (mass %) 0.5 1.0 1.5 2.0 2.5 2 4 6 8 10 Al (mass %) Sr (mg g–1) 0 2 4 6 8 10 2 4 6 8 10 Al (mass %) P (mass %) Yanou Lake (YAN) sediments Fildes Peninsula bedrock r = 0.89 P<0.0001 r = –0.63 P<0.0001 r = –0.90 P<0.0001 r = –0.94 P<0.0001 r = –0.88 P<0.0001 r = –0.90 P<0.0001 ARD: linear regression summary statistics 3 \| Scatterplots of element/aluminium ratios and comparison of C/N and C/P ratios. (a) Scatterplots of selected element/aluminium Lake and Yanou Lake sediments (this study), ornithogenic soils49 and local bedrock86. (b) Total Carbon/Total Nitrogen (TC/TN) and To n/Phosphorus (TC/P) ratios of Ardley Lake sediments, local lichens (for example, Usnea antarctica)50, liverworts (Cephalozia varians), mos ar plants (Deschampsia antarctica)50 and different types of ornithogenic soils49. The solid and the dashed lines show linear regression lines of ore ARD, and C/N and C/P ratios of phytoplankton according to the Redﬁeld ratio, respectively (Supplementary Data 3). ARTICLE and JRI atmospheric temperature (Fig. 4f)29. Overall, none of the guano phases were statistically related to warmer sea surface temperatures in the Palmer Deep (PD) record. Conversely, the non-guano phases were associated with signiﬁcantly warmer sea surface temperatures in the Palmer Deep record (PD-SST 0–200 m; Fig. 4e)48 (two-tailed Mann–Whitney U-tests: YAN-GDGT: P ¼ 0.58, JRI: P ¼ 0.57, PD-SST: P ¼ 0.01; Supplementary Discussion, Supplementary Palaeoenvironmental records. We then compared these ﬁve phases of elevated guano ﬂux with a range of local and regional records of past environments to identify the major drivers of changes in penguin populations. First, we found no signiﬁcant difference (5 or 10% level) in temperature data during guano and non-guano phases from cross-northern Peninsula 9–0 ka terrestrial temperature records, YAN-GDGT reconstructed mean summer air temperatures (MSAT) (Fig. 4a) Warm SST (0–200 m) Warm surface SST SST (°C) 0 2 Palmer Deep Temp. Record +Warm +Warm 0–200 m c Maxwell Bay Sea-ice Record TOC (%) 0.0 0.6 0.3 +Open water d Diatom ratio (F. curta/F. kerg.) Pelagic open water diatoms (%) 0 10 20 30 Anvers Shelf Sea-ice Record 0 1 2 3 1 2 4 5 3 Yanou Lake Temp. Record a GDGT-MSAT 6–0 ka Anomaly (°C ±1σ) +Warm (MSAT) Ardley Lake b e +Sea ice +Open water –1 0 1 PCA2μ-XRF (9%) T8? 0.00 0.05 0.10 ? T3b T1 0 10 20 SandDMAR (g cm–2 a–1) Fo.sed. DMAR (g cm–2 a–1) SandDMAR (g cm–2 a–1) T2 T4 T5 T6 T3a 0 0.05 Age (cal a BP) 0 2,000 4,000 6,000 8,000 10,000 g AP Warm periods RRR MCA? f James Ross Island Ice Core Temp. Record JRI temp. anomaly (°C ±1σ) –1 0 1 Airfall/ tephra and reworked sediment T7 6–0 ka upper bound on mean (95%) 100-year smoothed <10 °C 6–0 ka weighted mean MHH EHO to 11.5 ka 10% 0% % Guano (Fo.sed. % ±1σ) 95% conf. upper bound 0 50 100 Guano phases Guano phases 1 4 5 3 2 Volcanic eruptions 0.00 0.01 0.02 0.03 %Fo.sed. 100-year Fo.sed. ARTICLE DMAR –5 0 5 10 +Brackish conditions / >wind Glaciomarine to 11 ka Palaeoenvironmental data >6–0 ka upper bound on mean (95%): Open water 6-0 ka weighted mean 6–0 ka upper bound on mean (95%) 6–0 ka lower bound on mean (95%) >6–0 ka upper bound on mean (95%): Discontinuou >6–0 ka upper bound on mean (95%): Sustained w >2x 6–0 ka upper bound on mean (95%): Sustained AP composite warm periods Volcanic eruptions VEI>4 VEI>3 VEI< or =3 Weighted mean Eruption age and 95% min.–max. (cal a BP) Visible tephra No guano Ardley Lake post-eruption colony recovery interval ARD Guano phases GP-1 to GP-5 Penguin data ARD exceptionally elevated guano phases Published bone and egg ages (cal a ± 1σ) Diatom zone boundaries Modelled ARD bone ages (cal a ± 95% min.–max.) ARD bone–collagen ages (cal a ± 1σ) Marine 100% Marine 100% Lacustrine Brackish Brackish Colder C lder o e d o e Colder er Warme glaciating Deg RSL<5 m 5 m Penguin Record NATURE COMMUNICATIONS \| 8:14914 \| DOI: 10 1038/ncomms14914 \| www nature com/naturecom Yanou Lake Temp. Record a GDGT-MSAT 6–0 ka Anomaly (°C ±1σ) +Warm (MSAT) Ardley Lake b –1 0 1 PCA2μ-XRF (9%) T8? 0.00 0.05 0.10 ? T3b T1 0 10 20 SandDMAR (g cm–2 a–1) Fo.sed. DMAR (g cm–2 a–1) SandDMAR (g cm–2 a–1) T2 T4 T5 T6 T3a 0 0.05 Airfall/ tephra and reworked sediment T7 6–0 ka upper bound on mean (95%) 100-year smoothed <10 °C 6–0 ka weighted mean 10% 0% % Guano (Fo.sed. % ±1σ) 95% conf. upper bound 0 50 100 Guano phases 1 4 5 3 2 Volcanic eruptions 0.00 0.01 0.02 0.03 %Fo.sed. 100-year Fo.sed. DMAR –5 0 5 10 Glaciomarine to 11 ka Marine 100% Marine 100% Lacustrine Brackish Brackish Colder C lder o e d o e Colder er Warme glaciating Deg RSL<5 m 5 m Penguin Record Yanou Lake Temp. Record a Warm SST (0–200 m) Warm surface SST SST (°C) 0 2 Palmer Deep Temp. Record +Warm +Warm 0–200 m c Maxwell Bay Sea-ice Record TOC (%) 0.0 0.6 0.3 +Open water d Diatom ratio (F. curta/F. kerg.) Pelagic open water diatoms (%) 0 10 20 30 Anvers Shelf Sea-ice Record 0 1 2 3 1 2 4 5 3 e +Sea ice +Open water Age (cal a BP) 0 2,000 4,000 6,000 8,000 10,000 g AP Warm periods RRR MCA? NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14914 (a) rdley Lake and Yanou Lake sediments (this study), ornithogenic soils49 and local bedrock86. (b) T arbon/Phosphorus (TC/P) ratios of Ardley Lake sediments, local lichens (for example, Usnea anta b 0 10 20 30 40 50 0 2 4 6 8 P (mass %) TC (mass %) r = 0.89 P<0.0001 Redfield ratio Figure 3 \| Scatterplots of element/aluminium ratios and comparison of C/N and C/P ratios. (a) Scatterplots of selected element/aluminium ratios of Ardley Lake and Yanou Lake sediments (this study), ornithogenic soils49 and local bedrock86. (b) Total Carbon/Total Nitrogen (TC/TN) and Total Carbon/Phosphorus (TC/P) ratios of Ardley Lake sediments, local lichens (for example, Usnea antarctica)50, liverworts (Cephalozia varians), mosses50, vascular plants (Deschampsia antarctica)50 and different types of ornithogenic soils49. The solid and the dashed lines show linear regression lines of samples from core ARD, and C/N and C/P ratios of phytoplankton according to the Redﬁeld ratio, respectively (Supplementary Data 3). population models, guano phases GP-2, GP-3 and GP-4 corres- pond to a higher number of penguins present in the Ardley Lake catchment shown in Fig. 5b (see Methods, Supplementary Note 8, Supplementary Figs 16–19, Supplementary Table 9 for more details). A good correspondence between the Ardley Lake guano/ fossil record and the sub-fossil record of former penguin colonies from across the AP exists during these phases, and until the end of guano phase GP-5, c. 1.3 cal ka BP (Fig. 5). 5 NATURE COMMUNICATIONS \| 8:14914 \| DOI: 10.1038/ncomms14914 \| www.nature.com/naturecommunication NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14914 ARTICLE % ¼ 43.66±15.37 (1s) %), occurred between 3.4 and 3.0 cal ka BP when both the YAN-GDGT and JRI records were signiﬁcantly warmer than their 6–0 ka means (Fig. 4, Supplementary Fig. 18, Supplementary Tables 7,8). Second, we compared the guano phases with sea-ice and open water proxies, which impact on the penguins’ access to nesting sites and prey species. Although guano-phase GP-4 coincided with a sustained period of reduced spring/summer open water seasonality in the Anvers Trough record from c. 4 cal ka BP onwards (Fig. 4d), there were no signiﬁcant differences in open water conditions in Maxwell Bay (P ¼ 0.26) or at the Anvers Shelf (P ¼ 0.25) for guano and non-guano phases. More prolonged spring/summer open water conditions existed at the Anvers Trough shelf-edge in mid-Holocene guano phases (GP-2 and GP-3), but not during late Holocene guano phases (GP-4 and GP-5) (P ¼ 0.10). Volcanic activity. Third, we compared the guano phases with the combined record of volcanic tephra in the ARD and YAN lake sediment records. We identiﬁed seven phases of VEI ¼ 3 or VEI43 eruption activity (T1–T7) (Fig. 4a,b). Eruption T7 aside, Two other VEI ¼ 43 eruptions (T3a, T2) occurred at the same time as measurable, but not-signiﬁcant reductions in the Table 1 \| Summary statistics of guano phases GP-1 to GP-5 timing and duration and guano-inﬂuenced sediment percentage data. ARD ARD ARD Calibrated ages (cal a BP) Duration n % Guano-inﬂuenced sediment CONISS zone Guano phase Core depth min.–max. (cm) Weighted mean age min.–max. 95% conf. age range min.–max. (years±95% conf. diff.) [95% min.–max.] Weighted mean Fo.sed. (%±1r) 95% Conf. Fo.sed. Interval (lower–upper %) 12 5 13–35 1,290–2,670 1,160–2,830 1,380±145 [1,670] 11 23.57±10.74 16.35–30.79 9 4b 65–112 3,000–3,430 2,880–3,580 1,010±170 [1,350] 26 43.66±15.37 39.47–47.85 10 4a 112–155 3,430–4,010 3,270–4,230 26 12.95±2.76 11.41–14.49 7 3 175–190 4,310–4,510 4,090–4,640 200±175 [550] 7 26.33±12.16 18.92–33.74 5 2 284–310 5,300–5,750 5,180–6,020 450±195 [840] 9 29.38±9.90 21.95–36.82 3 1 320–326 6,270–6,670 5,820–7,360 400±570 [1,540] 3 26.77±9.49 5.31–48.23 Summary Whole record — 198 25.31±9.41 22.69–27.93 statistics Guano phases 690±250 [1,190] 82 34.19±12.19 31.06–37.32 Non-guano phases — 116 3.56±2.49 2.42–4.69 Late Holocene guano phases 1,200±160 [1,510] 62 36.57±12.73 32.73–40.40 Mid Holocene guano phases 350±310 [980] 20 27.77±10.72 23.66–31.88 All ages are in calibrated (equivalent to calendar) years before present (cal a BP), where BP is deﬁned as 1950 CE (see Supplementary Methods); [95% min. ARTICLE (cal years) is the average of the weighted mean minimum 95% conﬁdence minimum ages and the 95% conﬁdence maximum – weighted mean maximum ages; the duration of the 95% conﬁdence minimum to maximum age range is shown in square brackets. Guano-inﬂuenced sediment (Fo.sed.) weighted mean percentages ±1s, signiﬁcance level a ¼ 0.05.. Figure 4 \| Penguin occupation phases in the Ardley Lake record compared with key records of volcanic activity climate and sea-ice from the Antarctic Peninsula region for the last 10,000 years. In order from top: (a) Yanou Lake (YAN) sediment core palaeoenvironmental summary, reconstructed mean summer air temperature (MSAT) GDGT 100-year temperature anomaly data; 200 mm micro-XRF (m-XRF) Principal Component Analysis 2nd axis (PCA2, 9% variance explained) and sand Dry Mass Accumulation Rate (DMAR); main visible tephra deposits are black diagonally hatched zones, dotted lines show correlation of tephra layers between the ARD and YAN records, circle size reﬂects Volcanic Explosivity Index (VEI) of major eruptions and circle position is the weighted mean modelled age with 95% min.–max. conﬁdence age ranges (grey horizontal bars) based on 2.5% and 97.5% quantiles from Bayesian age-depth modeling (see Supplementary Notes 5–7 and Supplementary Methods). (b) Numbered, green-shaded guano phases GP-1 to GP-5 with the calculated fractions of ornithogenic sediments in the Ardley Lake record (Fo.sed. %±1s errors in grey) overlain by Fo.sed. dry mass accumulation rate (DMAR) (thick black line, 1s errors not shown for clarity, but in GP-4 and GP-5 these are on average c. 25-35%); red stippled areas are recovery intervals; white squares are radiocarbon-dated penguin bone-collagen extract ages; grey squares are modelled ages of bones in the ARD record. (c) Maxwell Bay (MB) percentage Total Organic Carbon (TOC) data (in grey) and 100-year interval negative exponential smoothed data (dark blue) used as sea-ice proxy52; data 46–0 ka upper bound on the mean (95% conﬁdence level) represents increased open water conditions (shaded dark blue). (d) Anvers Trough GC047/TC046 sediment core: the commonly used diatom-based ratio (Fragilariopsis curta: Fragilariopsis kerguelensis) with data 6–0 ka upper bound on the mean (95% conﬁdence level) shaded dark blue representing increased open-water conditions and percentage of total spring/summer pelagic open- water diatoms species (red line). ARTICLE f James Ross Island Ice Core Temp. Record JRI temp. anomaly (°C ±1σ) –1 0 1 MHH EHO to 11.5 ka Guano phases +Brackish conditions / >wind Palaeoenvironmental data >6–0 ka upper bound on mean (95%): Open water 6-0 ka weighted mean 6–0 ka upper bound on mean (95%) 6–0 ka lower bound on mean (95%) >6–0 ka upper bound on mean (95%): Discontinuous warmth >6–0 ka upper bound on mean (95%): Sustained warmth >2x 6–0 ka upper bound on mean (95%): Sustained warmth AP composite warm periods Volcanic eruptions VEI>4 VEI>3 VEI< or =3 Weighted mean Eruption age and 95% min.–max. (cal a BP) Visible tephra No guano Ardley Lake post-eruption colony recovery interval ARD Guano phases GP-1 to GP-5 Penguin data ARD exceptionally elevated guano phases Published bone and egg ages (cal a ± 1σ) Diatom zone boundaries Modelled ARD bone ages (cal a ± 95% min.–max.) ARD bone–collagen ages (cal a ± 1σ) \| \| \| NATURE COMMUNICATIONS \| 8:14914 \| DOI: 10.1038/ncomms14914 \| www.nature.com/naturecommunications 6 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14914 mid-late Holocene tephra layers had well-deﬁned basaltic- andesitic compositions, typical of Deception Island source com- positions (Supplementary Figs 20,21, Supplementary Table 10). Three VEI43 eruption events (T5a, T4, T3b) coincided with immediate and signiﬁcant reductions in guano deposition (that is, Fo.sed. to o10%) (Fig. 4a,b, Table 1). The most disruptive series of eruptions (T5a,b) recorded in both ARD and YAN sediments occurred during the mid-Holocene between c. 5.5–4.9 cal ka BP (combined ARD–YAN 95% maximum to mean age range; Fig. 4, Table 2, Supplementary Figs 12,16, Supplementary Table 3). Brackish conditions, greater catchment destabilization and increased erosion occurred for c. 1,000 years during the T5 eruption series (Fig. 4, Supplementary Fig. 16). The T4 (c. 4.5–4.2 cal ka BP) and T3b (c. 3.2–3.0 cal ka BP) eruptions that followed the T5 eruption series were smaller, but also coincided with immediate and signiﬁcant reductions in guano deposition. Colony recovery after the T5a eruption took 780±50 years [530– 1,130 years], and 410±30 [0–760] years and 480±60 [160–390] years after the T4 and T3b eruptions, respectively, with an overall weighted mean (±s.e.) recovery time of 570±110 years (Table 2). Table 8). Notably, the late Holocene guano Fo.sed. DMAR maxima in guano phase GP-4 (Fo.sed. NATURE COMMUNICATIONS \| 8:14914 \| DOI: 10.1038/ncomms14914 \| www.nature.com/naturecommunications ARTICLE max.] is the minimum and maximum 95% conﬁdence age range based on 2.5% and 97.5% quantiles from Bayesian age-depth modelling (see Supplementary Notes 5–7 and Supplementary Methods); durations are in calendar years with guano phase 4 shown as the combined duration of 4a and 4b; 95% conf. diff. (cal years) is the average of the weighted mean minimum 95% conﬁdence minimum ages and the 95% conﬁdence maximum – weighted mean maximum ages; the duration of the 95% conﬁdence minimum to maximum age range is shown in square brackets. Guano-inﬂuenced sediment (Fo.sed.) weighted mean percentages ±1s, signiﬁcance level a ¼ 0.05.. Table 1 \| Summary statistics of guano phases GP-1 to GP-5 timing and duration and guano-inﬂuenced sediment percentage data. Table 1 \| Summary statistics of guano phases GP-1 to GP-5 timing and duration and guano-inﬂuenced of guano phases GP-1 to GP-5 timing and duration and guano-inﬂuenced sediment percentage data. mmary statistics of guano phases GP-1 to GP-5 timing and duration and guano-inﬂuenced sediment per All ages are in calibrated (equivalent to calendar) years before present (cal a BP), where BP is deﬁned as 1950 CE (see Supplementary Methods); [95% min. max.] is the minimum and maximum 95% conﬁdence age range based on 2.5% and 97.5% quantiles from Bayesian age-depth modelling (see Supplementary Notes 5–7 and Supplementary Methods); durations are in calendar years with guano phase 4 shown as the combined duration of 4a and 4b; 95% conf. diff. (cal years) is the average of the weighted mean minimum 95% conﬁdence minimum ages and the 95% conﬁdence maximum – weighted mean maximum ages; the duration of the 95% conﬁdence minimum to maximum age range is shown in square brackets. Guano-inﬂuenced sediment (Fo.sed.) weighted mean percentages ±1s, signiﬁcance level a ¼ 0 05 All ages are in calibrated (equivalent to calendar) years before present (cal a BP), where BP is deﬁned as 1950 CE (see Supplementary Methods); [95% min. max.] is the minimum and maximum 95% conﬁdence age range based on 2.5% and 97.5% quantiles from Bayesian age-depth modelling (see Supplementary Notes 5–7 and Supplementary Methods); durations are in calendar years with guano phase 4 shown as the combined duration of 4a and 4b; 95% conf. diff. lation changes for the Ardley Island penguin colony compared with a summary of key environmental inﬂuences g \| p g p p g y p g y p y y during the Holocene. (a) Holocene relative sea level (RSL) changes for the South Shetland Islands (SSI)45 in metres above present mean sea level (m a.p.s.l.). (b) Modelled Ardley Lake (16 m a.p.s.l.) penguin colony population changes in guano phases GP-1 to GP-5 (this study) with major colony impact eruptions (circles), and post-eruption recovery intervals (red stipple); estimated modelled population combined errors for the 0.96% and 11% guano- delivery models are not shown for clarity, but typically 30–50% (Supplementary Fig. 19). (c) Sub-fossil penguin occupation record from the Antarctic Peninsula (AP) based on radiocarbon dates of remains at abandoned nesting sites (refs 10,12,53): 1 ¼ Ardley Lake colony (this study), 2 ¼ SSI and the Northwest AP, 3 ¼ Northeast AP, 4 ¼ Mid-southern AP (Supplementary Data 5). (d) Summarized timing of warmer phases and periods of greater open water around the SSI and the AP48,29,56 over the last 10,000 years, deﬁned as 46–0 ka upper bound on their respective means (95% conﬁdence level), where: 5 ¼ Yanou Lake mean summer air temperature (MSAT) (this study), 6 ¼ Anvers Trough open water record (this study), 7 ¼ Palmer Deep sea surface temperature (SST)48, 8 ¼ James Ross Island temperature anomaly29, 9 ¼ AP warm periods56 (Supplementary Data 4). (e) Legend. ARTICLE (e) Palmer Deep (PD) Sea Surface Temperature (SST) 100-year interval records integrated over 0–200 m depth48 and at the surface47 with data 46–0 ka upper bound on the mean (95% conﬁdence level) shaded in dark red; (f) James Ross Island (JRI) ice core temperature anomaly record (compared to 1961–1990 CE interval) with s.e. ranges of the isotope-temperature dependence shown as grey lines29. (g) AP regional palaeoclimate synthesis56 (Supplementary Data 4). 7 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14914 Age (cal a BP) 0 2,000 4,000 6,000 8,000 10,000 1 4 5 3 2 15 m 10 m 5 m 2 3 4 1 +Warm 0–200 m +Warm surface a c d RRR MCA? EHO MHH 8 7 6 5 9 T5a T7 T4 T3b Modelled penguin population (log scale) b 0 1 100 10,000 ,Min Max Warmer Colder Colder e Open water >6–0 ka upper bound (95%) >6–0 ka upper bound (95%): Discontinuous warmth >6–0 ka upper bound (95%): Sustained warmth >2x 6–0 ka upper bound (95%): Sustained warmth AP Composite warm periods Volcanic eruptions Palaeoenvironmental data Penguin data VEI>4 VEI>3 VEI< or =3 Weighted mean eruption age and 95% min.–max. (cal a BP) Ardley Lake post-eruption colony recovery interval ARD guano phases GP-1 to GP-5 ARD exceptionally elevated guano phases ARD catchment deglaciation Published bone and egg ages (cal a ± 1σ) Modelled ARD bone ages (cal a ± 95% min.–max.) ARD bone collagen ages (cal a ± 1σ) Colony population modelling 11% Guano-delivery (most likely minimum) 0.96% Guano-delivery (most likely maximum) 100% Guano-delivery (unlikely) Ellipsoid basin (sediment-focussed); <30° slope accum. area RSL (m a.p.s.l.) 0 8 16 gure 5 \| Modelled penguin population changes for the Ardley Island penguin colony compared with a summary of key environmental inﬂuences e NATURE COMMUNICATIONS \| 8:14914 \| DOI: 10.1038/ncomms14914 \| www.nature.com/naturecommunications Discussion percentage of guano in the sediments, while the T6 eruption c. 6.0–5.9 cal ka BP [6.4–5.7 cal ka BP max.–min. 95% conﬁdence interval age range] occurred c. 200 years after the guano phase GP-1 decline had begun (Fig. 4a,b, Supplementary Note 9, Supplementary Discussion, Supplementary Table 3). Eruptions in the early Holocene (T7) and in the last c. 2,000 years (T1,T2) did not have a measurable impact on the Ardley Lake penguin colony as the populations were already at, or near, minima during these times. percentage of guano in the sediments, while the T6 eruption c. 6.0–5.9 cal ka BP [6.4–5.7 cal ka BP max.–min. 95% conﬁdence interval age range] occurred c. 200 years after the guano phase GP-1 decline had begun (Fig. 4a,b, Supplementary Note 9, Supplementary Discussion, Supplementary Table 3). Eruptions in the early Holocene (T7) and in the last c. 2,000 years (T1,T2) did not have a measurable impact on the Ardley Lake penguin colony as the populations were already at, or near, minima during these times. Ardley Lake began accumulating sediments c. 8.8 cal ka BP [9.2–8.4 cal ka BP] following retreat of the SSI Ice Cap from this part of the Fildes Peninsula between 10.1 and 8.2 cal ka BP (ref. 52), driven by early Holocene thermal maximum conditions recorded in regional marine and ice core temperature records (the early Holocene Optimum (EHO) in Fig. 4g (refs 29,47,48)). The highly explosive, T7 eruption, c. 7 cal ka BP [6.0–7.7 cal ka BP], had a bi-modal basaltic-andesitic and trachydacitic-rhyolitic NATURE COMMUNICATIONS \| 8:14914 \| DOI: 10.1038/ncomms14914 \| www.nature.com/naturecommunications 8 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14914 Table 2 \| Major mid-late Holocene eruptions of the Deception Island volcano and Ardley Lake penguin colony recovery interval summary statistics. Volcanic eruption data Guano phase data Tephra layer Eruption size ARD & YAN Age 95% max.–mean (cal a BP) Decline to Fo.sed. o10% Recovery guano phase Recovery to Fo.sed. 410% Weighted mean Guano phase ended Weighted mean age [95% max.–min.] (cal a BP) Weighted mean age [95% max.–min.] (cal a BP) Recovery interval (years±95% conf. Discussion Warmer conditions have also been recorded at this time in lake and moss peat bank records from Livingston Island, Elephant Island, Beak Island56,58, some marine records from the AP48,56,59, Antarctic ice core composite records60 and stacked Southern Hemisphere temperature records61 (Fig. 4e–g, Supplementary Fig. 17e–i). These ‘more favourable’ climatic conditions for rearing juvenile penguins likely drove the Ardley Lake penguin colony to its Holocene population maxima, and led to the re-establishment of colonies in the mid-southern Peninsula region (Fig. 5c). Despite the gentoo penguins’ preference to feed near their colony, it appears that GP-4 was not adversely affected by the persistence of sea-ice in Maxwell Bay (Fig. 4c) and around the Anvers Shelf (Fig. 4d) and the cooler sea surface anomalies at PD (Fig. 4e). However, as with guano phases GP-2 and GP-3, guano phase GP-4 was abruptly terminated by a Deception Island eruption event (T3b). The peak in penguin populations during guano phase GP-1 (6.7–6.3 cal ka BP) coincided with a continuation of the local open water conditions in Maxwell Bay (Fig. 4c), but at the same time there were contrasting reductions in shelf-edge spring/ summer open water conditions 6.7–6.0 cal ka BP in the Anvers Trough (inferred from higher Fragilariopsis curta/F. kerguelensis diatom ratios and lower percentage of pelagic open water diatoms), and other records of ‘cooler’ and increased ‘sea-ice’ conditions around the SSI 7.3–5.2 cal ka BP (ref. 54). Local sea-ice conditions appear to be the most signiﬁcant factor in driving GP-1, which ended when there was a gradual increase in sea-ice in Maxwell Bay more than 200 years before the T6 eruption event. y y The following two (of three) prominent Ardley Lake guano dry mass accumulation maxima occurred during GP-2 and GP-3 c. 5.8–5.3 and 4.5–4.3 cal ka BP (Fig. 4b, Supplementary Table 7). In marked contrast to GP-1, both GP-2 and GP3 occurred during a period of greater sea-ice extent or seasonality in Maxwell Bay (Fig. 4c), despite the preference of gentoo penguins for ice-free conditions. These maxima also show no strong or consistent relationships with sea-ice conditions on the Anvers Shelf (Fig. 4d), ocean temperatures at PD (Fig. 4e) or atmospheric temperatures in the JRI ice core (Fig. 4f), although the peak of GP-3 corresponds with the onset of a ‘mid Holocene Hypsithermal’ (MHH in Fig. 4g) at c. 4.5 ka in several terrestrial cross-Peninsula records55,56. Discussion populations increasing during volcanically inactive periods and then experiencing abrupt catastrophic declines following major eruptions (Fig. 4b). p g After the T5a Deception Island eruption abruptly ended guano phase GP-2, the Ardley Lake colony struggled to fully re-establish itself for c. 800 years (Table 2) due to a series of closely spaced eruption events (Fig. 4b) and a phase of continued sea-ice presence in Maxwell Bay (Fig. 4c). Our analysis of longer-term (100-year interval) trends in the Maxwell Bay sea-ice reconstruc- tion52 (Fig. 4c) show that these cooler oceanographic conditions persisted well into the late Holocene (5.9–2.6 ka), corresponding to ‘cooler’ conditions and increased sea-ice in the Bransﬁeld Strait between c. 5 and 2 cal ka BP (ref. 57), and advancing glacier margins, increased local snow/ice and sea-ice and limited primary production in surface waters of King George Island54. Despite these cooler conditions the penguin population was able to re- establish itself during GP-3, but was then abruptly terminated by the T4 Deception Island eruption at c. 4.5–4.2 cal ka BP. Following the EHO, an extended period of generally ‘warmer’ interglacial conditions, with minor variations in temperature existed on the AP between 9.2 and 2.5 cal ka BP (refs 48,29) (Fig. 4f,g). These conditions, together with the extended periods of open water in Maxwell Bay (Fig. 4c) and on the Anvers Shelf (Fig. 4d) enabled the establishment of a small penguin colony on Ardley Island from c. 6.7 cal ka BP [7.4–5.8 cal ka BP]. This is the oldest in situ evidence of a Holocene penguin colony on the AP, and up to c. 1,000 years before the sub-fossil evidence of occupation for the mid-southern western AP from c. 5.8 cal ka BP [5.9–5.6 cal ka BP], and the northern AP from c. 5.5 cal ka BP [5.6–5.3 cal ka BP] (refs 12,53) (Fig. 5). p p The most sustained guano phase, GP-4 (c. 4.0–3.0 cal ka BP) (Table 1), occurred during the warm conditions of the late MHH (Fig. 4g), and is characterized by a marked increase in MSAT reconstructed in the Yanou Lake GDGT record (Fig. 4a), positive temperature anomalies at JRI (Fig. 4f) and a sustained period of signiﬁcantly warmer cross-Peninsula terrestrial temperatures from c. 3.8 cal ka BP (refs 29,48) (Fig. 4a,f,g, Supplementary Tables 7,8). Discussion diff.) [min.–max.] T5a VEI44 5,530–5,360 2 5,280 [5,430–5,160] 3 4,500 [4,630–4,300] 780±50 [530–1,130] T4 VEI43 4,480–4,230 3 4,310 [4,500–4,090] 4 3,900 [4,090–3,740] 410±30 [0–760] T3b VEI43 3,220–2,990 4 2,980 [3,090–2,860] 5 2,490 [2,700–2,160] 480±60 [160–390] Only eruptions that had a signiﬁcant impact on colony population levels are shown, where signiﬁcant is deﬁned as a reduction in the proportion of guano-inﬂuenced sediment (% Fo.sed.) to o10% within 200 years after an eruption event; the combined ARD and YAN 95% max. mean age was used to determine the most likely eruption age and is the maximum 95% conﬁdence age weighted mean age, derived from Bayesian age-depth modelling (see Supplementary Notes 5–7, Supplementary Methods); cal a BP is calibrated (or calendar) years before present, where BP is deﬁned as 1950 CE (Supplementary Methods); [95% max.- min.] is the maximum and minimum 95% conﬁdence age range based on 2.5% and 97.5% quantiles from Bayesian age-depth modelling; weighted mean recovery interval duration (cal years) ¼ weighted mean decline age - weighted mean recovery age; 95% conf. diff. is the average difference between the weighted mean recovery interval and its respective 95% max. or min. decline max. or min. recovery interval (cal years); [min.- max.] is the absolute minimum to maximum recovery interval duration (cal years), where the minimum recovery interval duration ¼ 95% min. decline - 95% max. recovery age and the maximum recovery interval duration ¼ 95% max. decline 95% min. recovery age; the 95% conf. diff. weighted average recovery interval duration (±s.e.) ¼ 570±110 cal years (1s±160 cal years; 95% lower-upper conﬁdence interval ¼ 90–1050 cal years; signiﬁcance level a ¼ 0.05; age data are rounded to the nearest 10 years, but rounding to the nearest 100 years provides a more realistic assessment of age-depth modelling errors and comparison to 100-year interval data in Figs 4 and 5. See Supplementary Table 3 for ARD and YAN tephra deposits age data. Table 2 \| Major mid-late Holocene eruptions of the Deception Island volcano and Ardley Lake penguin colony recovery interval summary statistics. Guano phase data composition, and dispersed tephra widely across the northern Peninsula and Scotia Sea (Supplementary Figs 20,21). This and other more widespread early Holocene eruptions occurred before the Ardley Lake colony was established, and could have disrupted early post-deglaciation penguin colonization across the north-western AP (Supplementary Discussion). NATURE COMMUNICATIONS \| 8:14914 \| DOI: 10.1038/ncomms14914 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14914 This resulted in an increase in the available area for nesting sites near the coast and may have driven the relocation of penguins from the Ardley Lake catchment to the western side of Ardley Island, where much of the present day colony is located (Fig. 6c). p y y g Although our new lake records suggest that warmer local atmospheric temperatures (Figs 4a,5) and the regional expression of the MHH led to increased penguin populations during guano phase GP-3 c. 4.5–4.3 cal ka BP and GP-4, c. 4.0–3.0 cal ka BP, our main ﬁnding is that large mid-late Holocene eruptions of the Deception Island volcano had a sporadic, but devastating, impact on the Ardley Lake penguin colony. All ﬁve phases of colony expansion occurred in the absence of large volcanic eruptions and three of the phases (GP-2, 3, 4) were abruptly ended by eruptions from the Deception Island volcano at c. 5.5–5.4 cal ka BP (T5a), 4.5–4.2 cal ka BP (T4) and 3.2–3.0 cal ka BP (T3b). Only guano phase GP-5 declined gradually, which we attribute to adverse ‘neoglacial’ conditions and migration away from nesting sites in the centre of the island towards newly emergent coastal areas. This guano phase appears to have been negatively impacted by the relatively minor T2 eruption event at c. 2.1 cal ka BP, but, in general, from c. 2,500 years onwards the Ardley Lake colony was too small to determine whether volcanic activity had a signiﬁcant impact. ( ) No signiﬁcant changes in the Ardley Lake colony have occurred in the last c. 1,000 years (Fig. 4), although we detected a minor increase in guano input into Ardley Lake from c. 500 years onwards (Fig. 5), which coincides with the onset of warming detected in north-eastern AP lake58 and ice core records29 from c. 500 years, after the northern AP ‘neoglacial’ minimum. Elsewhere, increases in guano input into Lake Y2 on Ardley Island (Fig. 6) and in Hope Bay records from the north- eastern AP10,16 have been linked to ‘warming’ associated with a ‘Medieval Climate Anomaly’43, inferred in some AP records between c. 1,200 and 600 years ago48,56,66. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14914 5a). After deglaciation, during the Early Holocene Warm Optimum (EHO) (11.5–9.5 ka)56, the land area available on Ardley Island was c. 0.6 km2, c. 30–35% less than the present day. The eastern half of the island, where the current penguin colony is located, was bordered by steep cliffs forcing early to mid Holocene colonies to nest in the centre of Ardley Island. During the mid to late Holocene, relative sea level (RSL) fell, increasing the land area available. The amount of guano deposited in Ardley Lake declined after c. 1,300 cal a BP as some colonies relocated to the Lake Y2 and Lake G catchments43. The eastern side of the island became more easily accessible when RSL fell below 5 m above present sea level (m a.p.s.l.) after c. 1,300 years ago. Future colony population increases could be accommodated in the central area of Ardley Island. See Supplementary Fig. 1 and Supplementary Data 6 for modern-day penguin population data; see Supplementary Fig. 22 for an extended version of this ﬁgure. This ﬁgure includes material copyright of DigitalGlobe, Inc., All Rights Reserved, used with permission under a NERC-BAS educational license and not included in the Creative Commons license for the article. BP. This ‘neoglacial’ is apparent in Peninsula-wide lake58, marine57,59 and ice core29 records (Fig. 4f), and marked by the episodic re-advance of local glaciers52,62 that slowed the rate of RSL decline on the SSI63,64. Although reconstructed temperatures in the YAN-GDGT record remained above average until c. 1.5 cal ka BP, sea-ice conditions in Maxwell Bay and the Bransﬁeld Strait52,57, and at some localities further south65, became progressively less favourable between c. 2.3 and 1.8 cal ka BP (ref. 10), likely constraining the growth of the Ardley Lake and Lake Y2 colonies. The relatively minor (VEIo3) T2 eruption event had a measurable impact on the Ardley Lake colony in the middle of guano-phase GP-5, c. 2.1 cal ka BP (Fig. 4b), but further deterioration in north-western AP climate into the ‘neoglacial’ minimum probably drove more progressive reductions in the Ardley Lake GP-5 and Lake Y2 colonies from c. 1.5 to 1.3 cal ka BP (ref. 43). Y2 sites during the last c. 1,000 years mirrors the increased rate of RSL fall from c. 10 to 0 m a.p.s.l. (refs 45,64,70) (Fig. 6). NATURE COMMUNICATIONS \| 8:14914 \| DOI: 10.1038/ncomms14914 \| www.nature.com/naturecommunications Discussion Instead, the main driver of these penguin population changes is volcanism, with colony We link the failure to re-establish another large and sustained colony of similar size and duration as guano phase GP-4 to the start of a marked ‘neoglacial’ decline in temperature at c. 2.5 cal ka 9 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14914 NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14914 Y2 G Y4 b 4,200–1,300 years ago <15–10 m a.p.s.l Ardley Lake <15–10 m a.p.s.l b 4,200–1,300 years ago b 4,200–1,300 years ago >15 m a.p.s.l. 9,000–4,200 years ago a 9,000–4,200 years ago a AD4 AD3 13.4–15 m 10.5–11.1 m 4.5–6.6 m Y2 Y4 G Raised beaches c <10–0 m a.p.s.l. 1,300 years ago–present day Ardley Lake d Main penguin landing sites Secondary penguin landing sites Penguin population >1,000 0 a b RSL (m a.p.s.l.) 0 16 0 2,000 4,000 6,000 8,000 10,000 c Age (cal a BP) d d c 1,300 years ago–present day c Ardley Lake Figure 6 \| Changes in land availability and colony population on Ardley Island over the last 9,000 years. (a) 9,000 to 4,200 years ago; (b) 4,200 to 1,300 years ago; (c) 1,300 years ago to the present day; (d) Legend and relative sea level (RSL) scenarios shown in (a)–(c) (see Fig. 5a). After deglaciation, during the Early Holocene Warm Optimum (EHO) (11.5–9.5 ka)56, the land area available on Ardley Island was c. 0.6 km2, c. 30–35% less than the present day. The eastern half of the island, where the current penguin colony is located, was bordered by steep cliffs forcing early to mid Holocene colonies to nest in the centre of Ardley Island. During the mid to late Holocene, relative sea level (RSL) fell, increasing the land area available. The amount of guano deposited in Ardley Lake declined after c. 1,300 cal a BP as some colonies relocated to the Lake Y2 and Lake G catchments43. The eastern side of the island became more easily accessible when RSL fell below 5 m above present sea level (m a.p.s.l.) after c. 1,300 years ago. Future colony population increases could be accommodated in the central area of Ardley Island. See Supplementary Fig. 1 and Supplementary Data 6 for modern-day penguin population data; see Supplementary Fig. 22 for an extended version of this ﬁgure. This ﬁgure includes material copyright of DigitalGlobe, Inc., All Rights Reserved, used with permission under a NERC-BAS educational license and not included in the Creative Commons license for the article. Figure 6 \| Changes in land availability and colony population on Ardley Island over the last 9,000 years. (a) 9,000 to 4,200 years ago; (b) 4,200 to 1,300 years ago; (c) 1,300 years ago to the present day; (d) Legend and relative sea level (RSL) scenarios shown in (a)–(c) (see Fig. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14914 AD4 AD3 13.4–15 m 10.5–11.1 m 4.5–6.6 m Y2 Y4 G Raised beaches ? ? ? Y2 G Y4 Y4 9,000–4,200 years ago a b c d 4,200–1,300 years ago <10–0 m a.p.s.l. >15 m a.p.s.l. <15–10 m a.p.s.l 1,300 years ago–present day Main penguin landing sites Secondary penguin landing sites Penguin population >1,000 0 a b RSL (m a.p.s.l.) 0 16 0 2,000 4,000 6,000 8,000 10,000 c Ardley Lake Ardley Lake Ardley Lake Age (cal a BP) Figure 6 \| Changes in land availability and colony population on Ardley Island over the last 9,000 years. (a) 9,000 to 4,200 years ago; (b) 4,200 to 1,300 years ago; (c) 1,300 years ago to the present day; (d) Legend and relative sea level (RSL) scenarios shown in (a)–(c) (see Fig. 5a). After deglaciation, during the Early Holocene Warm Optimum (EHO) (11.5–9.5 ka)56, the land area available on Ardley Island was c. 0.6 km2, c. 30–35% less than the present day. The eastern half of the island, where the current penguin colony is located, was bordered by steep cliffs forcing early to mid Holocene colonies to nest in the centre of Ardley Island. During the mid to late Holocene, relative sea level (RSL) fell, increasing the land area available. The amount of guano deposited in Ardley Lake declined after c. 1,300 cal a BP as some colonies relocated to the Lake Y2 and Lake G catchments43. The eastern side of the island became more easily accessible when RSL fell below 5 m above present sea level (m a.p.s.l.) after c. 1,300 years ago. Future colony population increases could be accommodated in the central area of Ardley Island. See Supplementary Fig. 1 and Supplementary Data 6 for modern-day penguin population data; see Supplementary Fig. 22 for an extended version of this ﬁgure. This ﬁgure includes material copyright of DigitalGlobe, Inc., All Rights Reserved, used with permission under a NERC-BAS educational license and not included in the Creative Commons license for the article. Y4 9,000–4,200 years ago a >15 m a.p.s.l. Ardley Lake ? ? ? Y2 G Y4 b d 4,200–1,300 years ago <15–10 m a.p.s.l Main penguin landing sites Secondary penguin landing sites Penguin population >1,000 0 a b RSL (m a.p.s.l.) 0 16 0 2,000 4,000 6,000 8,000 10,000 c Ardley Lake Age (cal a BP) ? ? ? Methods S l ll Sample collection and laboratory analyses. Sediment cores extracted from Ardley Lake (ARD) and Yanou Lake (YAN) were analysed for the concentration of elements associated with changing inputs of penguin guano and volcanic ash deposits using complementary multi-proxy biogeochemical techniques described in this section and in Supplementary Methods. Chronologies for the lake sediment cores were established using 18 (ARD) and 15 (YAN) AMS radiocarbon (14C) ages from, in order of preference: (1) moss macrofossil layers (consisting of hand-picked ﬁne strands of the aquatic moss Drepanocladus longifolius (Mitt.) Paris, but also occasional layers of Campylium polygamum (Schimp.) Lange & C.E.O. Jensen, and some unidentiﬁable/mixed species moss fragments—considered more likely to have been reworked; (2) terrestrial and/or lacustrine algae; (3) other intact macrofossils and sub-fossils, including bones (bone-collagen, where extractable); (4) other (macro)fossil fragments; (5) organic-rich bulk sediments and, near the base of each core and as a last resort, (6) bulk glaciolacustrine or glaciomarine sediments (Supplementary Table 2). Bulk sediments were only dated where macrofossils were absent, while paired macrofossil or bone-collagen and bulk sediment samples were measured in the surface sediment and wherever present to check for any systematic offsets between ages obtained from different carbon sources (see Supplementary Methods, Supplementary Notes 5 and 6 for more details). ) Physical disablement and poisoning are the most signiﬁcant potential hazards to penguins, even though they are well-adapted to elevated levels of F, Cu, Cd, and Hg through high dietary intake16. Prolonged exposure to the ﬁne abrasive glass particles in tephra while rearing chicks on land, or during feeding at sea, could adversely affect one (or all) of the major physiological processes of penguins and their prey (for example, respiration, digestion, immune function, vision; Supplementary Discussion). Colonies are more likely to be decimated if signiﬁcant amounts of ash fell during the breeding season onto unhatched eggs or recently hatched chicks, or if one parent failed to return from foraging. Mature individuals on long forages, outside the areas directly affected by ashfall, would have the best chance of survival. Our recovery interval calculations suggest that these individuals may have been displaced to alternative nesting sites such as those that were occupied in the mid-southern AP at c. 5.4 and 4.2 cal ka BP (Fig. 5c). NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14914 The increase in sub- fossil evidence of nesting sites across the AP in the last 1,000 years for all Pygoscelis spp., regardless of whether they are ‘ice-dependent’ or ‘ice-avoiding’ species, could be a response to the more favourable ‘Medieval Climate Anomaly’ and/or post-‘neoglacial’ conditions, or reﬂect migratory shifts to newly emerged, low-lying coastal areas created by declining Holocene RSLs across the Peninsula63,64,67–69 (for example, Fig. 5a, c). The absence of signiﬁcant colonies at both the Ardley Lake and Lake The remoteness of active volcanism in Antarctica means that modern-day analogues for volcanic impacts on penguin colonies are inevitably sparse and hard to quantify. Although the relative size of Deception Island eruptions is thought to have diminished in the late Holocene, comparatively small recent eruptions were locally hazardous, causing catastrophic ﬂooding and lahars20. Larger mid-late Holocene eruptions are thought to have generated pyroclastic density ﬂows and surges20,21, which could have been particularly destructive at ground level. Circumstantial evidence from the volcanically active South Sandwich Islands 10 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14914 shows that, close to an erupting volcano, penguins are largely absent from areas affected by recent ash fall (Supplementary Fig. 23). Proximal fallout of coarse-grained tephra could lead to high localized mortality rates and signiﬁcantly disrupt nesting activities, or drive migration to areas less affected by ash. The northern AP is deﬁned as having a ‘moderate to extremely high likelihood of supraglacial airfall by active volcanoes’, where a ‘signiﬁcant risk to life’ exists31, mainly because the deposition of far-travelled ﬁne black basaltic ash can lead to rapid snow-melt and (supraglacial) lahar formation71. Other indirect processes, such as burial by tephra, the generation of tsunamis, landscape destabilization, and the deposition of ﬁne ash layers and aerosols could pose a substantial risk to distal penguin colonies30, such as those on Ardley Island (Supplementary Fig. 12, Supplementary Discussion). nesting and foraging activities on Ardley Island. Moreover, the saw-toothed, asymmetric pattern of successive Ardley Lake penguin colony population maxima suggests that sustainable colony recovery following these mid-late Holocene volcanic eruptions was slow, taking, on average, between 400 and 800 years, but possibly as much as 1,100 years during the most disruptive phase of volcanism (T5) c. 5.5–4.6 cal ka BP. Methods S l ll For data replication and comparison purposes, the total inorganic carbon (TIC) of 69 (out of 385) samples from both cores was determined coulometrically by a CM 5012 CO2 coulometer coupled to a CM 5130 acidiﬁcation module (UIC, USA) while total organic carbon was then calculated as the difference between TC and TIC (TOC% ¼ TC% TIC%). Owing to the high correlation between TC and TOC (R240.9997) and the negligible TIC concentrations (av. 0.10 and o0.01 mass%) compared to TC values (av. 6.2 and 2.5 mass%) in both cores, TC is considered to reﬂect the amount of organic carbon in both lake sediments. Quantitative XRF analysis for major and trace elements (SiO2, Al2O3, CaO, K2O, Na2O, P2O5, As, Ba, Cu, Co, Ni, Sr, Y, Zn, Zr) was carried out with a conventional wavelength dispersive X-ray ﬂuorescence (WD-XRF) spectrometer (Philips PW 2400). For WD-XRF, glass beads were prepared following standard procedures74 and measurements undertaken in random order to avoid artiﬁcial trends. Trace element analysis (Cd, REE) of selected samples was performed by Inductively Coupled Plasma Mass Spectrometry (ICP-MS, Element 2 mass spectrometer, Thermo Scientiﬁc, Germany) at 2,500-fold dilution. For additional details concerning ICP-MS measuring conditions, see Schnetger75. Selenium was determined on acid digestions by graphite atomic absorption spectrometry (G-AAS) using a Unicam 939 QZ AA spectrometer and a Zeeman- effect background correction. A Milestone DMA-80 Direct Mercury Analyser was used for the measurement of mercury via cold vapour atomic absorption spectroscopy (CV-AAS). ICP-MS and G-AAS standard acid digestion procedures are described in Supplementary Methods. F ll i t bli h d d d i l d i ti lid d pp y Sub-samples for carbon, nitrogen, XRF and ICP-MS (quantitative, dry mass) bio-element analyses were taken at 1 cm intervals from ARD and YAN lake sediment cores, lyophilized and ground with an agate ball mill and manually with an agate pestle and mortar. ARD samples were analysed for total carbon (TC), total sulphur (TS) and total nitrogen (TN) using a CNS analyser (vario EL Cube, Elementar, Germany) equipped with a solid-state infrared and a heat conductivity detector. TC and TS measurements were conducted on YAN sediments by means of an ELTRA CS analyser. NATURE COMMUNICATIONS \| 8:14914 \| DOI: 10.1038/ncomms14914 \| www.nature.com/naturecommunications Methods S l ll The physical landscape disturbance and presence of tephra over Ardley Island prevented sustainable post-eruption reoccupation of the site for, on average, 570±110 years (weighted mean±s.e.), but as much as c. 800–1,100 years following the largest mid-late Holocene (T5a) eruption c. 5,500 years ago (Table 2). Measured radiocarbon ages from samples shown in Supplementary Table 2 were calibrated using SH13 and MARINE13 calibration curves and age-depth models were generated using Bayesian age-depth modelling techniques (Fig. 2, Supplementary Figs 3,9, Supplementary Methods and Supplementary Notes 5–7). All the ‘as measured’ (uncalibrated) radiocarbon age data shown in Supplementary Table 2 were used as input data for ﬁnal age-depth model runs (ARD-M5, YAN-M4, ANVERS-M1, where model run number is indicated by the -M sufﬁx). The weighted mean basal age of the ARD core was 8,750 cal a BP [8,410–9,230 min –max. 95% conﬁdence age range]. Error analysis shows that whole record mean 95% conﬁdence age-depth model uncertainties, rounded to the nearest 10 years with 95% conﬁdence minimum to maximum age ranges (in years at depths in cm) shown in square brackets are: ARD-M5: 410 years [0.6 years at 0 cm–1,430 years at 335 cm], YAN-M4: 600 years [160 years at 7.7 cm–1,990 years at 340 cm], and ANVERS-M1: 1,420 years [590 years at 0 cm–1,810 years at 375 cm]. Equivalent values for the late Holocene in each record are: ARD-M5: 640 years [0.6 years at 0 cm–750 years at 20 cm], YAN-M4: 680 years [160 years at 7.7 cm–760 years at 2.7 cm] and ANVERS-M1: 1,370 years [590 years at 0 cm–1,600 years at 155 cm] (Supplementary Note 7). Measured radiocarbon ages from samples shown in Supplementary Table 2 were calibrated using SH13 and MARINE13 calibration curves and age-depth models were generated using Bayesian age-depth modelling techniques (Fig. 2, Supplementary Figs 3,9, Supplementary Methods and Supplementary Notes 5–7). All the ‘as measured’ (uncalibrated) radiocarbon age data shown in Supplementary Table 2 were used as input data for ﬁnal age-depth model runs (ARD-M5, YAN-M4, ANVERS-M1, where model run number is indicated by the -M sufﬁx). The weighted mean basal age of the ARD core was 8,750 cal a BP [8,410–9,230 min –max. 95% conﬁdence age range]. Methods S l ll Error analysis shows that whole record mean 95% conﬁdence age-depth model uncertainties, rounded to the nearest 10 years with 95% conﬁdence minimum to maximum age ranges (in years at depths in cm) shown in square brackets are: ARD-M5: 410 years [0.6 years at 0 cm–1,430 years at 335 cm], YAN-M4: 600 years [160 years at 7.7 cm–1,990 years at 340 cm], and ANVERS-M1: 1,420 years [590 years at 0 cm–1,810 years at 375 cm]. Equivalent values for the late Holocene in each record are: ARD-M5: 640 years [0.6 years at 0 cm–750 years at 20 cm], YAN-M4: 680 years [160 years at 7.7 cm–760 years at 2.7 cm] and ANVERS-M1: 1,370 years [590 years at 0 cm–1,600 years at 155 cm] (Supplementary Note 7). ( ) In conclusion, while we cannot assess some factors that determine breeding success such as pressure from predators1, the ability to change diet72, relative changes in species composition, or recent anthropogenic impacts73, our detailed case study, using innovative methodologies, has revealed the key environmental factors inﬂuencing the Ardley Lake penguin colony through the Holocene. Once deglaciated, c. 8,500 years ago, climatic conditions on Ardley Island were sufﬁciently amenable throughout the rest of the Holocene for sustained penguin habitation. The Ardley Lake colony population maximum in ﬁrst half of the late Holocene, c. 4,000 to 3,000 years ago, occurred during a period of particularly ‘favourable’ regional climate (sustainably warmer than the 6–0 ka average, with fewer storms) and local-regional sea-ice conditions (near-average 6–0 ka sea-ice cover/seasonal extent), which enhanced nesting and foraging success. Conversely, ‘neoglacial’ conditions and falling sea levels during the second half of the late Holocene (c. 2,000 years ago to present) contributed to colony decline and/or migration away from the centre of Ardley Island. ( pp y ) Sub-samples for carbon, nitrogen, XRF and ICP-MS (quantitative, dry mass) bio-element analyses were taken at 1 cm intervals from ARD and YAN lake sediment cores, lyophilized and ground with an agate ball mill and manually with an agate pestle and mortar. ARD samples were analysed for total carbon (TC), total sulphur (TS) and total nitrogen (TN) using a CNS analyser (vario EL Cube, Elementar, Germany) equipped with a solid-state infrared and a heat conductivity detector. TC and TS measurements were conducted on YAN sediments by means of an ELTRA CS analyser. ARTICLE The target ions were m/z 1,302, 1,300, 1,298, 1,296 and 1,292 for the isoprenoid GDGTs (isoGDGTs) compounds and 1,050, 1,048, 1,046, 1,036, 1,034, 1,032, 1,022, 1,020 and 1,018 for the branched GDGT (brGDGTs) za ¼ zm 2 3 z : zm ð Þ0 =3½2 z : zm ð Þ0 1 Units : cm ð3Þ ð3Þ For a hyperboloid basin shape: SGðtÞhyper ¼ 2 3 1 z2 m 2zm z ð Þ2 dzG dtG Units : g cm 2a 1 ð4Þ where z ¼ core depth at time t; zm ¼ maximum depth of the original basin (that is, current lake depth þ sediment record depth); z : zm ð Þ0 ¼ ratio of the mean depth ðzÞto the maximum depth of the original basin (zm); dzG=dtG ð Þ ¼ guano dry mass accumulation rate ¼ Fo.sed DMAR in g cm 2 a 1. g Assuming Ardley Lake best approximates to an ellipsoid basin shape (that is, the ‘more likely’ basin shape scenario), the total basin-wide guano accumulated (IG) over the ‘more likely’ o30 degree slope accumulation area (Ao30) within Ardley Lake (Supplementary Fig. 2), corrected for the effects of sediment focussing, is then: Diatom analysis of the ARD and YAN records is as described in ref. 45 and summarized in Supplementary Fig. 14, while Anvers Shelf marine core (ANVERS) diatom analysis and chronology are described in Supplementary Methods and summarized in Supplementary Fig. 15. IGAo30ellipðtÞ ¼ SGðtÞellip: Ao30 ½ Units : g cm 2a 1cm2 ¼ g a 1 ð5Þ Data analysis. Bio-element assemblages are immobile in (Antarctic) lake sedi- ments43 and guano input to sediments leads to the formation and preservation of stable phosphates, such as struvite (Mg(NH4)PO4 x 6 H2O), leukoposphite and, in particular, hydroxylapatite (Ca5(PO4)3(OH)), which is one of the dominant compounds found in ornithogenic soils/sediments on King George Island. During the precipitation of apatites, an exchange between Ca2 þ, PO43 , F and OH and elements such as Ag, Br, Ba, Cd, Cu, Cr, I, Na, Mg, Mo, Pb, S, Se, Sr, U, V, Y and Zn is possible, coupled to microbial-mediated degradation of solid phases78 (see Supplementary Note 1 for more details). ARTICLE Contiguous 1 cm subsamples were taken in the top 20 cm of the YAN record, and at 2 cm intervals between 20 and 38 cm and 188 and 210 cm core depth (dated to c. 6.1 cal ka BP). Freeze dried and homogenized sediment samples weighing 0.2–4.3 g were microwave-extracted in DCM:Methanol (3:1, v/v). The total extracts were saponiﬁed and GDGTs isolated following ref. 76. Prior to analysis the GDGT extracts were ﬁltered through a 0.2 mm Whatman PTFE ﬁlter. GDGT analysis was undertaken using an Acquity Xevo TQ-S (triple quadrupole with step wave; Waters Ltd.) LC-MS set up with an atmospheric pressure chemical ionization source (Ion saber II) operated in positive ion mode. Analytical separation was achieved using a Grace Prevail Cyano HPLC column (3 mm, 150 2.1 mm i.d.) ﬁtted with an in-line ﬁlter (Waters Acquity UPLC in-line ﬁlter, 0.2 mm) at 40 C using a binary solvent gradient where eluent A was hexane and eluent B was propanol. The ﬂow rate of h b l h l h d ﬁl f To summarize, the basin-wide guano inﬂux rate, SG, at time t, and total Ardley basin maximum core depths of z (water depth þ sediment depth, in cm) at time t, was calculated as follows: For an ellipsoid-basin: SGðtÞellip ¼ 2 3 1 z2 a 2za þ z ð Þ2 dzG dtG Units : g cm 2a 1 ð2Þ ð2Þ the mobile phase was 0.2 ml per minute with a gradient proﬁle of 99% A 1% B (0–50 min); 98.2% A 1.8% B (50–55 min); 90% A 10% B (55–65 min) and ﬁnally 99% A 1% B (66–80 min). The LC-MS settings were: source offset 50 V, capillary and 1.5 kV, desolvation temperature 200 C, cone voltage 30 V, desolvation gas (N2). Detection was achieved using selected ion monitoring of targeted [M þ H] þ ions (dwell time 50 ms). ARTICLE The average relative proportion of guano or ornithogenically derived sediment in the Ardley Lake sediment matrix (Fo.sed.) was estimated by using the mixing equation (equation (1)), modiﬁed after Shultz and Calder80: Several studies have shown that catchment erosion and deposition in small closed-basins with a single catchment and non-complex inﬂow characteristics (for example, Ardley Lake) can be approximated by a linear relationship (see Several studies have shown that catchment erosion and deposition in small closed-basins with a single catchment and non-complex inﬂow characteristics (for example, Ardley Lake) can be approximated by a linear relationship (see Supplementary Note 8 for references); thus, a reasonable approximation of the total amount of sediment or, in this case, guano delivered (GD), by erosion (Ge), into the Ardley Lake basin, can be obtained, simply from ratio of lake-area to catchment- area, expressed as an estimated percentage: Fo:sed: rel:% ð Þ ¼ P El¼Cu;Sr;Zn;P El=Al ð Þsmp El=Al ð Þbgd El=Al ð Þo:sed: El=Al ð Þbgd 4 100 ð1Þ rel % ð Þ ¼ P El¼Cu;Sr;Zn;P El=Al ð Þsmp El=Al ð Þbgd El=Al ð Þo:sed: El=Al ð Þbgd ð1Þ example, Ardley Lake) can be approximated by a linear relationship (see Supplementary Note 8 for references); thus, a reasonable approximation of the total amount of sediment or, in this case, guano delivered (GD), by erosion (Ge), into the Ardley Lake basin, can be obtained, simply from ratio of lake-area to catchment- area, expressed as an estimated percentage: (El/Al)smp is the element/aluminium ratio of selected bio-elements (Cu, P, Sr, Zn) in Ardley Lake sediments and (El/Al)bgd (mean Ardley Island bedrock51) and (El/ Al)o.sed. (guano-bearing ornithogenic sediment and soils49) represent the respective ratios in both end members. In order to minimize misinterpretation of this proxy, we calculated the average fraction of ornithogenic sediment and soils in the lake sediments using a combination of four chemically unrelated El/Al ratios. This provided a buffering effect on possible variations in ornithogenic soil and bedrock composition over time. In the resultant Fo.sed. percentage plotted in Fig. 4b, for example, Fo.sed. ¼ 20%, means that 20% of the sediment sample is eroded ornithogenic sediment-soil and 80% eroded bedrock. Theoretically, all Fo.sed. ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14914 determine the precise position of tephra deposits (see Supplementary Methods, Supplementary Note 9, Supplementary Figs 3,7,10,12 for details). Electron Probe Micro-Analysis (EPMA) of 165 glass-shards from the most prominent T4–T7 tephra layers in the YAN, ARD and Beak 1 Lake records (Beak Island; Layers Ta–e (ref. 63)) was used to determine the eruption characteristics and source of mid-late Holocene eruptions that likely had the biggest environmental impacts. Results were compared with glass-shard analyses from age-equivalent tephra layers in marine cores PC460/461 from the Scotia Sea (new data; this study) and similarly analysed data in Moreton and Smellie23 from the Scotia Sea, northern Weddell Sea and Boyd Strait, and in Fretzdorff and Smellie22 from the Bransﬁeld Basin (see Supplementary Methods, Figs 20,21, Supplementary Table 10). 40% provide evidence for guano input above the local bedrock-terrigenous background level, but we used a weighted mean Fo.sed. value of 410% at the cut-off for CONISS-deﬁned geochemical zone to indicate the sustained presence of penguins around Ardley Lake (Table 1, Supplementary Table 7). We calculated post-eruption colony recovery intervals as the time taken until the percentage Fo.sed. value ﬁrst returned to 410% within the subsequent guano phase to represent the return of a sustained penguin presence around Ardley Lake. Measurement errors of o1% mean our deﬁnitions provide very conservative buffers of penguin presence during guano phases 1–5 (GP-1 to GP-5) (Table 2). Penguin population modelling results shown in Fig. 5 utilized dry mass Fo.sed.. accumulation rates (Fo.sed. DMAR in Fig. 4b), which were calculated following standard procedures81 (that is, multiplying Fo.sed. percentages by the dry mass accumulation rate, calculated from sedimentation rate and dry mass density data). We also used published penguin population parameters40,41 and the ‘most-realistic’ sediment focussed ellipsoid basin accumulation models81, assuming a o30 slope- angle accumulation area within Ardley Lake of 5,682 m2 (out of a maximum 7,274 m2 lake basin area) (see Supplementary Fig. 2). Further accumulation area scenarios, equations and references can be found in Supplementary Note 8. We considered the shape of Ardley Lake basin to approximate between a steep-sided hyperboloid basin and an ellipsoid basin (as below). Supplementary Methods, Figs 20,21, Supplementary Table 10). For the YAN-GDGT reconstructed temperature record, temperature-sensitive glycerol dialkyl glycerol tetraethers (GDGTs) biomarkers were extracted from 41 samples using a microwave-assisted solvent extraction76,77. ARTICLE These trace elements, which have naturally higher concentrations in penguin guano, are enriched in these phosphate phases and then immobilized in soils and sediment proﬁles during this substitution process. While the high correlation between Mg and P in ornithogenic soils from Vestfold Hills has been used to indicate the presence of struvite79, their lack of correlation in the Ardley Lake sediments means that Mg is likely derived from the bedrock lithology (Supplementary Fig. 3, Supplementary Table 4), and hydroxylapatite (Ca5(PO4)3(OH)) formation reﬂects guano input (Supplementary Fig. 6). where Ao30 ¼ 5; 682 568 m2 ¼ 5:68107cm2; SGðtÞellip ¼ the value for each 100-year interval obtained from equation (2). Using guano production rates of 84.5±21.1 g (25% error estimate applied) per penguin per day on Ardley Island, and a mean density of 0.31±0.19 gentoo penguins per m2 (for Signy Island)40,41, we estimated the total amount of guano delivered by erosion from the catchment into Ardley Lake as the guano yield (Gy) and the area required for the reconstructed population. As the main breeding season and ice/snow-free periods, when active erosion from catchments to lakes occurs on Ardley Island, last for approximately 3–4 months each year (91.3±22.8 days; 25% error estimate; equation (6) and Supplementary Table 9), we calculated the maximum amount of guano deposited per penguin (p 1), per year (a 1) inside the catchment and delivered to the lake as the maximum possible guano yield (Gy-100%) as follows: Gy100% ¼ D Pg ¼ 7; 716 2; 728 g a 1p 1 c:35% combined error ð Þ ð6Þ where D ¼ mean occupation time per year±estimated error ¼ 365.25/4 or 91.3±22.8 days (25% error applied); Pg ¼ amount, in grams (g) of guano deposited per year (a 1) per penguin (p 1). Methods S l ll For data replication and comparison purposes, the total inorganic carbon (TIC) of 69 (out of 385) samples from both cores was determined coulometrically by a CM 5012 CO2 coulometer coupled to a CM 5130 acidiﬁcation module (UIC, USA) while total organic carbon was then calculated as the difference between TC and TIC (TOC% ¼ TC% TIC%). Owing to the high correlation between TC and TOC (R240.9997) and the negligible TIC concentrations (av. 0.10 and o0.01 mass%) compared to TC values (av. 6.2 and 2.5 mass%) in both cores, TC is considered to reﬂect the amount of organic carbon in both lake sediments. Quantitative XRF analysis for major and trace elements (SiO2, Al2O3, CaO, K2O, Na2O, P2O5, As, Ba, Cu, Co, Ni, Sr, Y, Zn, Zr) was carried out with a conventional wavelength dispersive X-ray ﬂuorescence (WD-XRF) spectrometer (Philips PW 2400). For WD-XRF, glass beads were prepared following standard procedures74 and measurements undertaken in random order to avoid artiﬁcial trends. Trace element analysis (Cd, REE) of selected samples was performed by Inductively Coupled Plasma Mass Spectrometry (ICP-MS, Element 2 mass spectrometer, Thermo Scientiﬁc, Germany) at 2,500-fold dilution. For additional details concerning ICP-MS measuring conditions, see Schnetger75. Selenium was determined on acid digestions by graphite atomic absorption spectrometry (G-AAS) using a Unicam 939 QZ AA spectrometer and a Zeeman- effect background correction. A Milestone DMA-80 Direct Mercury Analyser was used for the measurement of mercury via cold vapour atomic absorption spectroscopy (CV-AAS). ICP-MS and G-AAS standard acid digestion procedures are described in Supplementary Methods. Overall, though, we found no consistent relationships between guano inputs into Ardley Lake and local-regional atmospheric and ocean temperatures or sea-ice conditions across the Holocene. Instead, the overriding driver of long-term penguin colony change on Ardley Island was volcanic activity from Deception Island. Three out of ﬁve mid-late Holocene guano- phases were interrupted by volcanic ash fallout from eruptions that were at least an order of magnitude greater than present-day eruptions. Mid-late Holocene volcanic eruptions deposited layer of volcanic ash across the landscape, which severely disrupted Following established procedures, we used visual descriptions, smear slides and contiguous micro-XRF (m-XRF) scanning, at 200 mm and 2 mm intervals, to 11 ARTICLE Contiguous 1 cm subsamples were taken in the top 20 cm of the YAN record, and at 2 cm intervals between 20 and 38 cm and 188 and 210 cm core depth (dated to c. 6.1 cal ka BP). Freeze dried and homogenized sediment samples weighing 0.2–4.3 g were microwave-extracted in DCM:Methanol (3:1, v/v). The total extracts were saponiﬁed and GDGTs isolated following ref. 76. Prior to analysis the GDGT extracts were ﬁltered through a 0.2 mm Whatman PTFE ﬁlter. GDGT analysis was undertaken using an Acquity Xevo TQ-S (triple quadrupole with step wave; Waters Ltd.) LC-MS set up with an atmospheric pressure chemical ionization source (Ion saber II) operated in positive ion mode. Analytical separation was achieved using a Grace Prevail Cyano HPLC column (3 mm, 150 2.1 mm i.d.) ﬁtted with an in-line ﬁlter (Waters Acquity UPLC in-line ﬁlter, 0.2 mm) at 40 C using a binary solvent gradient where eluent A was hexane and eluent B was propanol. The ﬂow rate of the mobile phase was 0.2 ml per minute with a gradient proﬁle of 99% A 1% B (0–50 min); 98.2% A 1.8% B (50–55 min); 90% A 10% B (55–65 min) and ﬁnally 99% A 1% B (66–80 min). The LC-MS settings were: source offset 50 V, capillary 1.5 kV, desolvation temperature 200 C, cone voltage 30 V, desolvation gas (N2). Detection was achieved using selected ion monitoring of targeted [M þ H] þ ions (dwell time 50 ms). The target ions were m/z 1,302, 1,300, 1,298, 1,296 and 1,292 for the isoprenoid GDGTs (isoGDGTs) compounds and 1,050, 1,048, 1,046, 1,036, 1,034, 1,032, 1,022, 1,020 and 1,018 for the branched GDGT (brGDGTs) compounds. GDGTs were identiﬁed and integrated using MassLynx software (v.4.1) and GDGT-derived temperatures were calculated using the Antarctic and sub-Antarctic GDGT-temperature calibration77 (see Data Analysis section). The Antarctic and sub-Antarctic GDGT-MSAT surface calibration data set (comprising 32 sites in total) includes surface sediments from Yanou Lake, Ardley Lake and three other lakes from Fildes Peninsula, along with two further lakes from Potter Peninsula and four lakes from the Trinity Peninsula, north-eastern AP (including Beak Island). pp y g pp y For the YAN-GDGT reconstructed temperature record, temperature-sensitive glycerol dialkyl glycerol tetraethers (GDGTs) biomarkers were extracted from 41 samples using a microwave-assisted solvent extraction76,77. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14914 NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14914 For Ardley Lake, the amount of guano delivered in grams (g) per year (a 1) per penguin (p 1) in the 11% guano (Gy–11%) yield scenario is given by: assess the uncertainties in cluster analysis. We used constrained incremental sum of squares (CONISS) stratigraphically constrained cluster analysis with broken stick analysis to deﬁne Diatom Zones (D) in the ARD, YAN and ANVERS records, Geochemical Zones (GZ) in the ARD and YAN records, and Guano phase GP-1 to GP-5 boundaries in the ARD record. Downcore cluster-zonation was undertaken on ecologically grouped diatom percentage data and square-root transformed geochemical data using the R packages vegan and rioja (see Supplementary Methods for software references). Downcore trends in diatom and geochemical data sets using Principal Components Analysis (PCA) were undertaken using square-root transformed percentage XRF data and m-XRF (Total Scatter Normalised (TSN)) percentage geochemical data and percentage diatom count data. Gy11% ¼ 0:11 DPg ¼ 847 300 g a 1p 1 ð8Þ ð8Þ Gy11% ¼ 0:11 DPg ¼ 847 300 g a 1p However, small lakes with restricted inﬂow and/or low rates of catchment erosion or slow sedimentation rates (that is, most typically high latitude, frozen lakes, for example, Yanou Lake during the late Holocene) are better represented by an exponential sediment delivery model, GDST (ref. 81), which, for Ardley Lake, produces GDST ¼ 0:96% as follows: GDST ¼ 0:36 A 0:2 max ¼ 0:36 7; 274 0:2 ¼ 0:96%: ð9Þ ð9Þ This allowed us to estimate the number of penguins (P) present in the catchment at time (t) for three guano-yield scenarios, Gy100%;11%;0:96%: This allowed us to estimate the number of penguins (P) present in the catchment at time (t) for three guano-yield scenarios, Gy100%;11%;0:96%: For climate and sea-ice comparisons, the Yanou Lake reconstructed GDGT temperature (YAN-GDGT) data (grey plot, with RMSE errors shaded grey in Supplementary Fig. 16a) and the Maxwell Bay measured TOC data (sea-ice proxy record)52 (grey plot in Fig. 4c) were smoothed using polynomial negative exponential regression analysis. Results were similar to smoothing by LOESS ﬁrst- order polynomial regression with tri-cube weighting (not shown). Reconstructed GDGT temperatures reﬂect MSAT (December, January, February or DJF) in the shallow and well-mixed open lake water environment of Yanou Lake76,77. ¼ Pðnumber of penguinsÞ To further validate our ﬁndings, we also calculated the catchment area required by the reconstructed colony population using published gentoo penguin density values40 and calculated mixed-species density values, partitioned according to published modern-day Ardley Island gentoo-Ade´lie-Chinstrap species compositions41 and the corresponding species density values40 (Supplementary Fig. 19b, Supplementary Table 9). After smoothing, all climate (YAN-GDGT, JRI29 and PD48); and sea-ice (Anvers Shelf and Maxwell Bay52) data sets were resampled at 100-year intervals (see Supplementary Methods). We then used 46–0 ka upper bound on the weighted mean (95% conﬁdence level) temperature, temperature anomaly, diatom open water ratio and smoothed TOC- and diatom-based sea-ice proxy data (Fig. 4) to deﬁne ‘warm’ and ‘sea-ice free’ (open-water) periods. We chose the 6–0 ka period and values greater than the upper bound on the weighted mean at 95% conﬁdence level of the weighted mean as this provides the best longer-term assessment of the mean temperature and variability that penguin colonies on Ardley Island would have encountered. It also corresponds to the time period covered by the terrestrial Yanou Lake GDGT-MSAT record. Using the Pre-industrial era (1,000–250 cal a BP) and its upper bound on the weighted mean value (95% conﬁdence) produced similar timings for ‘warm’ and ‘open-water’ intervals. y pp y g pp y The ‘most-likely’ minimum guano-delivery (yield) scenario assumes that 11% of the guano produced by penguins in the Ardley Lake catchment was eroded into Ardley Lake. This scenario produced a peak guano phase GP-4 colony population of 1,024±574 penguins, which occupied an area of 1,679–3,303 m2 out of the 66,249 m2 Ardley Lake catchment area (based on partitioned mixed species minimum density–gentoo penguin maximum density values40). Meanwhile, the 0.96% guano-yield scenario produced a maximum peak GP-4 colony population of 11,723±5,104, which corresponds to an occupied area of 20,686–37,815 m2 (Supplementary Fig. 19). Therefore, the c. 66,250 m2 Ardley Lake catchment area is easily large enough to accommodate all modelled population scenarios shown in Fig. 5. Assuming a hyperboloid basin shape for Ardley Lake would result in an average 33±25% reduction in the reconstructed population across all guano delivery scenarios. To compare variations in cross-Peninsula climate during guano and non-guano phases, we undertook normality tests and produced a set of descriptive statistics for the whole Ardley Fo.sed. ¼ Pðnumber of penguinsÞ ¼ Pðnumber of penguinsÞ Since the catchment colony would have produced and/or deposited guano outside the catchment area (for example, on foraging trips), and not all guano deposited in catchment areas is currently eroded into lake due to various factors, for example, utilization by vegetation, we consider the Gy–100% to be very unlikely. Therefore, we consider the Gy–11% and IGAo30ellip tð Þ to best represent a possible ‘most likely’ minimum population scenario as follows: Since the catchment colony would have produced and/or deposited guano outside the catchment area (for example, on foraging trips), and not all guano deposited in catchment areas is currently eroded into lake due to various factors, for example, utilization by vegetation, we consider the Gy–100% to be very unlikely. Therefore, we consider the Gy–11% and IGAo30ellip tð Þ to best represent a possible ‘most likely’ minimum population scenario as follows: MINPAo30ellipðtÞ ¼ IGAo30ellipðtÞ Gy11% Units : g a 1 g a 1p 1 ð11Þ ¼ Pðnumber of penguinsÞ ð11Þ ¼ Pðnumber of penguinsÞ ¼ Pðnumber of penguinsÞ ¼ Pðnumber of penguinsÞ As a reality check, the Gy-11% and IGAo30ellipðtÞ scenario recreated the near- absence of a penguin colony (o10 penguins) in the modern day catchment. Conversely, we consider that the Gy-0.96% and IGAo30ellipðtÞ scenario represents the ‘most likely’ maximum population scenario: As a reality check, the Gy-11% and IGAo30ellipðtÞ scenario recreated the near- absence of a penguin colony (o10 penguins) in the modern day catchment. As a reality check, the Gy-11% and IGAo30ellipðtÞ scenario recreated the near- absence of a penguin colony (o10 penguins) in the modern day catchment. y y pð Þ absence of a penguin colony (o10 penguins) in the modern day catchment. Conversely, we consider that the Gy-0.96% and IGAo30ellipðtÞ scenario represents the ‘most likely’ maximum population scenario: Conversely, we consider that the Gy-0.96% and IGAo30ellipðtÞ scenario represents the ‘most likely’ maximum population scenario: MAXPAo30ellipðtÞ ¼ IGAo30ellipðtÞ Gy0:96% Units : g a 1 g a 1p 1 ð12Þ ¼ Pðnumber of penguinsÞ ð12Þ ¼ Pðnumber of penguinsÞ ð12Þ These two end-member scenarios are shown in Fig. 5, but since sedimentation rates in Ardley Lake have varied signiﬁcantly through time, it is possible that, when erosion rates are high, for example, between c. 5.5 and 4.5 cal ka BP, the Ardley Island colony population would be better estimated by a guano-yield scenario somewhere between 11 and 100%. ARTICLE ARTICLE NATURE COMMUNICATIONS \| 8:14914 \| DOI: 10.1038/ncomms14914 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14914 For the YAN record, whole YAN-GDGT data set 5th and 95th percentile outliers (o 0.33 C and 48.61 C) 11.34 C and 13.84 C at 1,164 cal a BP and 3,135 cal a BP, respectively, were excluded by smoothing analysis (Fig. 4a,c, Supplementary Fig. 17a). These values were also greater than the 10.3 C maximum limit of the surface sediment-MSAT Antarctic and sub-Antarctic GDGT-MSAT calibration (ANT-GDGT) data set77. All reconstructed GDGT temperatures were greater than the 2.2 C minimum limit of the ANT-GDGT calibration data set. The YAN- GDGT o10 C data set was used in 6–0 ka weighted mean temperature anomaly calculations, statistical analyses and hypothesis testing. The mean±1s 6–0 ka reconstructed YAN-GDGT o10 C data set weighted mean MSAT value of þ 2.39±2.67 C [95% conﬁdence interval: 1.52–3.25 C] (Bootstrapping, n ¼ 10,000, þ 2.38±2.61 C [1.52–3.25 C]; 5th/95th percentile ¼ 0.51/ þ 7.96 C; weighted by Antarctic GDGT calibration data set RMSE value of 1.45 C; Supplementary Table 7) encompasses the modern-day (1968–2015 CE) observational mean summer (DJF) temperature from Bellingshausen Research Station on Fildes Peninsula of þ 1.09±0.56 C (2s 95% measured range ¼ 0.03 to 2.21 C; n ¼ 48, 1968–2015 CE, where year refers to December) (Marshall, pers. comm.; SCAR-READER database: http://www.antarctica.ac.uk/met/READER/). On a regional to global-scale modern-day reconstructed lake sediment GDGT- temperatures broadly reﬂect changes in air temperature77. At the local, small lake scale, water temperature might decouple from observed MSAT due to factors such as the thickness and length of seasonal lake ice-cover and the volume of meltwater input, both of which suppress lake water temperatures. Conversely, since water bodies store excess heat generated during extended ice-free seasons, small and/or shallow lakes (o10 m deep) reach temperatures of 6 C or more (for example, Sombre Lake, Signy Island)82. As the focus of this paper is penguin colony reconstruction, we investigate these aspects further in a forthcoming paper. Gy100% ¼ 7; 716 2; 728 g a 1p 1 Gy11% ¼ 847 300 g a 1p 1 Gy0:96% ¼ 74 26 g a 1p 1 as follows as follows PAo30ellipðtÞ ¼ IGAo30ellipðtÞ Gy100%;11%;0:96% Units : g a 1 g a 1p 1 ð10Þ ¼ Pðnumber of penguinsÞ ð10Þ ¼ Pðnumber of penguinsÞ ð1 ARTICLE values GD ¼ Gy100% Ge ¼ Alake Acatch ¼ 7; 274 66; 249 m2 m2 ¼ 1 : 9 ¼ 11% ð7Þ ð7Þ where GD is the ratio, expressed as a percentage, of the total guano produced in the catchment (Gy-100%) and the total guano-eroded from the catchment (Ge) into the lake, which, here we approximate to the lake area: lake catchment ratio, Alake=Acatch. Alake=Acatch. NATURE COMMUNICATIONS \| 8:14914 \| DOI: 10.1038/ncomms14914 12 ARTICLE 22) and is present in the JRI ice core29. Neither the 1976 or 1970 eruptions formed visible ash layers near the top of the ARD core, probably because only up to 1 mm of ash was deposited on Fildes Peninsula21. Therefore, we broadly equated major VEI ¼ 3 eruptions from Deception to visible tephra deposits o1 cm thick in the YAN and ARD records, and airfall ash deposits 41 cm thick to VEI43 eruptions. Since the post-deglaciation (after c. 8–7.5 cal ka BP) lithogenic deposition style in YAN and ARD is predominantly ﬁne-grained silt-clay, the sand dry mass accumulation rate (DMAR) provided a useful proxy for eruption size and the volume of tephra deposited onto Ardley Island and King George Island during VEI 3 i (Fi 4 b S l Fi 12 20 21) hil l d C /Ti 18. Li, C. et al. Two Antarctic penguin genomes reveal insights into their evolutionary history and molecular changes related to the Antarctic environment. Gigascience 3, 27 (2014). g 19. Levy, H. et al. Population structure and phylogeography of the Gentoo Penguin (Pygoscelis papua) across the Scotia Arc. Ecol. Evol. 6, 1834–1853 (2016). 20. Bartolini, S., Geyer, A., Martı´, J., Pedrazzi, D. & Aguirre-Dı´az, G. Volcanic hazard on Deception Island (South Shetland Islands, Antarctica). J. Volcanol. Geotherm. Res. 285, 150–168 (2014). 21. Pedrazzi, D., Aguirre-Dı´az, G., Bartolini, S., Martı´, J. & Geyer, A. The 1970 eruption on Deception Island (Antarctica): eruptive dynamics and implications for volcanic hazards. J. Geol. Soc. London 171, 765–778 (2014). 22. Fretzdorff, S. & Smellie, J. L. Electron microprobe characterization of ash layers in sediments from the central Bransﬁeld basin (Antarctic Peninsula): evidence for at least two volcanic sources. Antarct. Sci. 14, 412–421 (2003). 23. Moreton, S. G. & Smellie, J. L. Identiﬁcation and correlation of distal tephra layers in deep-sea sediment cores, Scotia Sea, Antarctica. Ann. Glaciol. 27, 285–289 (1998). Data availability. Key data sets for this study are in Supplementary Data ﬁles 1–7. All data produced by this study and associated IMCOAST/IMCONET projects are available from the corresponding author (sjro@bas.ac.uk) and the NERC Polar Data Centre (BAS) (https://www.bas.ac.uk/data/uk-pdc/) and Pangaea Data Publisher website (AWI) (https://www.pangaea.de/). Antarctic location maps and spatial data in Fig. 1 are the LIMA (Landsat Image Mosaic of Antarctica) downloaded from https://lima.usgs.gov/index.php and the SCAR Antarctic Digital Database http://www.add.scar.org/, used under a Creative Commons license. The basemap in Fig. References Nature 535, 411–415 (2016). 37. Meredith, M. P. & King, J. C. Rapid climate change in the ocean west of the Antarctic Peninsula during the second half of the 20th century. Geophys. Res. Lett. 32, 1–5 (2005). 8. Cook, A. J. et al. Ocean forcing of glacier retreat in the western Antarctic Peninsula. Science 353, 283–286 (2016). 38. Trivelpiece, W. Z. et al. 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Ser. 265, 1–15 (2003). 32. Lynch, H. J., Naveen, R., Trathan, P. N. & Fagan, W. F. Spatially integrated assessment reveals widespread changes in penguin populations on the Antarctic Peninsula. Ecology 93, 1367–1377 (2012). g 4. Forcada, J., Trathan, P. N., Reid, K., Murphy, E. J. & Croxall, J. P. Contrasting population changes in sympatric penguin species in association with climate warming. Glob. Chang. Biol. 12, 411–423 (2006). gy 33. Dunn, M. J. et al. Population size and decadal trends of three penguin species nesting at Signy Island, South Orkney Islands. PLoS ONE 11, e0164025 (2016). 34. Lynch, H. J., Fagan, W. F. & Naveen, R. Population trends and reproductive success at a frequently visited penguin colony on the western Antarctic Peninsula. Polar Biol. 33, 493–503 (2010). 5. Lynch, H. J., Fagan, W. F., Naveen, R., Trivelpiece, S. G. & Trivelpiece, W. Z. Differential advancement of breeding phenology in response to climate may alter staggered breeding among sympatric pygoscelid penguins. Mar. Ecol. Prog. Ser. 454, 135–145 (2012). 35. Cimino, M. A., Lynch, H. J., Saba, V. S. & Oliver, M. J. Projected asymmetric response of Ade´lie penguins to Antarctic climate change. Sci. Rep. 6, 28785 (2016). 6. Southwell, C. et al. Spatially extensive standardized surveys reveal widespread, multi-decadal increase in East Antarctic Ade´lie penguin populations. PLoS ONE 10, e0139877 (2015). 36. Sun, L. et al. Vertebrate records in polar sediments: biological responses to past climate change and human activities. Earth-Science Rev. 126, 147–155 (2013). 7. Turner, J. et al. Absence of 21st century warming on Antarctic Peninsula consistent with natural variability. ARTICLE 1a and Supplementary Figs 20,23 is the ESRI Ocean Basemap (http://goto.arcgisonline.com/maps/Ocean_Basemap) used under a BAS-NERC software license. Satellite images and image derivatives in Figs 1c,d and 6 and Supplementary Figs 2,22,23 are copyright of DigitalGlobe, Inc. All Rights Reserved (Catalogue numbers: 1030010020C0C900 and 11MAR04115702), reproduced at low resolution in a static format under a BAS-NERC educational license, and are not included in the Creative Commons license for the article. 24. Smellie, J. L. Lithostratigraphy and volcanic evolution of Deception Island, South Shetland Islands. Antarct. Sci. 13, 188–209 (2001). 25. Newhall, C. G. & Self, S. The volcanic explosivity index (VEI) an estimate of l i it d f hi t i l l i J G ph R 87 1231 (1982) 25. Newhall, C. G. & Self, S. The volcanic explosivity index (VEI) an estimate of explosive magnitude for historical volcanism. J. Geophys. Res. 87, 1231 (1982). 26. Ben-Zvi, T. et al. The P-wave velocity structure of Deception Island, Antarctica, from two-dimensional seismic tomography. J. Volcanol. Geotherm. Res. 180, 67–80 (2009). 27. Toro, M. et al. Chronostratigraphy of the sedimentary record of Limnopolar Lake, Byers Peninsula, Livingston Island, Antarctica. Antarct. Sci. 25, 198–212 (2013). 28. Bjo¨rck, S., Håkansson, H., Zale, R., Karlen, W. & Jo´nsson, B. L. A late Holocene lake sediment sequence from Livingston Island, South Shetland Islands, with palaeoclimatic implications. Antarct. Sci. 3, 61–72 (1991). 29. Mulvaney, R. et al. Recent Antarctic Peninsula warming relative to Holocene climate and ice-shelf history. Nature 489, 141–144 (2012). ¼ Pðnumber of penguinsÞ data set (weighted by 1s error), the YAN-GDGT o10 C temperature data set, the JRI temperature anomaly data set29, and the PD 0–200 m sea-surface temperature (PD-SST) record48 (weighted by published errors, where available). Using a ¼ 5% and 10% signiﬁcance levels for three hypothesis test scenarios A–C shown in Supplementary Table 8, we ﬁrst performed Fisher’s F-test Variance Ratio analysis. Since some data sets were not normally distributed, we then used the Mann–Whitney U-statistic to test the hypothesis that temperature and temperature anomaly distributions in the YAN-GDGT o10 C, JRI, PD-SST records were statistically different during the ﬁve guano-inﬂuenced phases (Scenario 1A in Supplementary Table 8) compared to the eight non-guano phases (Scenario 2A). We then repeated this process comparing guano phases of the mid- Holocene (8.2–4.2 ka; 1B) to those of the late Holocene (4.2–0 ka; 2B), and for late Holocene guano phases (1C) versus non-guano phases (2C). We also undertook similar hypothesis testing using the Maxwell Bay sea-ice and Anvers Shelf open- water data sets to determine whether sea-ice conditions within guano and non- guano were statistically different in the three scenarios (A–C) described above. Results are summarized in Supplementary Table 8. It is important to note that the apparent increase in the amount of guano deposited into Ardley Lake over the last 500 years is less than the signiﬁcant 10% Fo.sed. level we used to determine colony presence elsewhere in its Holocene record, and only equates to a total of 65±48 penguins (using the 11% guano-delivery scenario). This also illustrates why the Ardley Lake guano phase colony was too small after c. 2,500 years for us to determine whether volcanic activity had a signiﬁcant impact. Correlation analysis of geochemical data was undertaken using R 2.15.2 (R Foundation for Statistical Computing). Hierarchical R-mode cluster analyses were conducted with the R package ‘Pvclust’ (version 1.2-2) using average linkage and a correlation-based dissimilarity matrix (see Supplementary Methods for software references). Based on multi-scale bootstrap resampling (number of bootstraps: 10,000), approximately unbiased P values were further calculated to 13 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14914 Apart from one recent minor eruption, all visible (that is, 42 mm thick) Holocene-age tephra deposits in lake, marine and ice core records of the northern Antarctic Peninsula (N-AP), SSI and Scotia Sea have been linked to VEI ¼ 3 or VEI43 eruptions from Deception Island20–23,27–29,58,63,83,84 (Supplementary Note 9, Supplementary Figs 20, 21, Supplementary Tables 3, 10). Tephra from even relatively small 1967 and 1970 CE Deception Island eruptions (VEI ¼ 3) reached more than 150 km (ref. 22) and is present in the JRI ice core29. Neither the 1976 or 1970 eruptions formed visible ash layers near the top of the ARD core, probably because only up to 1 mm of ash was deposited on Fildes Peninsula21. Therefore, we broadly equated major VEI ¼ 3 eruptions from Deception to visible tephra deposits o1 cm thick in the YAN and ARD records, and airfall ash deposits 41 cm thick to VEI43 eruptions. Since the post-deglaciation (after c. 8–7.5 cal ka BP) lithogenic deposition style in YAN and ARD is predominantly ﬁne-grained silt-clay, the sand dry mass accumulation rate (DMAR) provided a useful proxy for eruption size and the volume of tephra deposited onto Ardley Island and King George Island during VEI43 eruptions (Fig. 4a,b, Supplementary Figs 12,20,21), while elevated Ca/Ti ratios and PCA2 values from combined 2 mm and 200 mm m-XRF scanning data and the reliable single-species aquatic moss age-depth model provided precise stratigraphic constraints on the positions (and ages) of tephra deposits in the YAN record (Supplementary Note 9, Supplementary Figs 9–11,16, Supplementary Table 3). Apart from one recent minor eruption, all visible (that is, 42 mm thick) Holocene-age tephra deposits in lake, marine and ice core records of the northern 16. Emslie, S. D., Polito, M. J., Brasso, R., Patterson, W. P. & Sun, L. Ornithogenic soils and the paleoecology of pygoscelid penguins in Antarctica. Quat. Int. 352, 4–15 (2014). g p p Antarctic Peninsula (N-AP), SSI and Scotia Sea have been linked to VEI ¼ 3 or VEI43 eruptions from Deception Island20–23,27–29,58,63,83,84 (Supplementary Note 17. Clucas, G. V. et al. A reversal of fortunes: climate change ‘winners’ and ‘losers’ in Antarctic Peninsula penguins. Sci. Rep. 4, 5024 (2014). 9, Supplementary Figs 20, 21, Supplementary Tables 3, 10). Tephra from even relatively small 1967 and 1970 CE Deception Island eruptions (VEI ¼ 3) reached more than 150 km (ref. NATURE COMMUNICATIONS \| 8:14914 \| DOI: 10.1038/ncomms14914 \| www.nature.com/naturecommunications Acknowledgements This study forms part of the ESF-funded IMCOAST project, AP-6 led by S.J.R., and the EU project IMCONet (FP7404 IRSES, action no. 319718; coordinated by the Alfred Wegener Institute, Helmholtz Centre for Polar and Marine Research, Germany) with additional funding from the Natural Environmental Research Council (NERC), and the German Research Foundation (DFG project no. BR 775/25-1) and logistic support from the NERC-British Antarctic Survey (BAS), HMS Endurance and 892 Naval Air Squadron, the Alfred Wegner Institute (AWI) and the Instituto Anta´rtico Argentino (IAA). We thank NERC for funding studentship NE/J500173/1 (BAS and Newcastle University) to L.C.F. (supervisors S.J.R., E.J.P., D.A.H., S.J.). We thank colleagues at the Chinese Great Wall Station and Russian Bellingshausen Bases on Fildes Peninsula for their generous hospitality, H. Biester and T. Riedel from Braunschweig University for help with Hg analyses, technical assistants at the Institute for Chemistry and Biology of the Marine Environment (ICBM), Hilary Blagbrough and Paul Geissler at BAS, S. Xu at the SUERC AMS Facility, Chris Hayward (EPMA, University of Edinburgh), the Natural History Museum (Tring) for access to, and advice about, their avian fossil-bone collection, Helen Talbot and Frances Sidgwick for running samples on the LC-MS at Newcastle University, and Huw Grifﬁths (BAS) for assistance with ARC-GIS. We thank Phil Trathan, Claire Waluda and three reviewers for their constructive comments. 61. Marcott, S. A., Shakun, J. D., Clark, P. U. & Mix, A. C. A reconstruction of regional and global temperature for the past 11,300 years. Science 339, 1198–1201 (2013). 62. Domack, E. W. et al. Chronology of the Palmer Deep site, Antarctic Peninsula: a Holocene palaeoenvironmental reference for the circum-Antarctic. Holocene 11, 1–9 (2001). 63. Roberts, S. J. et al. 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Holocene glacial and climate history of Prince Gustav Channel, northeastern Antarctic Peninsula. Quat. Sci. Rev. ARTICLE 31, 93–111 (2012). 86. Machado, A. et al. Geochemistry constraints of Mesozoic–Cenozoic calc- alkaline magmatism in the South Shetland arc, Antarctica. J. South Am. Earth Sci. 18, 407–425 (2005). 59. Allen, C. S., Oakes-Fretwell, L., Anderson, J. B. & Hodgson, D. A. A record of Holocene glacial and oceanographic variability in Neny Fjord, Antarctic Peninsula. Holocene 20, 551–564 (2010). 60. Masson-Delmotte, V. et al. A comparison of the present and last interglacial periods in six Antarctic ice cores. Clim. Past 7, 397–423 (2011). Acknowledgements A geochemical record of late Holocene palaeoenvironmental changes at King George Island (maritime Antarctica). Antarct. Sci. 23, 255–267 (2011). 67. Whitehouse, P. L., Bentley, M. J. & Le Brocq, A. M. A deglacial model for Antarctica: geological constraints and glaciological modelling as a basis for a new model of Antarctic glacial isostatic adjustment. Quat. Sci. Rev. 32, 1–24 (2012). Author contributions S.J.R., P.M., L.C.F., D.A.H. and M.J.B wrote the paper. Fieldwork was carried out by S.J.R., D.A.H., E.P.H., M.J.B. and P.F.; P.M., S.J.R., L.C.F., E.J.P., S.J., J.L., B.S., H.-J.B. undertook analytical work and data analysis and contributed to the manuscript; S.J.R. and S.J.D. undertook and interpreted core scan data, A.R.H. and C.S.A. collected and conducted diatom analyses on marine cores from the Anvers Shelf; L.I. and P.F. per- formed satellite vegetation mapping analysis; R.O. identiﬁed mosses used for radiocarbon dating; S.G.M. undertook SUERC radiocarbon dating analyses and provided his pre- viously analysed Deception Island EPMA data. All authors contributed to the inter- pretations and commented on the manuscript. 68. Argus, D. F., Peltier, W. R., Drummond, R. & Moore, A. W. The Antarctica component of postglacial rebound model ICE-6G_C (VM5a) based on GPS positioning, exposure age dating of ice thicknesses, and relative sea level histories. Geophys. J. Int. 198, 537–563 (2014). histories. Geophys. J. Int. 198, 537–563 (2014). 69. Hodgson, D. A. et al. Late Quaternary environmental changes in Marguerite Bay, Antarctic Peninsula, inferred from lake sediments and raised beaches. Quat. Sci. Rev. 68, 216–236 (2013). ´ 70. O´ Cofaigh, C. et al. Reconstruction of ice-sheet changes in the Antarctic Peninsula since the Last Glacial Maximum. Quat. Sci. Rev. 100, 87–110 (2014). References Stable isotope analysis of ancient and modern gentoo penguin egg membrane and the krill surplus hypothesis in Antarctica. Antarct. Sci. 25, 213–218 (2013). 44. Liu, X. D., Sun, L. G., Xie, Z. Q., Yin, X. B. & Wang, Y. H. A 1300-year record of penguin populations at Ardley Island in the Antarctic, as deduced from the 14 NATURE COMMUNICATIONS \| 8:14914 \| DOI: 10.1038/ncomms14914 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| 8:14914 \| DOI: 10.1038/ncomms14914 \| www.nature.com/ NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14914 NATURE COMMUNICATIONS \| 8:14914 \| DOI: 10.1038/ncomms14914 \| www.nature.com/naturecommunications Additional information 71. Van Daele, M. et al. The 600 yr eruptive history of Villarrica Volcano (Chile) revealed by annually laminated lake sediments. Geol. Soc. Am. Bull. 126, 481–498 (2014). Supplementary Information accompanies this paper at http://www.nature.com/ naturecommunications 15 NATURE COMMUNICATIONS \| 8:14914 \| DOI: 10.1038/ncomms14914 \| www.nature.com/naturecommunications URE COMMUNICATIONS \| 8:14914 \| DOI: 10.1038/ncomms14914 \| www.nature.com/naturecommunications r The Author(s) 2017 NATURE COMMUNICATIONS \| 8:14914 \| DOI: 10.1038/ncomms14914 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14914 NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14914 Competing interests: The authors declare no competing ﬁnancial interests. 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/ Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ How to cite this article: Roberts, S. J. et al. Past penguin colony responses to explosive volcanism on the Antarctic Peninsula. Nat. Commun. 8, 14914 doi: 10.1038/ ncomms14914 (2017). Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. r The Author(s) 2017 NATURE COMMUNICATIONS \| 8:14914 \| DOI: 10.1038/ncomms14914 \| www.nature.com/naturecommunications 16
W2284225525.txt	https://www.nature.com/articles/ncomms12890.pdf	en		A physical zero-knowledge object-comparison system for nuclear warhead verification	Nature communications	2,016	cc-by	6,703	ARTICLE Received 27 Feb 2016 \| Accepted 12 Aug 2016 \| Published 20 Sep 2016 DOI: 10.1038/ncomms12890 OPEN A physical zero-knowledge object-comparison system for nuclear warhead veriﬁcation Sébastien Philippe1, Robert J. Goldston2, Alexander Glaser1 & Francesco d’Errico3,4 Zero-knowledge proofs are mathematical cryptographic methods to demonstrate the validity of a claim while providing no further information beyond the claim itself. The possibility of using such proofs to process classiﬁed and other sensitive physical data has attracted attention, especially in the ﬁeld of nuclear arms control. Here we demonstrate a non-electronic fast neutron differential radiography technique using superheated emulsion detectors that can conﬁrm that two objects are identical without revealing their geometry or composition. Such a technique could form the basis of a veriﬁcation system that could conﬁrm the authenticity of nuclear weapons without sharing any secret design information. More broadly, by demonstrating a physical zero-knowledge proof that can compare physical properties of objects, this experiment opens the door to developing other such secure proof-systems for other applications. 1 Department of Mechanical and Aerospace Engineering, Princeton University, E-Quad, Olden Street, Princeton, New Jersey 08544, USA. 2 Princeton Plasma Physics Laboratory, Princeton University, PO Box 451, MS 41, Princeton, New Jersey 08540, USA. 3 School of Engineering, University of Pisa, Largo Lazzarino 1, 56126 Pisa, Italy. 4 School of Medicine, Yale University, 300 Cedar Street, New Haven, Connecticut 06520, USA. Correspondence and requests for materials should be addressed to S.P. (email: sp6@princeton.edu). NATURE COMMUNICATIONS \| 7:12890 \| DOI: 10.1038/ncomms12890 \| www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12890 T he next round of arms-control agreements may require a trusted veriﬁcation mechanism to conﬁrm the authenticity of items presented as nuclear warheads. Proliferation and national security concerns require, however, that such a veriﬁcation mechanism reveals no information about the composition or design of these warheads1–3. Considerable research efforts have been directed towards the development of engineered information barriers to address this paradox4,5. These barriers consist of automated measurement and analysis systems that process sensitive information but only display the results of internal analysis in a binary manner (valid object and invalid object). These systems are, by their nature, at risk of electronic tampering and snooping—and their trusted implementation has so far proved difﬁcult to realize6–8. Glaser, Barak and Goldston (GBG)6 have recently proposed a different approach based on a zero-knowledge interactive proof, or protocol, for warhead veriﬁcation. Physical zero-knowledge protocols can be used to perform comparisons or computations on physical data such that sensitive information is never measured and does not need to be protected afterwards9. They rely on the concept of interactive zero-knowledge proofs, originally developed for computational cryptographic applications10, which have the property of yielding no knowledge beyond the validity of the assertion being proven. This property remains guaranteed only if the prover follows the protocol11. Attempts at cheating open the possibility of information leakage, a potential deterrent for a range of attacks6,7. The timely demonstration of a trusted zeroknowledge warhead inspection system would represent a breakthrough towards a veriﬁcation regime targeted at deeper and multilateral cuts in nuclear arsenals. Other critical applications of physical zero-knowledge proofs could include any system where classiﬁed or personal data need to be protected, such as in forensic DNA analysis9. We have devised a physical zero-knowledge system, using a non-electronic neutron differential radiography technique, to perform reproducible object comparison without ever acquiring data about the objects being compared. We employ this system to demonstrate experimentally the validity of key aspects of the protocol for warhead veriﬁcation proposed by GBG. In a real inspection, this protocol requires the existence of at least one reference warhead, for example, retrieved from active delivery systems12,13 at the inspector’s designation. Using simple radiographic test objects, we ﬁnd experimentally that items practically identical to the reference item can be conﬁrmed to be genuine while inspectors gain no knowledge about their geometry and composition. Furthermore, items differing signiﬁcantly from the reference item can be discriminated. Results A zero-knowledge object-comparison system. Our technique compares the neutron radiographic proﬁle of an inspected item against that of a reference item at various energies (in particular 14 MeV) to conﬁrm that the items are identical within experimental accuracy (Fig. 1). In the case of nuclear weapons, such data are highly sensitive. We circumvent this problem by recording the radiographs of the inspected items on sets of previously exposed superheated emulsion (bubble) detectors14 preloaded with the ‘complement’ of the transmission image of the reference item (or any item claimed to be similar to the reference item; Fig. 1a). In this case, the ‘complement’ implemented by the host, the owner of the items, is deﬁned as the preload that will result in the total ﬂuence recorded in each detector being the same as if no item had been present when the inspected item is the same as the reference item—we call this total 2 count Nmax. Such a radiograph with a ﬂat proﬁle of Nmax and Poisson noise conveys zero information. We emphasize that the inspector is not present while the host prepares the preloads. In the GBG protocol the inspector is offered multiple sets of detectors preloaded by the host and then randomly selects which to use either with the reference item(s) or with the inspected item(s). This prevents the host from matching with certainty a modiﬁed preload to a spoof warhead. The use of non-electronic detectors in the protocol also avoids the risks of electronic tampering by the host once the preload has been selected and of electronic snooping by the inspector to attempt to learn the preload pattern. The inspection protocol. We envision that the GBG protocol could be realized the following way: ﬁrst, the host declares (n 1) items as ‘treaty accountable items’ that are to be inspected and compared with a reference item (for a total of n items). Here we assume a single reference item, but there could be more than one in principle. The host claims that all n items are identical. Then, the parties must agree to the appropriate level of statistics for the inspection. This includes agreeing on Nmax as well as any statistical test to be applied to the data. Probabilities of passing a test depend on random choices made by the inspector and the measurement noise, which are independent between each invocation of the inspection test. Finally, we assume that parties have agreed on procedures for chain of custody, perimeter monitoring and other security measures related to the conduct of the inspections. Initialization 1. The host produces a collection of detectors. He measures and records their neutron efﬁciency curves. The inspector then chooses a subset of these detectors to be used in the inspections, and can take home the others and independently conﬁrm their calibration curves. 2. The host takes a large number of radiographs of non-reference items at m different angles, and l different incoming neutron energies minimizing any systematic errors in the preloads. The radiographs can be taken on non-reference items to avoid difﬁculties associated with maintaining chain of custody for the reference item, which is the only item inspectors believed to be genuine before inspections start. Preparation of preloads. The host, without the inspector present, prepares n preloaded detector sets pki;j with k ¼ 1yn, each corresponding to the complement image of the treaty accountable items (claimed to be identical to the reference) at a particular angle yi, for i ¼ 1ym, and particular energy Ej, for j ¼ 1yl. Inspection 1. The inspector calls for an orientation yi of the items and energy Ej of the neutron beam. 2. The host presents the corresponding n preloaded detector sets p1i;j ; . . . ; pni;j to the inspector (including the associated calibration data). The inspector chooses a random assignment of the detector sets to the reference item and (n 1) inspected items. 3. The items are exposed to the neutron source and their radiographs are recorded on the preloaded detectors. Both parties monitor the source ﬂuence—each with their own apparatus. This ensures the inspector that a measurement is NATURE COMMUNICATIONS \| 7:12890 \| DOI: 10.1038/ncomms12890 \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12890 a b + = Valid Complement (Accept) = + Spoof Complement c Single-pixel bubble detector (Reject) Collimated neutron beam (14-MeV DT generator) AL AL Reference item AL SS SS AL AL SS AL SS d Staging area (with reference item) 1 2 3 4 5 6 7 Detector position Detector array Figure 1 \| Experimental realization of a physical zero-knowledge object comparison system. (a) Concept of zero-knowledge differential neutron radiography using superheated droplet detectors. Items are exposed to a neutron beam and their 2D transmission radiographs are recorded on detectors preloaded with the complement radiograph (including Poisson noise) of a reference item. If the item is valid (identical to the reference), the ﬁnal radiograph is identical to the expected exposure if no object had been present (in the ideal implementation the root mean square deviation from the expected bubble count, Nmax, is solely because of Poisson noise, (Nmax)0.5). If the item is a spoof with an experimentally signiﬁcantly different radiograph, some characteristic features appear in the ﬁnal radiograph (the results are no longer zero-knowledge) and the inspector rejects the proof. Each pixel represents the bubble count from a single superheated droplet (bubble) detector. (b) Picture of an irradiated superheated droplet (bubble) detector. Some metastable droplets vapourized and expanded into macroscopic bubbles after a neutron interaction. The bubble count reﬂects the total ﬂuence delivered to the detector. (c) One-dimensional experimental realization with superheated droplet detector array, reference item (black box hood not shown) on staging area and aperture of a 14 MeV collimated neutron beam. The set-up is placed in a room shielded with borated concrete walls. A fast neutron counter (not shown) monitored the source ﬂuence along the axial direction. (d) Composition and pattern of the reference item with detector positions (AL, aluminium; SS, stainless steel). taking place, and that the agreed ﬂuence corresponding to Nmax has been provided. 4. After the inspection of each item, the number of macroscopic bubbles in each detector is counted. Both parties compute the result of the agreed upon test for the hypothesis that the distribution of the counts is n independent Poisson random variables each with expectation Nmax. 5. Some detectors, as selected by the inspector, are recompressed (to remove the bubbles, preparing them for re-use) and reexposed to the source to verify their functionality and speciﬁed calibration. Test repetition. The inspection phase can be repeated to increase arbitrarily the soundness of the proof. The inspector can decide to re-test items at the same angle and energy (for example, if a particular item has failed the test), or continue to test the items at another (yi, Ej) pair. By this means, the protocol can meet arbitrarily stringent overall false-positive and -negative rates by accumulating statistics. Strategies to minimize the effort to achieve speciﬁc overall type I and type II errors will be the subject of a future publication. In this realization, we assume that the inspector follows the protocol described here. All actions of the inspector, unlike those of the host, are performed in public view of both parties. In particular, the inspector has only access to the bubble count in each detector using an agreed-upon bubble-counting technique. She does not, for example, have any possibility to measure the neutron-induced activation level of the bubble detectors. Properties of the protocol. Zero knowledge: At the end of the protocol, if the host behaved honestly, we expect the total bubble count in each detector to be Poisson-distributed with expectation value Nmax, the sum of a matching preload and radiograph— equal to the total count if no item had been present between the source and the detectors. For the zero-knowledge property to be conserved, neither the signal nor the noise may carry information. For a particle beam experiment, where many particles would strike a detector for every recorded interaction, and in the case where discrete counts are independent, random and occurring at a constant rate, the counts should be distributed according to a NATURE COMMUNICATIONS \| 7:12890 \| DOI: 10.1038/ncomms12890 \| www.nature.com/naturecommunications 3 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12890 Poisson distribution15–17. Recalling that the sum of two independent Poisson random variables is a Poisson random variable18, we obtain that the sum of the radiograph and its matching complement is Poisson-distributed with parameter Nmax—that is, both the mean and variance of this distribution are Nmax, in the limit of high accuracy of calibration and preload as discussed infra. Therefore, as long as the items are identical, the veriﬁer observes n independent draws from a Poisson(Nmax) random variable. Since the inspector could have sampled this a priori, she does not learn anything. Soundness and completeness: The soundness and completeness of the protocol are based on the assumption that two items with signiﬁcantly different geometry and/or composition result in a difference in the expected bubble counts. ‘Signiﬁcant differences’ are deﬁned by the parties within the treaty protocol. Neutron counts in the detectors are affected by the geometry and physical properties of the item. For example, neutron opacity depends on the total neutron cross-section, which is energy- and nuclide-dependent; neutron intensities and their distributions at large angles depend on elastic and inelastic scattering, ﬁssion and (n,2n) reaction cross-sections, again energy- and nuclidedependent; geometry and ﬁssile material composition affect the neutron multiplication factor, which is also energy-dependent. Exposing the inspected item to different neutron energies allows verifying that both geometry and material composition are not signiﬁcantly different. Energies at which neutrons are highly penetrating (such as 14-MeV neutrons) or where large differences in ﬁssion cross-sections19, or large resonances in the total crosssection20 exist, are of particular interest. It is possible to probe the object at an energy lower than the detector energy threshold for bubble nucleation to emphasize differences in the ﬁssion signature21. Formally, we assume that if an item X is not signiﬁcantly different from the reference item, then it passes inspections at all angle–energy (yi, Ej) pairs with probability of at least (1 a), where a is the false-positive rate. Furthermore, we make the physical assumption that there exists b such that for any reference item X and any object Y created by the adversary, if Y signiﬁcantly differs from X, then there exists an orientation– energy pair (yi, Ej) such that testing with these parameters the inspector will declare the item to pass the inspection with probability of at most b. If the host is cheating, at least sZ1 spoofs are presented for inspection. Suppose the host also provide ps spoof preloads (deﬁned as the complement of the spoof item) and (n ps) valid preloads (complement of the reference item). One of the following cases must apply: In the ﬁrst case, s 6¼ ps , then either at least one spoof is tested against a valid preload or at least one valid item is tested against a spoof preload. In either case, the cheating host evades detection with probability of at most b. In the second case, s ¼ ps, one of the items will be tested against 1 the wrong type of preload with probability 1 Cðn;s Þ where C(n,s) is the number of s combinations from the set of n items. Thus, the adversary evades detection with probability of at most 1=C ðn; sÞ þ bð1 1=Cðn; sÞÞ. In this case, the probability of evasion is always greater than b. Therefore, such a cheating strategy should always be preferred over s 6¼ ps . Since s n 1, the probability of evasion is maximized when s ¼ ps ¼ 1 or s ¼ ps ¼ n 1. Since s is under host control, we assume that the host will present n 1 spoofs and their associated spoof preloads. In this case, the probability to evade detection is bounded by 1=n þ bð1 1=nÞ. Therefore, for the protocol to be sound 1=n þ bð1 1=nÞo1 a, which can also be written as bo1 an=ðn 1Þ. In the worst case, n ¼ 2 4 (only one reference and one inspected item), bo1 2a, which also means that ao1/2. As noted above, the probability of evading detection can be arbitrarily reduced by re-testing. This would also detect cases where the host provides n preloads with counts between those of the spoof and the reference item. The closer b is to 1 an=ðn 1Þ, the more repetition will be needed to ensure a high soundness level, however. Efﬁcient and acceptable testing strategies based on the values of a, b and the soundness and completeness levels negotiated between parties will need to be developed. For n ¼ 10 and b ¼ 0.1, the probability of evasion would be equal to 0.19, which may be sufﬁcient to deter the host from using the strategies we described. Experimental realization. For this proof of principle, and to provide a physical basis to the GBG protocol, we inspected simple items in one orientation with one neutron beam energy ﬁxed at 14 MeV. We represented the reference warhead and the inspected items by combinations of 2-inch cubes of aluminium and steel. The cubes attenuate differently 14 MeV neutrons (44% and 27% for 2.54 cm of steel and aluminium, respectively) and can be arranged in different patterns. Figure 1c shows the experimental set-up, as well as the cube pattern used to represent the internal components of the reference item. The item also included a ‘black box’ aluminium hood to mimic the fact that inspected objects would likely be in containers, hiding their appearance. In a real implementation, to prevent disclosure of the preload, the detectors would also be covered with visually opaque material until the irradiation of the items was complete. In addition, a fast neutron counter monitors the source ﬂuence to assure that it is the agreed value to produce the total number of bubbles expected in the detectors in the absence of a test item. To prove the validity and usefulness of our technique, we tested both its ability to convey zero knowledge in cases where the item presented for inspection is identical to the reference item (valid case) and its robustness to detect cases in which the cube pattern had been altered, referred to as spoofs. For an interactive zero-knowledge proof applied to nuclear warhead veriﬁcation, the burden of proof22 falls on the host (the owner of the inspected items) to demonstrate that his claim is robust while his secrets are protected. As mentioned earlier, to prepare the inspection, the host must calibrate the measurement system and appropriately characterize the reference item. The calibration will be checked by inspectors through test irradiations in the absence of interposed items. In our demonstration, after characterizing the ﬂuence response function, f, of the detector system, we acquired 10 radiographs of the reference item and summed the results at each detector position. Each exposure lasted B4 min; without an interposed item, the corresponding ﬂuence would produce a total value of NmaxB1,200 bubbles. Once the reference item is fully characterized, the host can produce the necessary complementary radiographs, by irradiating the bare detectors to a ﬂuence equal to f 1 ðNmax Þ f 1 Nref ;i where f 1 is the inverse of the detector response function (counted bubbles as a function of counts from the reference fast neutron detector), and Nref,i the bubble count obtained from the radiograph of the reference item at every position i ¼ {1,...,7}. f is a nonlinear function of the number of bubbles present in a bubble detector. The nonlinearity is because of bubbles occulting one another in the photographic images used for bubble counting (see Fig. 1b). Individual exposure was kept at NB120 to avoid large nonlinear bubble-counting response. The analysis reported hereafter is based on the number of bubbles present in the detectors, which is somewhat larger than the NATURE COMMUNICATIONS \| 7:12890 \| DOI: 10.1038/ncomms12890 \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12890 Experimental results. We performed measurements for ﬁve different scenarios, one with an inspected item identical to the reference item and four scenarios with spoofs whose cube patterns differed from that of the reference item. During the inspections, the patterns were placed under the ‘black box’ hood and their radiographs recorded on a set of seven bubble detectors preloaded with the complement radiograph of the reference item. Each scenario was repeated 10 times and the bubble counts were summed to achieve an effective Nmax ¼ 1,191. The use of 10 measurements allowed us to use the variance of the mean to determine the error bars in the measurement experimentally. In general, this variance was found to be indistinguishable from the measurement as expected from Poisson statistics (see Methods). Figure 2a summarizes all our experimental results (the results uncorrected for occultation are shown in Methods). Figure 2b shows good agreement between Monte–Carlo neutron transport simulations of the experiment that calculated the neutron ﬂuence in the detectors using the code Monte Carlo N-particle23, and the experimental results. These data also support the validity of our nonlinear occultation model. In the ‘valid’ case, data are randomly distributed around Nmax and the measured mean variance from P7 ðN Nmax Þ2 ¼ 2646, is in good agreement with the Nmax, i¼1 7 P7 ð2Nmax þ Nref Þ0:5 2 Þ ¼ 2964. This is consistent predicted value of i¼1 ð 7 with an inspector gaining no knowledge about the transmission proﬁle of the reference item from this measurement. In the ﬁrst diversion scenario (Spoof1), the cubes making up the inspected item are swapped from left to right compared with the reference item. This change can be easily identiﬁed in the data. The next two diversion scenarios (Spoof2 and Spoof3) consisted of altering the reference item pattern by either adding (increased attenuation) or removing (decreased attenuation) a single steel cube. Again, both cases are clearly identiﬁed as different from the reference item with very high conﬁdence. Finally, in the last case (Spoof4), an aluminium cube replaced a steel cube on the right side (small a 2,000 Experimental results (bubbles) 1,800 1,600 1,400 1,200 1,000 800 600 Valid b Experimental results (bubbles) number counted, obtained by correcting the data for bubble occultation (See Methods). In practice, when preparing detectors for individual exposure, we used a linear approximation to f, which introduced a o2% error in the preloads. It is necessary to correct for the slight difference in gain between bubble detectors and for the effect of the location of any given detector. The number of bubbles is not exactly Nmax in each detector exposed to the same ﬂuence when no item is present. Using data from 10 different exposures of the detectors at a ﬂuence f 1(Nmax), we found that our detectors differed in efﬁciency with 3.7% 1 s dispersion. We corrected our results to take these variations into account. This calibration itself introduces a variance of Nmax in the ﬁnal experimental results. The total expected variance for our measurements consists of (1) this calibration variance, Nmax, (2) the variance of the radiograph of the reference item (or valid item), Nref, (3) the variance of the preload, Nmax Nref and (4) the variance of the subsequent exposure of the inspected item, Nitem. The calibration variance (1) and the reference radiograph variance (2) can be driven down as low as desired by the inspector and the host, respectively. The former will improve the sensitivity of the measurement, while the latter will prevent this variance from being a source of information leakage when a large number of valid items is inspected. In the limit where only variance sources (3) and (4) play a role, and an valid item is presented, therefore, Nitem ¼ Nref, then the expected distribution is Poisson with the mean value Nmax and variance Nmax. Spoof 1 Spoof 2 Spoof 3 Spoof 4 2,000 1,500 1,000 500 0 Predicted by occultation model Expected linear relationship Observed data Data corrected for occultation 0 500 1,000 1,500 MCNP simulations (bubbles) 2,000 Figure 2 \| Experimental evidence of a practical physical zero-knowledge proof. (a) Experimental results for the ﬁve inspection scenarios investigated (valid item and four spoofs denominated Spoof1 to Spoof4). The item patterns are represented for each scenario with white as empty, light grey as aluminium and dark grey as steel. The results are corrected for the nonlinear counting effect. Error bars represent one s.e.m. calculated at each detector position from the 10 measurements performed for each scenario and the calibration data (obtained from 10 measurements with no item). The light grey band around Nmax represents the expected error from a valid item (2Nmax þ Nref)0.5. The dark grey band corresponds to the minimum achievable value with Nmax bubbles, (Nmax)0.5. The test statistic P ðN Nmax Þ2 T ¼ 7i¼1 2N was compared with a w2 distribution with 7 degrees of max þ Nref freedom, giving the following results for each scenario: Valid, T ¼ 6.60, P ¼ 0.474; Spoof 1, T ¼ 453.35, Po10 16; Spoof 2, T ¼ 132.72, Po10 16; Spoof 3, T ¼ 87.80, P ¼ 3. 10 16; Spoof 4, T ¼ 17.88, P ¼ 0.013. (b) Observed and deduced true bubble count, from all scenarios at every position with interposed objects, versus corresponding Monte Carlo simulations obtained from the computational model of the experiment. The red dashed curve is obtained from our bubble occultation model using the calibration data with no interposed objects (see Methods). Error bars represent one s.e.m. calculated from measurements and calibration data. reduction of the attenuation). This case could be considered an acceptable spoof detection, with P ¼ 0.013, depending on the agreement between host and inspector. The data, however, suggest that a tighter criterion or repeated testing be used for this magnitude of P. NATURE COMMUNICATIONS \| 7:12890 \| DOI: 10.1038/ncomms12890 \| www.nature.com/naturecommunications 5 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12890 Methods Experimental set-up. The radiographs of the test items are performed using 14 MeV neutrons from a D–T neutron generator (Thermoﬁsher B320) with a yield of B108 neutrons per second. The generator is placed inside a borated polyethylene cylinder (1 m high 1.2 m in diameter) with a 5 cm high and 17 degreewide fan-shaped collimating slot. A single row of seven specially made superheated droplet neutron (or ‘bubble’) detectors are placed B110 cm away from the source, behind the test item. The items are mounted on a stand afﬁxed to a honeycomb aluminium optical table allowing precise and reproducible positioning (staging area in Fig. 1). A fast neutron counter (Eljen EJ-410 zinc sulﬁde scintillator) monitors the generator output during irradiation by detecting neutrons emerging from a channel at the bottom of the polyethylene cylinder. The complete apparatus is placed in a room shielded by borated concrete walls at the Princeton Plasma Physics Laboratory (Plainsboro, USA). The detectors developed for this application14,19 consist of glass tubes ﬁlled with emulsions of superheated octaﬂuorocyclobutane (C4F8, ﬂuorocarbon C-318) 100 mm diameter droplets homogeneously dispersed in a viscous aqueous gel matrix (4,000 per cm3). At 21 °C the detectors are only sensitive to neutrons above 1 MeV and insensitive to gamma rays. When a metastable droplet vapourizes, because of a sufﬁciently energetic neutron interaction, it expands into a stable bubble about six times larger in diameter. The detectors can be irradiated several times in series, recording the total ﬂuence to which they were exposed (pre-loadable property). If an isostatic pressure of 500 p.s.i. is applied to the aqueous gel for B10 min, the bubbles are recondensed into superheated droplets. All bubble detectors used in this study were manufactured from the same batch. Fourteen detectors were chosen out of twenty-one after being irradiated several times at every position for B6 min to compare their relative behaviour. The chosen detectors exhibited a 3.7% 1 s dispersion and were calibrated to allow determination of their absolute efﬁciency (of the order of 4 10 4 bubbles per crossing neutron in an active volume of 5.3 cm3). Data were acquired in two ways to study the effect of recompression on the detector response. One detector batch corresponding to 5,000, 10,000, up to 25,000, fast neutron counts were irradiated, measured, recompressed and then irradiated to the next higher level. A second batch was irradiated, counted and then further irradiated to provide data at 2,500, 7,500, and so on, counts. The recompression was shown to have no detectable effect on the measurements. Because the energy threshold of bubble nucleation is temperature-dependent, we monitored the temperature of the detectors throughout the study. The experimental hall was temperature-controlled and the detector temperature exhibited minor variations over all measurements (20.79±0.34 °C). After irradiation, photographs of the detectors were taken using a commercial instrument (BDR-III reader, Bubble Technology Industries (BTI)) at multiple angles. The bubbles were then counted with the associated BTI software with settings modiﬁed to accommodate our detector tubes. The results were crosschecked with visual counting using large printed copies of the images. The main challenge of these techniques stems from the correct segmentation of bubbles that overlap in the two-dimensional (2D) image. Bubble occultation model. Before starting any measurements, we carefully characterized the ﬂuence response of our detector system, bvis ¼ f(n), where bvis is the number of bubbles recorded by the reading system and n the counts from the fast neutron detector. A nonlinear behaviour emerged, because of the bubblecounting method used in the experiments. Bubbles are counted from a 2D (y,z) projection of the volume perpendicular to the x axis (direction of light) and parallel to the vertical z axis of the cylindrical detector. Thus, bubbles at the same z and y positions may occult one another. (Note that these data do not necessarily imply that BTI bubble detectors read with BTI equipment produce the level of nonlinearity we observed. These results apply only to our own detectors with tailored 6 1,600 Uncorrected results (bubbles) 1,400 1,200 1,000 800 600 400 Valid Spoof1 Spoof2 Spoof3 Spoof4 Scenarios Figure 3 \| Uncorrected experimental results. Uncorrected experimental results for the ﬁve inspection scenarios investigated and deﬁned in the Results section (valid item and four diversions denominated Spoof1 to Spoof4). Data points are presented with one s.e.m. error bars calculated at each detector position from the 10 measurements performed for each scenario and the calibration data (obtained from 10 measurements with no item). The grey band is ð2Nmax Þ0:5 centred on Nmax. 7 6 Log (variance bubbles) Discussion This experiment demonstrates the key aspects of the viability of non-electronic differential fast neutron radiography to perform zero-knowledge object comparison, establishing a physical basis for the nuclear warhead veriﬁcation approach proposed by GBG. It constitutes the demonstration of a physical zero-knowledge proof protocol for a practical high-security application that can compare physical properties of objects. We have shown that the combination of pre-loading and direct measurement on an honest object gives the desired null result, with a well-understood level of noise. We have identiﬁed areas where the noise can be reduced to improve the technique. We have also explored the capability of this technique at the level of Nmax ¼ 1,200 bubbles for detecting diversions. Further research, at higher bubble counts, with different neutron energies, and with special nuclear materials, will be needed to assess the capabilities of the system for the full range of relevant diversion scenarios. 5 4 3 2 Expected relationship Min 2 fit Bootstrapped data 1 0 0 1 2 3 4 Log (corrected bubbles) 5 6 7 Figure 4 \| Analysis of the superheated droplet detector variance. Plot of the logarithm of the variance versus the logarithm of the average number of true (corrected) bubbles. The data and one s.d. error bars were obtained by bootstrapping the corrected experimental results (1,000 samples per data point). settings to accommodate our speciﬁc material and geometric conditions.) To account for this nonlinear effect, we used a simple particle interaction model. For an observer placed far away from the detector and looking in the x direction, a new bubble will be visible only if its image does not cross an old bubble. Thus, we can br sd assume that for each new bubble, on average the observer only sees e V bubbles, where V is the counting volume, d is a characteristic width of the cylinder along the line of sight, s the effective occultation cross-section and br the real number of bubbles at the time a new bubble is created. Averaging over the cylinder indicates that d should be taken to be 4/p times the cylinder radius. We expect the number of real bubbles to be a linear function of the neutron count in the fast neutron detector because only a very small fraction of the droplets have vapourized (B0.6% for 4 min irradiation), and the fractional volume occupied by the vapourized droplets is very small (B0.25% volume displacement). Thus, we r assume that db dn ¼ e. This gives us the rate at which visible bubbles accumulate as a br sd function of neutron irradiation, dbdnvis ¼ ee V , and integrating this expression, using ensd V br ¼ en, we obtain bvis ¼ sd ð1 e V Þ. This model has efﬁciency e at low ﬂuence V . and saturates, when br goes to inﬁnity, at bvis ¼ sd NATURE COMMUNICATIONS \| 7:12890 \| DOI: 10.1038/ncomms12890 \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12890 Calibration data, obtained at increasing exposures without interposed items, were ﬁtted to this model to obtain the true number of bubbles br from bvis. (In practice, this calibration procedure also compensates for the small effects because of droplet vapourization and occupied volume.) Data obtained later from the detector preloads conﬁrmed the good ﬁt and reproducibility of this effect. We ﬁnd that the effective cross-section s is equivalent to a disk with a radius of the order of twice the radius of a single bubble (B300 mm), effectively corresponding to two bubbles touching one another. Uncorrected inspection results. In the main section, the inspection results were corrected for the nonlinear effect introduced by the reading technique. Figure 3 presents the uncorrected data. These results were directly obtained from the readout, and calibrated to account for spatial and detector gain differences as explained in the main text. Detector variance. The zero-knowledge property of our protocol depends on its ability to carry information neither in the signal nor in the noise of the measurements. Since we are measuring radiation, the sum of the detector counts (preload and radiograph of inspected item) should follow Poisson statistics. Thus, at each position we expect the data obtained for all detectors to have a variance pﬃﬃﬃﬃ equal to their bubble count. To verify that the noise of our detector varies as N , we generated synthetic data from our experimental results using the bootstrapping method24. The bootstrapping method consists of estimating population parameters by randomly sampling with replacement from an approximating distribution (here the empirical population obtained from our experimental data). We used 10 measurements for each inspection scenario (there are 5) as well as 10 radiographs of the reference item and 10 preloads (complement radiographs) as starting data sets. A measurement corresponds to 7 data points for each position. We generated 1,000 synthetic measurements for each case to obtain the bootstrapped mean and variance with their respective error pﬃﬃﬃﬃ bars. The results presented in Fig. 4 shows good agreement with the expected N noise from Poisson statistics. Note that here we are using br deduced from the nonlinear model. The good agreement indicates that the occultation process and its correction do not introduce signiﬁcant additional noise at the level of nonlinearity explored. Data availability. Source data for ﬁgures are available from the corresponding author upon request. References 1. Comley, C. et al. Conﬁdence, Security & Veriﬁcation: the Challenge of Global Nuclear Weapons Arms Control (Atomic Weapons Establishment, 2000). 2. National Academy of Sciences CISAC, Monitoring Nuclear Weapons and Nuclear-Explosive Materials: an Assessment of Methods and Capabilities (National Academies Press, 2005). 3. Stone, R. Not-seeing is believing. Science 344, 1436–1437 (2014). 4. Spears, D. (ed.) Technology R&D for Arms Control (US Department of Energy, Ofﬁce of Nonproliferation Research and Engineering, 2001). 5. Evans, N. Software Development and Authentication for Arms Control Information Barriers. In FM 2015: formal Methods 581–584 (Springer International Publishing, 2015). 6. Glaser, A., Barak, B. & Goldston, R. A zero-knowledge protocol for nuclear warhead veriﬁcation. Nature 510, 497–502 (2014). 7. Philippe, S., Barak, B. & Glaser, A. Designing protocols for nuclear warhead veriﬁcation. In Proc. 56th Annual INMM Meeting (Indian Wells, CA, USA, 2015). 8. Fisch, B., Freund, D. & Naor, M. Secure Physical Computation using Disposable Circuits. In Theory of Cryptography 182–198 (Springer, 2015). 9. Fisch, B., Freund, D. & Naor, M. Physical Zero-Knowledge Proofs of Physical Properties. In Advances in Cryptology—CRYPTO 2014 313–336 (Springer, 2014). 10. Goldwasser, S., Micali, S. & Rackoff, C. The knowledge complexity of interactive proof-systems. SIAM J. Comput. 18, 186–208 (1989). 11. Goldreich, O. Foundations of Cryptography: Basic Tools (Cambridge University Press, 2000). 12. Marleau, P. et al. Report on a zero-knowledge protocol tabletop exercise (SAND2015-5075, Sandia National Laboratory, 2015). 13. Yan, J. & Glaser, A. Nuclear warhead veriﬁcation: a review of attribute and template systems. Sci. Global Security 23, 157–170 (2015). 14. d’Errico, F. Radiation dosimetry and spectrometry with superheated emulsions. Nucl. Instrum. Methods Phys. Res. B 184, 229–254 (2001). 15. Knoll, G. F. Radiation Detection and Measurement (John Wiley & Sons, 2010). 16. Lyons, L. Statistics for Nuclear and Particle Physicists (Cambridge University Press, 1986). 17. James, F. Statistical Methods in Experimental Physics 2nd edn (World Scientiﬁc, 2006). 18. Lehmann, E. L. & Romano, J. P. Testing Statistical Hypotheses, 3rd edn (Springer, New York, 2005). 19. Goldston, R. J. et al. Zero-knowledge warhead veriﬁcation: system requirements and detector technology. In Proc. 55th Annual INMM Meeting (Atlanta, GA, USA, 2014). 20. Chen, G & Lanza, R. C. Fast neutron resonance radiography for elemental imaging: theory and applications. IEEE Trans. Nucl. Sci. 49, 1919–1924 ð2002Þ: 21. Yan, J. & Glaser, A. Two-color neutron detection for zero-knowledge nuclear warhead veriﬁcation. In Proc. 56th Annual INMM Meeting (Indian Wells, CA, USA, 2015). 22. Kaplow, L. Burden of proof. Yale L.J 121, 738–859 (2012). 23. A general Monte Carlo N-particle (MCNP) transport code, version 6.1.0. Los Alamos National Laboratory, mcnp.lanl.gov. 24. Efron, B. & Tibshirani, R. J. An Introduction to the Bootstrap (CRC press, 1994). Acknowledgements Princeton University, Yale University and the Princeton Plasma Physics Laboratory received support for this research through DOE/NNSA’s Consortium for Veriﬁcation Technology, DE-NA 0002534. Financial support was also provided by the John D. and Catherine T. MacArthur Foundation and the Carnegie Corporation of New York. We thank B. Barak for his advice on the protocol construction and assumptions, A. Carpe and the Health Physics team of the Princeton Plasma Physics Laboratory for their aid during the conduct of the experiments, M. Gattas-Sethi (Yale University) for manufacturing the detectors, Z. Mian, M. Kütt and three reviewers for providing helpful comments on the manuscript. All simulations were run on Princeton University’s High Performance Cluster. Author contributions S.P. designed and built the apparatus, simulated and conducted the laboratory experiments. R.J.G. and A.G. mentored S.P. and contributed, along with S.P., to the elaboration of the protocol and the analysis of the data. F.d’E. developed the detectors and participated in their characterization. All contributed to the manuscript. Additional information Competing ﬁnancial interests: The authors declare no competing ﬁnancial interests. Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ How to cite this article: Philippe, S. et al. A physical zero-knowledge objectcomparison system for nuclear warhead veriﬁcation. Nat. Commun. 7:12890 doi: 10.1038/ncomms12890 (2016). 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/ r The Author(s) 2016 NATURE COMMUNICATIONS \| 7:12890 \| DOI: 10.1038/ncomms12890 \| www.nature.com/naturecommunications 7
https://openalex.org/W3166398090	https://gfzpublic.gfz-potsdam.de/pubman/item/item_5007544_2/component/file_5007549/5007544.pdf	English	null	Constructive and Destructive Processes During the 2018–2019 Eruption Episode at Shiveluch Volcano, Kamchatka, Studied From Satellite and Aerial Data	Frontiers in earth science	2,021	cc-by	21,637	Edited by: Stephen Self, Edited by: Stephen Self, University of California, Berkeley, United States Dome-building volcanoes often develop by intrusion and extrusion, recurrent destabilization and sector collapses, and renewed volcanic growth inside the collapse embayment. However, details of the structural architecture afﬁliated with renewed volcanic activity and the inﬂuences of regional structures remain poorly understood. Here, we analyze the recent activity of Shiveluch volcano, Kamchatka Peninsula, characterized by repeated episodes of lava dome growth and destruction due to large explosions and gravity-driven collapses. We collect and process a multisensor dataset comprising high- resolution optical (aerial and tri-stereo Pleiades satellite), radar (TerraSAR-X and TanDEM- X satellites), and thermal (aerial and MODIS, Sentinel-2, and Landsat 8 satellites) data. We investigate the evolution of the 2018–2019 eruption episode and evaluate the morphological and structural changes that led to the August 29, 2019 explosive eruption and partial dome collapse. Our results show that a new massive lava lobe gradually extruded onto the SW ﬂank of the dome, concurrent with magmatic intrusion into the eastern dome sector, adding 0.15 km3 to the lava dome complex. As the amphitheater inﬁlled, new eruption craters emerged along a SW-NE alignment close to the amphitheater rim. Then, the large August 29, 2019 explosive eruption occurred, followed by partial dome collapse, which was initially directed away from this SW-NE trend. The eruption and collapse removed 0.11 km3 of the dome ediﬁce and led to the formation of a new central SW-NE-elongated crater with dimensions of 430 m × 490 m, a collapse scar at the eastern part of the dome, and pyroclastic density currents that traveled ∼12 km downslope. This work sheds light on the structural architecture dominated by a SW-NE lineament and the complex interplay of volcano constructive and destructive processes. We develop a conceptual model emphasizing the relevance of structural trends, namely, 1) a SW- NE-oriented (possibly regional) structure and 2) the inﬁlled amphitheater and its decollement surface, both of which are vital for understanding the directions of growth Reviewed by: David Matthew Hyman, Cooperative Institute for Meteorological Satellite Studies (CIMSS), United States Matt Pritchard, Cornell University, United States Nick Varley, University of Colima, Mexico Reviewed by: David Matthew Hyman, Cooperative Institute for Meteorological Satellite Studies (CIMSS), United States Matt Pritchard, Cornell University, United States Nick Varley, University of Colima, Mexico *Correspondence: Alina V. Edited by: Stephen Self, Shevchenko alinash@gfz-potsdam.de Specialty section: This article was submitted to Volcanology, a section of the journal Frontiers in Earth Science Received: 13 March 2021 Accepted: 25 May 2021 Published: 08 June 2021 Specialty section: This article was submitted to Volcanology, a section of the journal Frontiers in Earth Science Received: 13 March 2021 Accepted: 25 May 2021 Published: 08 June 2021 ORIGINAL RESEARCH published: 08 June 2021 doi: 10.3389/feart.2021.680051 1German Research Centre for Geosciences GFZ, Potsdam, Germany, 2Institute of Volcanology and Seismology FEB RAS, Petropavlovsk-Kamchatsky, Russia, 3Institute of Photogrammetry and GeoInformation, Leibniz University Hannover, Hannover, Germany, 4Department of Earth Sciences, University of Turin, Turin, Italy INTRODUCTION In contrast, if the lavas have a lower viscosity, they do not lead to the formation of steep domes but rather tend to ﬂow over long distances, thereby forming thick and massive coulees, as those at Merapi, Indonesia (Walter et al., 2013), and Kizimen, Russia (Dvigalo et al., 2013). Lava domes can grow endogenously by the intrusion of new material into their bodies or exogenously by the extrusion of separate lava lobes onto their surfaces through cracks or vents (Williams and McBirney, 1979; Hale et al., 2009). The accumulation of coherent volcaniclastic deposits together with unconsolidated ejecta results in the growth of dome-building volcanoes and the formation of composite volcanoes (Karátson et al., 2010), where the transition from a lava dome to a stratovolcano can be observed (Shevchenko et al., 2020). On the other hand, volcanoes are destroyed by explosive eruptions that produce craters and excavate part of the ediﬁce. For example, a large explosion at Bezymianny in 2006 produced a summit crater in the center of the dome (Girina, 2013; Shevchenko et al., 2020). Additionally, subsidence in the summit areas of active volcanoes results in the formation of collapse calderas (Geshi et al., 2002) or in the deepening of already existing depressions (Walker, 1984), and these features may be obscured in the future by growing lava domes (Tatsumi et al., 2018). Furthermore, sector collapses can destroy large parts of a volcanic ediﬁce (Watt, 2019). For instance, the catastrophic sector collapses that occurred at Bezymianny in 1956 (Gorshkov, 1959; Belousov and Belousova, 1998), at Mount St. Helens in 1980 (Lipman and Mullineaux, 1981) and at Anak Krakatau, Indonesia, in 2018 (Walter et al., 2019) resulted in the formation of large and wide-open collapse amphitheaters that span 0.5–2 km in width; in contrast, smaller- Remote sensing is an efﬁcient and safe method for monitoring the evolution of a volcano; effective observation techniques include thermal remote sensing (Coppola et al., 2020), synthetic aperture radar (SAR) analysis from spaceborne or airborne platforms (Schaefer et al., 2016), and multisensor approaches (Corradini et al., 2016). Freely available satellite data and smart data science allow the monitoring of deformation ﬁelds, thermal anomalies, and gas emissions in near real time (e.g., Valade et al., 2019; Massimetti et al., 2020). In this study, we use a multisensor remote sensing approach to investigate Shiveluch, one of the most active volcanoes in Kamchatka (Figures 1A,B). INTRODUCTION scale partial dome collapses, e.g., those that occurred at Soufriere Hills, Montserrat, in 1997 (Voight et al., 2002) and 2010 (Stinton et al., 2014) and at Merapi, Indonesia, in 2006 (Ratdomopurbo et al., 2013), produce narrower collapse scars and are more common. Volcanic ediﬁces develop as a result of interactions between volcano growth and mass wasting - constructive and destructive processes, respectively. A volcano is constructed as a result of the deposition of lava ﬂows, lava domes, cinder- or stratocones, shallow magma intrusions, among others. Volcanoes are destroyed again by the formation of craters, collapse scars, landslides, and calderas. Important volcano architectures comprising detachment surfaces, fault zones, and lithological contrasts evolve in association with these activities, and these structures may control the future evolution of a volcano and its magma pathways (Gudmundsson, 2020). Such constructive and destructive processes may act simultaneously or in alternating ways. Both types of processes are largely affected by existing and evolving tectonic structures, both of which are related to the regional tectonics and local volcano-tectonics (Gudmundsson, 2020). Sector collapses evolving along decollements yield open amphitheaters that are inﬁlled by renewed volcanism until a repeat collapse occurs (Walter and Schmincke, 2002), and the original sliding plane is preserved as a mechanically weak zone that affects future activities. Furthermore, tectonic alignments may control the direction of growth (such as the preferred geometry of dike intrusions) and the direction of collapse (directed away from fault zones), potentially allowing the location of such a collapse to be inferred indirectly (Merle et al., 2001; Acocella, 2005). While inﬁlled sliding planes and former decollements may be represented by inclined to horizontal lithologies, exposed tectonic faults may be nearly vertical. Nevertheless, since constructive and destructive processes may interfere with regional and local tectonic processes, details regarding their structural alignments are commonly difﬁcult to decipher. The constructive and destructive dynamics of volcanoes have been analyzed for various volcano types and are particularly dramatic at dome-building volcanoes. Some volcanoes are constructed through the accumulation of viscous lava, resulting in the formation of lava domes; examples include Mount St. Helens, United States (Swanson and Holcomb, 1990), Bezymianny, Russia (Gorshkov and Bogoyavlenskaya, 1965), Unzen, Japan (Nakada et al., 1999), and Popocatépetl, Mexico (Gómez-Vazquez et al., 2016). Citation: Shevchenko AV, Dvigalo VN, Zorn EU, Vassileva MS, Massimetti F, Walter TR, Svirid IY, Chirkov SA, Ozerov AY, Tsvetkov VA and Borisov IA (2021) Constructive and Destructive Processes During the 2018–2019 Eruption Episode at Shiveluch Volcano, Kamchatka, Studied From Satellite and Aerial Data. Front. Earth Sci. 9:680051. doi: 10.3389/feart.2021.680051 June 2021 \| Volume 9 \| Article 680051 Frontiers in Earth Science \| www.frontiersin.org 1 The 2018–2019 Shiveluch Volcano Eruption Shevchenko et al. and collapse and for assessing the potential hazards at both Shiveluch and dome-building volcanoes elsewhere. and collapse and for assessing the potential hazards at both Shiveluch and dome-building volcanoes elsewhere. INTRODUCTION FIGURE 1 \| Shiveluch volcano and its deposit ﬁeld (A) Optical image from the Dove satellite (Planet Labs, Inc.https://api.planet.com) acquired on September 13, 2019 showing the location of the active lava dome encircled by the 1964 collapse amphitheater (red dotted line), from which all subsequent eruptive deposits (labeled 1964 2005 2010 and 2019) spread outward toward the south The scattered deposits from the 2019 eruption are visible and marked by black arrows The upper right FIGURE 1 \| Shiveluch volcano and its deposit ﬁeld (A) Optical image from the Dove satellite (Planet Labs, Inc.https://api.planet.com) acquired on September 13, 2019 showing the location of the active lava dome encircled by the 1964 collapse amphitheater (red dotted line), from which all subsequent eruptive deposits (labeled 1964, 2005, 2010, and 2019) spread outward toward the south. The scattered deposits from the 2019 eruption are visible and marked by black arrows. The upper right inset map shows the location of the volcano (red square) on the Kamchatka Peninsula (B) Oblique helicopter ﬂight view to the north showing the Shiveluch lava dome on August 22, 2019. The 1964 collapse amphitheater is delineated by a red dotted line. The map was generated using ArcGIS 10.2.1 in the WGS84 coordinate system (C) Data used in the investigation. The high-resolution optical data (diamonds) acquired before and during the eruption were used for topographic analysis and compared with the TSX satellite radar data (green triangles). The thermal emissions were analyzed using the Sentinel 2 and Landsat 8 satellites (red bars). Blue lines with circles summarize the 2018–2019 VRP time series (in watts) provided by the MIROVA system (www.mirovaweb.it). Black stars and dotted lines mark the occurrence of the two main explosions on December 30, 2018 and August 29, 2019. the remnants of Old Shiveluch (Stariy Shiveluch), a stratovolcano visible to the NE that was destroyed by a giant collapse at 30 ka (Ponomareva et al., 1998), and 2) Young Shiveluch (Molodoy Shiveluch), which is visible to the SW and is currently active (Dvigalo, 1988; Dirksen et al., 2006; Shevchenko et al., 2015). major destructive event was soon followed by the new construction of an ediﬁce located atop the former decollement plane and gradual inﬁlling of the amphitheater. Fifteen years following this event, in 1979, the ﬁrst photogrammetric aerial survey of Shiveluch’s collapse amphitheater was performed by the Institute of Volcanology and Seismology (IVS). INTRODUCTION Shiveluch, which can be characterized by long-term interactions between constructive (dome growth) and destructive (explosions, dome collapses) processes, is located at the junction of the Aleutian and Kuril-Kamchatka volcanic arcs in the northern part of the Central Kamchatka depression. The volcano is situated to the north of the Klyuchevskaya volcanic group, which comprises the active volcanoes Tolbachik, Bezymianny, and Klyuchevskoy (Koulakov et al., 2020). The details of the structural trend at Shiveluch remain unclear, but the volcano appears to be associated with a regional SW–NE- aligned tectonic architecture that is traceable by an alignment of monogenic cones in this direction (Koulakov et al., 2020). The age of Shiveluch is estimated to reach 60–70 ka (Melekestsev et al., 2003). Its ediﬁce consists of two parts: 1) Frontiers in Earth Science \| www.frontiersin.org June 2021 \| Volume 9 \| Article 680051 2 The 2018–2019 Shiveluch Volcano Eruption Shevchenko et al. FIGURE 1 \| Shiveluch volcano and its deposit ﬁeld (A) Optical image from the Dove satellite (Planet Labs, Inc.https://api.planet.com) acquired on September 13, 2019 showing the location of the active lava dome encircled by the 1964 collapse amphitheater (red dotted line), from which all subsequent eruptive deposits (labeled 1964, 2005, 2010, and 2019) spread outward toward the south. The scattered deposits from the 2019 eruption are visible and marked by black arrows. The upper right inset map shows the location of the volcano (red square) on the Kamchatka Peninsula (B) Oblique helicopter ﬂight view to the north showing the Shiveluch lava dome on August 22, 2019. The 1964 collapse amphitheater is delineated by a red dotted line. The map was generated using ArcGIS 10.2.1 in the WGS84 coordinate system (C) Data used in the investigation. The high-resolution optical data (diamonds) acquired before and during the eruption were used for topographic analysis and compared with the TSX satellite radar data (green triangles). The thermal emissions were analyzed using the Sentinel 2 and Landsat 8 satellites (red bars). Blue lines with circles summarize the 2018–2019 VRP time series (in watts) provided by the MIROVA system (www.mirovaweb.it). Black stars and dotted lines mark the occurrence of the two main explosions on December 30, 2018 and August 29, 2019. Data and Methods This study of the 2018–2019 eruptive activity at Shiveluch is based on remote sensing data acquired by optical sensors complemented by SAR and thermal sensors. While optical sensors provide a high spatial resolution and permit the three- dimensional mapping, SAR sensors possess the high temporal resolution required to detect and measure the processes involved. Accordingly, the methods section describes the techniques used to process and analyze the optical, radar, and thermal sensor data. The limitations of these methods are discussed in the Supplementary Material Limitations section. g g During the 2018–2019 period, the Kamchatka Volcanic Eruption Response Team (KVERT) (http://www.kscnet.ru/ivs/ kvert/index?langen; Gordeev and Girina, 2014) and the online Middle Infrared Observation of Volcanic Activity (MIROVA) system (https://www.mirovaweb.it; Coppola et al., 2016) suggested pronounced two-stage activity consisting of a constructive-destructive episode at Shiveluch volcano. Additionally, the regional seismic network operated by the Kamchatka Branch of the Uniﬁed Geophysical Service (http:// www.emsd.ru; Chebrov et al., 2013) detected evidence reﬂecting the culminating activity of this episode. The MIROVA system showed that the 2018 thermal activity was weak and discontinuous and consisted of low-power anomalies, indicating that hot exposed magmatic material was absent and that these anomalies were related just to moderate gas and steam. Changes in the thermal emissions of the volcano were observed in late December 2018, when an explosive event that marked the beginning of a new eruptive phase was identiﬁed by KVERT, which issued a Volcano Observatory Notice for Aviation (VONA) on December 30, 2018 (VONA note 20201229/ 0050Z). Thereafter, the thermal ﬂux continued to be high but gradually declined throughout 2019, suggesting ongoing shallow activity and the production of hot ejecta over the dome ediﬁce. Another large explosive eruption occurred on August 29, 2019 (VONA note 20190829/0339Z), marking another peak in thermal emissions on September 02, 2019 that then declined by the end of 2019. This trend in volcanic radiative power (VRP) was monitored by the online MIROVA system, which revealed abrupt changes after the explosion and suggested an important role of explosive events in the shallow volcanic behavior and the production of diverse volcanic products and styles. According to data from the Kamchatka Branch of the Uniﬁed Geophysical INTRODUCTION A comparison of the 1979 data with the data obtained during the second survey in 1980 revealed precursors of further activity: surface deformation of the amphitheater ﬂoor, fractures, and the disappearance of thermal lakes that were previously observed (Kirsanova, 1970; Dvigalo, 1988; Dvigalo, 2000). The evolving activity was further monitored and in August 1980, the growth of a new lava dome On 12 November 1964, a catastrophic event occurred at Young Shiveluch (Belousov, 1995) in association with 1) the collapse of a complex of lava domes, 2) the formation of a collapse amphitheater open to the SSW with dimensions of 1.8 km × 3.5 km, 3) the emplacement of a debris-avalanche covering an area of 104 km2 (Dvigalo and Shevchenko, 2015) at the southern foot of the volcano, and 4) a Plinian eruption caused by decompression due to the collapse (Belousov, 1995). This June 2021 \| Volume 9 \| Article 680051 Frontiers in Earth Science \| www.frontiersin.org 3 The 2018–2019 Shiveluch Volcano Eruption Shevchenko et al. was observed within the amphitheater; this growth continued over several intervals to the present (2021). During the ﬁrst two growth periods in 1980–1981 and 1993–1995, the dome grew endogenously (Dvigalo, 1988; Melekestsev et al., 2004). However, the activity shifted in 2001 (Fedotov et al., 2001) with the manifestation of exogenous growth as lava lobes began to extrude and crease structures formed (Shevchenko et al., 2015). These constructive episodes continued, but the dome ediﬁce became unstable due to the growing load, uneven distribution of the extrusive bodies, episodic endogenous growth, and increased explosive activity (Shevchenko et al., 2015). As a consequence, the construction of the Shiveluch dome was repeatedly interrupted by destructive events (Shevchenko and Svirid, 2014): the two largest events occurred in 2005 and 2010, which left collapse scars on the SSW and SSE ﬂanks of its ediﬁce, respectively, and the pyroclastic density currents (PDCs) from these collapses extended distances of ∼19 and ∼16 km, respectively (Dvigalo et al., 2011; Ramsey et al., 2012). The eruption columns from the explosive events reached a height of 15 km (Ozerov et al., 2020). To the best of our knowledge, the Shiveluch lava dome has not been visited for direct study since 2008 because it is hazardous to access and exhibits unpredictable activity. Therefore, the remote sensing techniques employed in this work are relevant for studying the ongoing evolution of the Shiveluch dome in detail. INTRODUCTION was observed within the amphitheater; this growth continued over several intervals to the present (2021). During the ﬁrst two growth periods in 1980–1981 and 1993–1995, the dome grew endogenously (Dvigalo, 1988; Melekestsev et al., 2004). However, the activity shifted in 2001 (Fedotov et al., 2001) with the manifestation of exogenous growth as lava lobes began to extrude and crease structures formed (Shevchenko et al., 2015). These constructive episodes continued, but the dome ediﬁce became unstable due to the growing load, uneven distribution of the extrusive bodies, episodic endogenous growth, and increased explosive activity (Shevchenko et al., 2015). As a consequence, the construction of the Shiveluch dome was repeatedly interrupted by destructive events (Shevchenko and Svirid, 2014): the two largest events occurred in 2005 and 2010, which left collapse scars on the SSW and SSE ﬂanks of its ediﬁce, respectively, and the pyroclastic density currents (PDCs) from these collapses extended distances of ∼19 and ∼16 km, respectively (Dvigalo et al., 2011; Ramsey et al., 2012). The eruption columns from the explosive events reached a height of 15 km (Ozerov et al., 2020). To the best of our knowledge, the Shiveluch lava dome has not been visited for direct study since 2008 because it is hazardous to access and exhibits unpredictable activity. Therefore, the remote sensing techniques employed in this work are relevant for studying the ongoing evolution of the Shiveluch dome in detail. Service, the seismicity exceeded the background level on August 29, 2019, with 53 events occurring across the ediﬁce. Continuous spasmodic volcanic tremors up to 0.5 μm/s were detected, and from 00:57 to 06:00 (GMT), a series of shallow events were recorded, possibly accompanying a powerful explosive eruption with ash emissions reaching 12 km above sea level (a.s.l.). In this study, we utilize a set of multisensor data (Figure 1C; Shevchenko et al., 2021) to explore these events in the frame of the complex constructive-destructive behavior that Shiveluch typically exhibits with regard to its volcanic activity. Moreover, we discuss the inﬂuence of structural control, possibly manifesting as a tectonic SW–NE-oriented structure together with a local decollement. To better understand the relationship between these constructive and destructive events and to shed some light on the structural architecture of the volcano, we investigated the 2018–2019 activity and revealed an interesting and complex relationship among the different intrusive, extrusive, explosive, and thermal characteristics of the dome. Photogrammetry and Digital Elevation Model Analysis TABLE 1 \| Main geometric parameters summarizing the initial construction and later destruction of the Shiveluch dome. The volumes include the talus and deposits in the western (2005) and eastern (2010) collapse scars. The volume changes and rates were calculated between two consecutive survey dates. aResults from Shevchenko et al., 2015. Heights are given in the WGS84 datum; in the previous study (Shevchenko et al., 2015), heights were given in the Baltic system of heights. The relative heights and dome volumes were measured from the 1979 DEM of the amphitheater ﬂoor prior to the growth of the dome (Dvigalo, 1988; Shevchenko et al., 2015). while a rational polynomial coefﬁcient block adjustment, which is a transformation from pixel information to latitude, longitude, and height, was automatically employed for the interior and exterior orientations. After constraining the image orientation, we obtained a photogrammetric model with a total root mean square error (RMSE) of 0.2 m. By using the Enhanced Automatic Terrain Extraction (eATE) module with the normalized cross- correlation algorithm implemented in Erdas Imagine, we were able to extract a 2-m resolution point cloud (PC) referenced to the World Geodetic System 1984 (WGS84) coordinate system (UTM zone 57). This PC was ﬁltered with the CloudCompare v2.9.1 noise ﬁlter and then manually cleaned with the CloudCompare segmentation tool. As strong volcanic steam emissions caused a large gap in the PC in the NE part of the dome, we used a 5-m resolution DEM constructed from TanDEM-X data (see SAR Data and Monitoring the Material Redistribution (d)) to ﬁll the gap and obtain the missing topography. emissions and by atmospheric clouds were ﬁlled in by collecting manual points using the anaglyph stereo mode of Photomod 5, which was performed by placing a ﬂoating mark on the visible surface and storing the XYZ coordinates of each point similar to (Schilling et al., 2008; Shevchenko et al., 2020). g The aerial PCs had the same spatial scale as the Pleiades PC but were geopositionally shifted due to the different coordinate systems. Hence, to compare the PCs, we aligned the aerial PCs to the Pleiades PC with several points along the rim of the amphitheater using the CloudCompare alignment tool. The RMSEs of the alignment vary from 2.3 to 3.1 m (Supplementary Table S1). Thus, we obtained four stacked PCs in WGS84 UTM57 and were able to calculate the differences between them. Photogrammetry and Digital Elevation Model Analysis We outlined the areas of the lava dome, including the talus, separately for each date and calculated the volumes within these speciﬁc areas between two consecutive PCs. The total volumes of the dome were calculated relative to the 1979 DEM with the topography of the amphitheater ﬂoor prior to the dome growth (Dvigalo, 1988; Shevchenko et al., 2015). The time- averaged growth rates were obtained by dividing each volume difference by the time interval between the two corresponding consecutive acquisitions. Helicopter surveys allowed us to acquire nadir and oblique aerial images during overﬂights on July 12, 2012 with a conventional Canon EOS 20D digital camera (focal length: 14.183 mm, resolution: 3,504 px × 2,336 px) and on August 22, 2019 and October 22, 2019 with a PhaseOne IXA 160 digital aerial camera (focal length: 28 mm, resolution: 8,984 px × 6,732 px). The average ﬂying height was 4,000 m a.s.l. for the nadir survey and 3,200 m a.s.l. for the oblique survey. We processed the images in Agisoft Metashape 1.5.2. To ascertain the interior orientation, we set the camera parameters (focal length and sensor size), and the relative orientation was performed automatically by aligning the images and calculating tie points. To perform the exterior orientation and assign ground control points (GCPs), we used coordinates taken from stable, prominent topographic peaks identiﬁed in the 1979 photogrammetric model (Dvigalo, 1988; Dvigalo, 2000), which is referenced to the USSR State Geodetic Network coordinate system (Chumachenko, 1966); the same technique was employed to georeference the previous photogrammetric dataset of Shiveluch volcano (Shevchenko et al., 2015). The total RMSEs of the aerial models orientation vary from 1.5 to 2 m (Supplementary Table S1). As a result of processing, we obtained three aerial PCs with an average resolution of 2 m; these PCs were then ﬁltered and cleaned in the same way described above. The gaps caused by volcanic steam To estimate the uncertainties in the calculated volumes, we distributed the RMSE for each acquisition date (Supplementary Table S1) over the corresponding area of the dome (Table 1). For the volume derived from the Pleiades DEM, we used the RMSE of the photogrammetric model orientation; for the volumes derived from the aerial DEMs, we used the total RMSEs, which includes the orientation errors and the errors of alignment to the Pleiades PC. Photogrammetry and Digital Elevation Model Analysis The maximum volume error is 12 × 106 m3 on August 22, 2019, which is 1.6% of the total dome volume (Table 1). The uncertainties in the growth rates were calculated by dividing the volume errors by the time interval between two neighboring dates. The maximum growth rate error amounts to 31,000 m3/ day (0.36 m3/s) or 8.5% for the August 22, 2019 data (Table 1; Supplementary Table S1). Photogrammetry and Digital Elevation Model Analysis An initial overview was obtained by utilizing satellite imagery acquired by the Dove satellites, the largest satellite constellation dedicated to imaging the Earth, and distributed by Planet Labs, Inc. (https://api.planet.com). We downloaded the available Planet data and imported the 3-m resolution imagery as background information in a geoinformatics framework using ArcGIS 10.2.1 to aid the interpretation of our higher-resolution photogrammetric dataset. The photogrammetric dataset was acquired by processing high-resolution aerial and satellite optical imagery. Photogrammetry was conducted to extract the pre-eruption topography for July 18, 2018 and to monitor the co-eruption topography on August 22, 2019 and October 22, 2019. We also reprocessed the July 12, 2012 data described in (Shevchenko et al., 2015) with new techniques to achieve a better resolution and used the data in this work to continue the topographic chronology recorded for 2001–2012 (Shevchenko et al., 2015). The optical satellite data were tri-stereo panchromatic 1-m resolution imagery acquired on July 18, 2018 with the Pleiades satellite PHR1B sensor. We processed the data in Erdas Imagine 2015 v15.1 similar to (Bagnardi et al., 2016; Shevchenko et al., 2020). To determine the relative orientation of the images, 37 tie points were calculated automatically with a manual correction, Frontiers in Earth Science \| www.frontiersin.org June 2021 \| Volume 9 \| Article 680051 4 The 2018–2019 Shiveluch Volcano Eruption Shevchenko et al. TABLE 1 \| Main geometric parameters summarizing the initial construction and later destruction of the Shiveluch dome. Date Dome elevation (m) Dome relative height (m) Dome area (m2) Volume change (km3) Dome volume (km3) Dome volume error (%) Growth rate (m3/day)/ (m3/sec) Growth rate error (%) July 12, 2012 2,670 526a 2,602,585 0.09a 0.63a 1.3 150,000a/1.74 9.2 July 18, 2018 2,757 613 3,265,734 0.016 0.65 0.1 7,200/0.083 4.1 August 22, 2019 2,755 611 3,457,470 0.146 0.79 1.6 365,000/4.22 8.5 October 22, 2019 2,729 585 3,551,788 -0.11 0.68 1.6 – – The volumes include the talus and deposits in the western (2005) and eastern (2010) collapse scars. The volume changes and rates were calculated between two consecutive survey dates. aResults from Shevchenko et al., 2015. Heights are given in the WGS84 datum; in the previous study (Shevchenko et al., 2015), heights were given in the Baltic system of heights. The relative heights and dome volumes were measured from the 1979 DEM of the amphitheater ﬂoor prior to the growth of the dome (Dvigalo, 1988; Shevchenko et al., 2015). SAR Data and Monitoring the Material Redistribution The image cross- correlation and pixel offset methods have been used to solve other geodetic problems for many years; for instance, these techniques have been applied to optical imagery and SAR data of glaciers (Evans, 2000), to analyze the displacements of landslides and man-made activities (Sun and Muller, 2016), and to monitor seismogenic and volcanic terrains (Funning et al., 2005; Casu et al., 2011). The pixel offset method has also been used to measure the subpixel deformations of dome-building volcanoes at high resolution (Salzer et al., 2016; Zorn et al., 2020). Unlike optical data, SAR pixel offset vectors are measured in the LOS (range) direction and in the satellite’s along-track ﬂight direction, where the data row and column (range and azimuth) offsets between two consecutive acquisitions are determined (Pathier et al., 2006). These pixel offset measurements are ideally scaled to units of meters because even submeter displacements may be determined (Casu et al., 2011). Here, we derived an approximate uniform pixel-to-meter scale only, which is based on the dimensions of known, prominent cliff faces (0.59 px/m for the ascending view and 0.73 px/m for the descending view). We thus focused on qualitatively interpreting the pixel-scale range/azimuth displacements. However, as the image perspective is the same between images, we can still quantify the pattern, the relative magnitude, and the approximate direction of the displacements. Features are tracked on the coregistered images with repeat passes of decreasing subwindows, twice with 128 px × 128 px and 50% overlap and once with 68 px × 68 px and 75% overlap. (c) g g y (b) Second, we performed a more quantitative analysis of the shadowing effects located in areas containing approximately N–S-oriented topographic valleys and ridges with slopes at an angle higher than the SAR line-of-sight (LOS) incidence angle (similar to Wadge et al., 2011; Arnold et al., 2017; Arnold et al., 2019). In other words, radar shadows develop in deeply eroded ravines but disappear if these ravines are reﬁlled by new volcanic materials. SAR Data and Monitoring the Material Redistribution Tasking the TerraSAR-X (TSX) satellite allows us to select a set of 6 ascending and 26 descending amplitude images of Shiveluch Frontiers in Earth Science \| www.frontiersin.org June 2021 \| Volume 9 \| Article 680051 5 The 2018–2019 Shiveluch Volcano Eruption Shevchenko et al. for the different dates by dividing the deposition extents obtained by visual digitization using the SAR images into polygonal subareas (i.e., Voronoi diagrams) based on the deposit thicknesses calculated at given points. Polygons of different sizes varying between hundreds of square meters and hundreds of thousands of square meters were generated; for each polygon, a constant thickness was assumed. A sketch of this method is illustrated in Supplementary Figure S1. volcano from December 5, 2018 to December 25, 2019. The data were acquired in spotlight mode, which allows the redistribution of the dome material to be monitored at a ∼1-m spatial resolution with a temporal resolution reaching 11 days (for descending data). The SAR data were then analyzed in the following ways: (a) First, we visually described the amplitude imagery used for interpretation in radar coordinates similar to the method previously applied to other volcanoes (e.g., Arnold et al., 2019). For this purpose, we mainly used the descending data, as their acquisition was more regular and the viewing geometry was more favorable with fewer foreshortening and shadowing effects than the ascending data. We loaded all available data into a coregistered stack using ENVI SARScape v. 5.5.3 and applied a 3 × 3 kernel ﬁlter to reduce the speckle effect and visualize the reﬂectance changes observed in the imagery. of this method is illustrated in Supplementary Figure S1. c) As small-scale (on the order of millimeter-to centimeter- level) deformations were not detectable using the differential interferometric SAR method due to decorrelation and coherence loss, we focused on investigating large-scale (meter-level) deformations using the pixel offset method. Accordingly, we determined pixel offsets by applying the particle image velocimetry (PIV) technique using the LaVision DaVis v10.0.5 software package to track and visualize changes in the coregistered SAR images. This method vectorizes pixel offsets in the image plane based on the intensity value of the image. SAR Data and Monitoring the Material Redistribution The radar shadow width is proportional to the relative height h of the topographic feature (h wloscosϕ/tanθ), where wlos is the radar shadow width in the LOS direction and is measured as a distance in the geocoded image; ϕ is the angle between the satellite LOS direction and the slope direction based on the DEM and is measured as an angle between two lines in the geocoded image; and θ is the radar incidence angle, a given parameter related to the SAR acquisition geometry. For this purpose, we geocoded the coregistered and ﬁltered TSX images and resampled them to 1 × 1 m2 pixels using the 2018–2019 optical DEMs (see Photogrammetry and Digital Elevation Model Analysis). Then, we applied the abovementioned radar shadow method to indirectly derive the minimum deposit thickness during this period of constructive and destructive activity assuming that shadows change by altering the depths of the ravines. We derived a time series of the minimum deposition thickness by comparing the shadow width between a reference image and a speciﬁc date. We used the QGIS v. 3.16.3 software package to perform the distance and angle measurements and further analysis. We measured wlos and ϕ at several points along the slopes where the shadow was projected, and obtained a pointwise measure of the deposit thickness. We note that this shadow method allows us to obtain only a minimum value of the thickness at some points, for instance, where the accumulated material exceeds the slope height casting the shadow. Accordingly, we can estimate the thickness up to the total height of the topographic features, but cannot measure how much deposited material has accumulated above that height. Finally, we estimated approximate deposit volumes (d) (d) Finally, we used the SAR images to construct a 5-m resolution DEM from the July 10, 2018 descending TanDEM-X (TDX) data to ﬁll the gap in the 2018 Pleiades DEM (see Photogrammetry and Digital Elevation Model Analysis). TDX is a bistatic SAR mission formed by adding a second, almost identical spacecraft to TSX June 2021 \| Volume 9 \| Article 680051 Frontiers in Earth Science \| www.frontiersin.org 6 The 2018–2019 Shiveluch Volcano Eruption Shevchenko et al. 2020). The reliability of the applied algorithm has already been successfully veriﬁed on a variety of different volcanological thermal-emitting phenomena worldwide (Valade et al., 2019; Massimetti et al., 2020). SAR Data and Monitoring the Material Redistribution The combined thermal datasets allow major hot volcanic thermal processes and their effects to be tracked, particularly those in a complex volcanic context such as Shiveluch volcano with a variety of volcanic products and styles. (Kubanek et al., 2021). Therefore, this mission allows the acquisition of two simultaneous SAR images over the same area, thereby eliminating possible temporal decorrelations between the images and maintaining a normal baseline between 250 and 500 m, which is suitable for SAR interferometry and the generation of a DEM. We used the interferometric module in ENVI SARscape to build the interferogram, perform the unwrapping step, and ﬁnally convert the result into height information using a forward transformation from radar to geographic coordinates. The RMSE of the generated DEM was evaluated to be approximately 5 m based on the coherence value, i.e., the quality of the interferogram. (b) y (b) We also acquired thermal infrared images of the lava dome from a helicopter in conjunction with the optical survey (see Photogrammetry and Digital Elevation Model Analysis) before and after the August 29, 2019 eruption. On the ﬁrst ﬂight, we used a FLIR Tau 2 camera with a 9 mm lens and a TEAX ThermalCapture frame grabber set to a sampling rate of 8 Hz. The resulting images had a resolution of 640 px × 512 px and provided radiometric temperature data. The images were processed and exported using Thermoviewer (v3.0.4) assuming a constant emissivity of 0.95, a transmissivity of 0.7 and environmental and path temperatures of 10°C, which are comparable to those in similar studies (Stevenson and Varley, 2008; Walter et al., 2013a; Zorn et al., 2020). For the second ﬂight, we used a ThermaCAM P640 camera at a resolution of 640 px × 480 px, and the images were exported and processed with FLIR Tools (v.5.13) while assuming the same environmental parameters as before. Thermal Infrared Data and Determination of Migrating Temperature Anomalies Satellite-acquired thermal infrared data were also used in this study and were validated during our ﬁeldwork overﬂights using handheld cameras. (a) The satellite images used for this study were acquired by different infrared satellite platforms, including Moderate Resolution Imaging Spectroradiometer (MODIS) images from the Terra and Aqua satellites, Multispectral Instrument (MSI) images from Sentinel-2 (S2), and Landsat 8 (L8) Operational Land Imager (OLI) images (Coppola et al., 2020; Plank et al., 2021). These products are complementary, as, for instance, MODIS acquires images with a near-daily temporal resolution at a low spatial resolution of 1 km, whereas the S2 and L8 sensors provide data at spatial resolutions of 20 and 30 m, respectively, although they have temporal resolutions of only 2–5 days and 8–16 days, respectively (Ramsey and Harris, 2013; Coppola et al., 2016). The MODIS data were processed by the MIROVA system in the mid-infrared (3.9 µm) region, providing the VRP (in watts) emitted by volcanic activity at magmatic temperatures (T > 500 K) with an average error of ±30% (Coppola et al., 2016). MODIS infrared data have a 1 km spatial resolution and a high revisit frequency (up to four times daily) for volcanic monitoring purposes. High- spatial resolution thermal satellite datasets, including S2 and L8, have been investigated by applying a novel hotspot detection algorithm based on ﬁxed ratios in the shortwave infrared (SWIR) regions with a contextual threshold derived from a statistical distribution of hotspot pixel clusters (Massimetti et al., 2020), as SWIR signals, in particular, record almost purely thermal emissions produced by hot emitting bodies (Blackett, 2017). Images were analyzed considering the SWIR top-of-atmosphere (TOA) reﬂectances in the ρ12 (2.19 µm), ρ11 (1.61 µm), and ρ8A (0.86 µm) bands for the S2 MSI and the ρ7 (2.11–2.29 µm), ρ6 (1.57–1.65 µm), and ρ5 (0.85–0.88 µm) bands for the L8 OLI. The algorithm works on both imagery datasets and detects the number of “hot” pixels (S2-L8 number of pixels), where a hotter area is superﬁcially exposed, with an overall estimate of 2–4% false alerts detected (Massimetti et al., ) The satellite images used for this study were acquired by different infrared satellite platforms, including Moderate Resolution Imaging Spectroradiometer (MODIS) images from the Terra and Aqua satellites, Multispectral Instrument (MSI) images from Sentinel-2 (S2), and Landsat 8 (L8) Operational Land Imager (OLI) images (Coppola et al., 2020; Plank et al., 2021). Thermal Infrared Data and Determination of Migrating Temperature Anomalies These products are complementary, as, for instance, MODIS acquires images with a near-daily temporal resolution at a low spatial resolution of 1 km, whereas the S2 and L8 sensors provide data at spatial resolutions of 20 and 30 m, respectively, although they have temporal resolutions of only 2–5 days and 8–16 days, respectively (Ramsey and Harris, 2013; Coppola et al., 2016). The MODIS data were processed by the MIROVA system in the mid-infrared (3.9 µm) region, providing the VRP (in watts) emitted by volcanic activity at magmatic temperatures (T > 500 K) with an average error of ±30% (Coppola et al., 2016). MODIS infrared data have a 1 km spatial resolution and a high revisit frequency (up to four times daily) for volcanic monitoring purposes. High- spatial resolution thermal satellite datasets, including S2 and L8, have been investigated by applying a novel hotspot detection algorithm based on ﬁxed ratios in the shortwave infrared (SWIR) regions with a contextual threshold derived from a statistical distribution of hotspot pixel clusters (Massimetti et al., 2020), as SWIR signals, in particular, record almost purely thermal emissions produced by hot emitting bodies (Blackett, 2017). Images were analyzed considering the SWIR top-of-atmosphere (TOA) reﬂectances in the ρ12 (2.19 µm), ρ11 (1.61 µm), and ρ8A (0.86 µm) bands for the S2 MSI and the ρ7 (2.11–2.29 µm), ρ6 (1.57–1.65 µm), and ρ5 (0.85–0.88 µm) bands for the L8 OLI. The algorithm works on both imagery datasets and detects the number of “hot” pixels (S2-L8 number of pixels), where a hotter area is superﬁcially exposed, with an overall estimate of 2–4% false alerts detected (Massimetti et al., Morphological Changes Derived From a Comparison of the Optical DEMs For a base map, we used a photogrammetric dataset that was acquired on July 12, 2012 and has already been described earlier (Shevchenko et al., 2015). This base map shows the collapse amphitheater resulting from the 1964 collapse, the central dome that grew inside the amphitheater, and two smaller scars that formed in 2005 and 2010 (highlighted in Figure 2A). Two lava lobes can be identiﬁed on the dome: the northern lobe that partially overlaps the NW rim of the 2010 scar and the SSE lobe with a crater on top (see Figure 3 and the details in Shevchenko et al., 2015). Reprocessing the data at a higher resolution allowed us to identify a SW–NE-striking lineament with an azimuth of 027°N (dashed line in Figure 3A). This lineament is also traceable through complex topography; because it notably bisects the lava lobes, the crater, and the talus in the NE sector of the amphitheater and is visible in both the optical and the topographic data, this lineament may represent an important structural feature (Figures 2A, 3A). The DEM that we constructed from the July 18, 2018 Pleiades dataset (Figures 2B, 3B) shows the morphology of the volcano several months prior to the beginning of the studied extrusive eruption (which commenced in December 2018). The western to SW sector of the dome currently accommodates a new collapse scar (800 m × 2,000 m) originating from the September 18, 2016 partial collapse. This scar outcrops the core parts of the previously June 2021 \| Volume 9 \| Article 680051 Frontiers in Earth Science \| www.frontiersin.org 7 The 2018–2019 Shiveluch Volcano Eruption Shevchenko et al. photos of Young Shiveluch constructed from aerial (A,C,D) and Pleiades satellite (B) data with the main destruction features indicated by the FIGURE 2 \| Orthophotos of Young Shiveluch constructed from aerial (A,C,D) and Pleiades satellite (B) data with the main destruction features indicated by the colored lines. FIGURE 2 \| Orthophotos of Young Shiveluch constructed from aerial (A,C,D) and Pleiades satellite (B) data with the main destruc colored lines. formed lava lobes and narrows towards the SSW into an avalanche chute within the borders of the 2005 scar. Another new, smaller scar (370 m × 700 m) can be observed on the NE ﬂank of the dome, where it narrows into a chute following the eastern rim of the amphitheater. Morphological Changes Derived From a Comparison of the Optical DEMs At the summit of the dome, a central crater with a diameter of approximately 350 m is identiﬁed (red outline in Figure 2B). The 2012 lava lobe on the SSE ﬂank persists without any major morphological changes; it was destroyed only at the very summit due to the formation of the scar and crater. The main accumulation of material is detected June 2021 \| Volume 9 \| Article 680051 Frontiers in Earth Science \| www.frontiersin.org 8 The 2018–2019 Shiveluch Volcano Eruption Shevchenko et al. in the northern sector of the dome near the former amphitheater rim (Figures 3B, 4A). A peak-like remnant of the northern part of the dome, which was destroyed by the two collapses (blue outlines in Figures 2B, 3B), had an elevation of 2,757 m and almost reached the highest point of Young Shiveluch, the Fourth Su (Chetvertaya Vershina), which is 2,782 m in height. abovementioned SW–NE-striking structural lineament is observed here, where it bisects the peak-like remnant an FIGURE 3 \| Hillshade maps of the Young Shiveluch dome for the 2012 base map and three acquisitions covering the 2018–2019 construction-destruction period. Note the formation of new central craters (red circles) and short-lived explosion craters (green circles). The DEMs were derived from aerial photogrammetric data (A,C and from Pleiades satellite data locally complemented by TDX data (B). Shevchenko et al. The 2018–2019 Shiveluch Volcano E FIGURE 3 \| Hillshade maps of the Young Shiveluch dome for the 2012 base map and three acquisitions covering the 2018–2019 construction-destruction period. Note the formation of new central craters (red circles) and short-lived explosion craters (green circles). The DEMs were derived from aerial photogrammetric data (A,C,D) and from Pleiades satellite data locally complemented by TDX data (B). FIGURE 3 \| Hillshade maps of the Young Shiveluch dome for the 2012 base map and three acquisitions covering the 2018–2019 construction-destruction period. Note the formation of new central craters (red circles) and short-lived explosion craters (green circles). The DEMs were derived from aerial photogrammetric data (A,C,D) and from Pleiades satellite data locally complemented by TDX data (B). reached the highest point of Young Shiveluch, the Fourth Summit (Chetvertaya Vershina), which is 2,782 m in height. The abovementioned SW–NE-striking structural lineament is also observed here, where it bisects the peak-like remnant and the reached the highest point of Young Shiveluch, the Fourth Summit (Chetvertaya Vershina), which is 2,782 m in height. Morphological Changes Derived From a Comparison of the Optical DEMs The abovementioned SW–NE-striking structural lineament is also observed here, where it bisects the peak-like remnant and the in the northern sector of the dome near the former amphitheater rim (Figures 3B, 4A). A peak-like remnant of the northern part of the dome, which was destroyed by the two collapses (blue outlines in Figures 2B, 3B), had an elevation of 2,757 m and almost Frontiers in Earth Science \| www.frontiersin.org June 2021 \| Volume 9 \| Article 680051 9 The 2018–2019 Shiveluch Volcano Eruption Shevchenko et al. northern half of the central crater (Figure 3B). The relative height of the dome was 613 m. By July 2018, the northern and NE rims of the amphitheater had been almost completely buried under new unconsolidated material. The 2010 collapse scar at the SE foot of the dome was also ﬁlled with new material. By comparing the DEMs from 2012 to 2018, we estimate that the dome volume increased by 16 × 106 m3, with a total dome volume of approximately 646 × 106 m3 (Figure 4A). Hence, the average growth rate over the 2012–2018 period was 7,200 m3/day (0.08 m3/s), and further details are provided in Table 1. scar decreased by approximately 5 × 106 m3, while that within the eastern scar decreased by 84 × 106 m3. The height of the dome decreased by 26 m due to the partial destruction of the northern peak and the formation of the northern crater, yielding a new height of 585 m. The total volume of the dome decreased by 110 × 106 m3 (including the scars); thus, the total dome volume became 682 × 106 m3. The August 29, 2019 eruption produced a PDC that traveled 12.3 km towards the SE, and the majority of the volume was deposited on top of the 2010 PDC deposits, albeit with a shorter run-out distance. A visual inspection and measurements of the Planet satellite image (Figure 1A) suggest that the August 29, 2019 PDC was deposited in several scattered ﬁelds along its path over a total area of 12 km2. A small amount of material also descended along the western (2005) scar and was deposited at the SW foot over an area of 0.5 km2, but this deposition was not captured during our aerial survey. Morphological Changes Derived From a Comparison of the Optical DEMs To further analyze the main August 2019 deposition ﬁeld, we inspected the aerial orthophoto and performed a comparison of the optical DEMs (Figure 5). Material deposition occurred within an area of 7.2 km2 (Figure 5A), reaching a maximum thickness of almost 30 m and an average thickness of approximately 12 m (Figure 5B). We estimate that the volume of the main deposits was 87 × 106 m3, which is 23 × 106 m3 lower than the volume estimated to have been removed from the source ediﬁce. The differences between the volumes of the source material and the deposits can be explained by the scattered regions of deposition in other areas not captured during the aerial survey, by the lack of dense rock equivalent (DRE) measurements, and by the dispersion of ﬁne particles to more distant locations. A geometric analysis of the deposition area implies that this was the third-largest PDC that occurred during the current period (since 2001), after the 2005 and 2010 deposits, which extended over distances of 19 and 16 km, respectively, and spanned areas of 25 km2 (Ramsey et al., 2012) and 27 km2 (Dvigalo et al., 2011). ( ) p During the December 2018 – August 2019 period, a new extrusive eruption emplaced a massive lava lobe (see Chronology Derived From the TSX Amplitude Images). By August 2019, this lava lobe occupied the entire central part of the dome and covered its western ﬂank (Figures 2C, 3C) with dimensions of 700 m × 1,080 m and a volume (as determined by DEM differencing) of 51 × 106 m3. The lava lobe might be the largest extrusive body that emerged on the dome over the current period of regrowth (since 2001). Furthermore, the data allow the recognition of two new craters: one in the NE sector of the lobe (approximately 180 m in diameter) and another along the northern rim of the amphitheater (130 m × 200 m). During the observation period (July 2018 – August 2019), the thickness of added loose material along the 2005 (western) collapse scar reached 90 and 100 m within the 2010 (eastern) scar. We also estimated the volumes of added material since July 2018 to be approximately 19 × 106 m3 in the 2005 scar and 33 × 106 m3 in the 2010 scar. Morphological Changes Derived From a Comparison of the Optical DEMs The height of the dome remained almost the same at 611 m, as its highest northern part did not exhibit any morphological changes that can be identiﬁed in the DEMs. The dome volume increased by 146 × 106 m3 after July 2018, which includes the talus and deposits within the two collapse scars (Figure 4B). Ultimately, the total dome volume on August 22, 2019 was 792 × 106 m3. Thus, the average extrusion rate over 1 year was 365,000 m3/day (4.22 m3/s). y y ( ) On August 29, 2019, a large explosive eruption occurred at Shiveluch, which led to a secondary partial dome collapse (see Chronology Derived From the TSX Amplitude Images). Analysis of the August 2019 (Figure 2C) and October 2019 (Figure 2D) aerial orthophotos allows us to describe details of the morphological changes that occurred in association with this eruption. A new large central summit crater is identiﬁed in the October 2019 dataset with dimensions of 430 m × 490 m, and a new extrusion is observed inside this crater with a blocky appearance and dimensions of 220 m × 250 m. At the northern part of the dome, another large crater 300 m in diameter can be recognized. We identify four new small side craters (Figure 3D) with diameters varying from 45 to 90 m. In the eastern and NE parts of the dome, a newly formed scar is visible, which narrows into an avalanche chute in the south; the size of this scar is 600 m × 1,100 m, and its depth varies from shallow (up to 50 m) in its southern sector to deep (up to 270 m) in its NE sector. The western (2005) and eastern (2010) scars also appear to have lost material (Figure 4C). The October 2019 dataset suggests that after the August 29, 2019 eruption, the western scar became deeper by 15 m, while the eastern scar deepened by 40 m on average. The volume within the western Topographic proﬁles help to further visualize the effects of the constructive and destructive processes at Shiveluch volcano over the studied period (Figure 6). The 2018 proﬁles show signiﬁcant enlargement of the ediﬁce in the northern part of the dome relative to 2012 but, in contrast, the loss of material in the central and western parts due to the 2016 collapse and formation of the summit crater (Figures 4A, 6B,C). Material Redistribution Observed by Satellite Radar Observations Chronology Derived From the TSX Amplitude Images The analysis of TSX amplitude images provides a more continuous overview of the deformation, as the active system onboard TSX can penetrate dense clouds and thus provide ground scattering observations that can display morphological details. Here, we examine descending orbit images only; however, the ascending imagery is provided in Supplementary Figure S2. The TSX data reveal that the morphology of the dome remained almost the same throughout the year from January 2018 to December 2018 (Figure 7A; Supplementary Movie S1) until December 27, 2018 (Figure 7B), when the new lava lobe became visible at the top of the Shiveluch dome (see Morphological Changes Derived From a Comparison of the Optical DEMs). This new lobe seems to have extruded from the summit crater and spread laterally towards the SW ﬂank. The image from January 7, 2019 shows a further extension of the new lobe (Figure 7C) that covers the older lithology, including the 2012 lava lobe. By January 18, 2019, two sectors of the new lobe had slumped (Figure 7D). In the January 29, 2019 TSX image (Figure 7E), we identify a new half- circular darkened area resulting from low signal backscatter located along the northern rim of the amphitheater and the adjacent outer slope. This darkening is possibly associated with the shadowing effects caused by steepening topography. By February 20, 2019, we can identify the beginning of the gradual destruction of the front of the new lava lobe (Figure 7F). The March 14, 2019 image shows a new small crater (see Morphological Changes Derived From a Comparison of the Optical DEMs) at the top of the lobe in its NE sector (Figure 7G). On April 16, 2019, another new small crater (see Morphological Changes Derived From a Comparison of the Optical DEMs) can be seen in our TSX imagery at the northern rim of the amphitheater (Figure 7H) where the half-circular pp y The most profound changes are identiﬁed in the ﬁrst post- eruption (i.e., after August 29, 2019) TSX image, which was acquired on September 17, 2019 (Figure 7K); this image reveals the large central crater (see Morphological Changes Derived From a Comparison of the Optical DEMs) that opens eastward into the deep collapse scar, which rotates southward. Morphological Changes Derived From a Comparison of the Optical DEMs By August 22, 2019, the ediﬁce height had increased with respect to that in 2012 in the central part and notably surpassed the 2018 height in the eastern sector of the dome (Figures 4B, 6B,C). Because of the substantial loss of material due to the August 29, 2019 eruption, the central dome height decreased even in comparison with the 2012 height; relative to the 2019 pre-eruption morphology, this decrease in height exceeded 250 m in the eastern part of the dome (Figures 4C,D, 6B,C). The accumulation of loose material alternated with its removal within the western (2005) and eastern (2010) scars (Figures 6D,E). In 2018, there was a signiﬁcant loss of material in the western scar due to the 2016 collapse; then, new material was added in 2019 and eroded again during the 2019 explosive eruption and collapse (Figures 4A–C, 6D). In the eastern scar, deposits accumulated signiﬁcantly after 2012 but were June 2021 \| Volume 9 \| Article 680051 Frontiers in Earth Science \| www.frontiersin.org 10 The 2018–2019 Shiveluch Volcano Eruption Shevchenko et al. FIGURE 4 \| Incremental height difference maps generated from the base DEM and the 2018–2019 DEMs. Green indicates no major change, blue indicates mater oss (destruction), and red indicates material addition (construction). (A) Comparison between the July 2012 and July 2018 DEMs; (B) comparison between t December 2018 and post-August 2019 extrusive eruption DEMs; (C) comparison between the pre- and post-August 2019 explosive eruption DEMs; (D) com between the July 2012 and October 2019 DEMs (covering the whole period of study). GURE 4 \| Incremental height difference maps generated from the base DEM and the 2018–2019 DEMs. Green indicates no major change, blue indicate FIGURE 4 \| Incremental height difference maps generated from the base DEM and the 2018–2019 DEMs. Green indicates no major change, blue indicates material loss (destruction), and red indicates material addition (construction). (A) Comparison between the July 2012 and July 2018 DEMs; (B) comparison between the pre- December 2018 and post-August 2019 extrusive eruption DEMs; (C) comparison between the pre- and post-August 2019 explosive eruption DEMs; (D) comparison between the July 2012 and October 2019 DEMs (covering the whole period of study). June 2021 \| Volume 9 \| Article 680051 Frontiers in Earth Science \| www.frontiersin.org 11 The 2018–2019 Shiveluch Volcano Eruption Shevchenko et al. Morphological Changes Derived From a Comparison of the Optical DEMs FIGURE 5 \| Orthophoto (A) and height difference map (B) of the main PDC deposit ﬁeld from the August 29, 2019 eruption. The location of the ﬁeld is shown in Figure 1. FIGURE 5 \| Orthophoto (A) and height difference map (B) of the main PDC deposit ﬁeld from the August 29, 2019 eruption. The location of the ﬁeld is shown in Figure 1. FIGURE 5 \| Orthophoto (A) and height difference map (B) of the main PDC deposit ﬁeld from the August 29, 2019 eruption. The location of the ﬁeld is shown in Figure 1 subsequently removed due to the 2019 destructive events (Figures 4A–C, 6E). darkened area had previously been located (Figure 7E). The image from May 8, 2019 shows the continuation of the gradual collapse of the lobe’s front and a noticeable increase in the deposition of loose material along the eastern rim of the amphitheater (Figure 7I). Subsequently, no major morphological changes are revealed, and the image from August 26, 2019 (Figure 7J) shows a very similar morphology, with the exception of the continued minor collapse of the lobe’s front and the deposition of loose material, as well as a new small area of darkening visible at the NE rim of the amphitheater. The extrusion of the lava lobe continued throughout almost the whole observation period, being more intense from the end of December 2018 to the end of April 2019, when the lava lobe virtually ceased growing and was visible only as minor deformations in the vent area (further details are provided in Lava Dome Deformations Revealed by PIV; Supplementary Movie S1). Material Redistribution Observed by Satellite Radar Observations One new small side crater is visible close to the NE rim of the amphitheater exactly where the area of darkening was previously observed (Figure 7J). The other two craters identiﬁed in the aerial data (see Morphological Changes Derived From a Comparison of the Optical DEMs) are not visible in the TSX images due to distortion and shadowing. The southern ﬂank of the dome did not collapse. The image from October 20, 2019 (Figure 7L) shows extrusion occurring from the central crater and destruction of the upper part of the peak (see Morphological Changes Derived From a Comparison of the Optical DEMs) at the northern sector of the dome coincident with the formation of a second large crater. Therefore, although the TSX amplitude images are geometrically distorted, important chronological details of morphological changes can be distinguished, including the relatively precise timing of lava lobe extrusion and the appearance of new craters and collapse scars. June 2021 \| Volume 9 \| Article 680051 Frontiers in Earth Science \| www.frontiersin.org 12 The 2018–2019 Shiveluch Volcano Eruption Shevchenko et al. GURE 6 \| Topographic proﬁles of the Shiveluch lava dome (B,C) and collapse scars (D,E). The proﬁle lines are shown in the insert map (A). The blue shading resents a negative difference between the pre- and post-2019 destructive events. FIGURE 6 \| Topographic proﬁles of the Shiveluch lava dome (B,C) and collapse scars (D,E). The proﬁle lines are shown in the insert map (A). The blue shading GURE 6 \| Topographic proﬁles of the Shiveluch lava dome (B C) and collapse scars (D E) The proﬁle lines are shown in the insert map (A) The blue shading FIGURE 6 \| Topographic proﬁles of the Shiveluch lava dome (B,C) and collapse scars (D,E). The proﬁle lines are shown in the insert map (A). The blue shading represents a negative difference between the pre- and post-2019 destructive events. Frontiers in Earth Science \| www.frontiersin.org June 2021 \| Volume 9 \| Article 680051 13 The 2018–2019 Shiveluch Volcano Eruption Shevchenko et al. E 7 \| Time series of TSX descending orbit amplitude images (in radar coordinates: azimuth and range) showing the chronology of the eruptive events an orphological changes that occurred over the studied period. Low-amplitude features (craters and scars) are shown with dotted lines; high-amplitude fe ve bodies) are shown with dashed lines. Material Redistribution Observed by Satellite Radar Observations Arrows point to the features labeled in the bottom left corners of the images. FIGURE 7 \| Time series of TSX descending orbit amplitude images (in radar coordinates: azimuth and range) showing the chronology of the eruptive events and main morphological changes that occurred over the studied period. Low-amplitude features (craters and scars) are shown with dotted lines; high-amplitude features (extrusive bodies) are shown with dashed lines. Arrows point to the features labeled in the bottom left corners of the images. Frontiers in Earth Science \| www.frontiersin.org June 2021 \| Volume 9 \| Article 680051 14 The 2018–2019 Shiveluch Volcano Eruption Shevchenko et al. FIGURE 8 \| Selected PIV tracking maps from the TSX data in ascending (A–D) and descending (E–H) orbits. The yellow dashed lines outline the new lava lobe; the yellow dotted lines indicate the outlines of motion before the partial destruction of the dome (C,F) and the shape of the area affected by destruction (D,G). The approximate scales are 0.59 px/m for the ascending view and 0.73 px/m for the descending view, but these scales should be used with caution (see the Data and Methods). The full time series of the tracking results can be found in the supplementary material (Supplementary Figure S3). For the base images, please refer to Figure 7 for the descending data and to Supplementary Figure S2 for the ascending data. FIGURE 8 \| Selected PIV tracking maps from the TSX data in ascending (A–D) and descending (E–H) orbits. The yellow dashed lines outline the new lava lobe; the yellow dotted lines indicate the outlines of motion before the partial destruction of the dome (C,F) and the shape of the area affected by destruction (D,G). The approximate scales are 0.59 px/m for the ascending view and 0.73 px/m for the descending view, but these scales should be used with caution (see the Data and Methods). The full time series of the tracking results can be found in the supplementary material (Supplementary Figure S3). For the base images, please refer to Figure 7 for the descending data and to Supplementary Figure S2 for the ascending data. Lava Dome Deformations Revealed by PIV Th T l d d ll d d Lava Dome Deformations Revealed by PIV The TSX amplitude data allowed us to determine pixel offsets. Material Redistribution Observed by Satellite Radar Observations As regular TSX acquisitions were realized in descending orbit, they captured the period covered in this study better than the acquisition in ascending orbit, as the ascending view was less regular and available only during the timeframe preceding the August 29, 2019 eruption and dome collapse. The tracking results are highlighted in Figure 8, and the full tracking sequence can be found in Supplementary Figure S3. The results are displayed in radar coordinates to preserve the constant-pixel dimensions, where the displacement vectors combine range and azimuth offsets. The tracking initially shows the emplacement of the lava lobe in January 2019, presenting a mostly decorrelated surface at the top and in the western sector of the dome due to the intense extrusion of new material. With the gradual slowing of extrusion, increased vector lengths can be observed along the ESE dome ﬂank, which was not covered by the new lava lobe. These offsets started in early February 2019 and persisted through February–March 2019 (Figures 8A,B,E). The main area of motion was then restricted to the summit dome, which presented deformations of the upper part of the lava lobe; however, slower motion of the lower ﬂanks persisted (Figures 8C,F). No deﬁnitive statement can be made regarding the exact growth direction (the range depends on both horizontal and vertical motions); however, since both the ascending and the descending views appear to show predominantly SE motion, the dominant growth direction was likely toward the SE with little or no vertical motion. The pixel offsets were measured at varying rates, which were highest during the ﬁrst measurement in March 2019 (Figure 8E) at nearly 0.65 px/day (orthogonal to the radar LOS). The rates then decreased and varied between 0.30 and 0.45 px/day, being higher only between mid-June 2019 and mid-July 2019. In the weeks before the large explosive eruption and dome collapse, the offsets returned to their previous levels. In the ascending view, the pixel offsets similarly varied between 0.31 and 0.42 px/day, which are slower rates compared to those during the same timespan in the descending view, likely due to the different perspectives. The decorrelated area after the explosion and collapse events on August 29, 2019 is consistent with the area of high deformation preceding the collapse, both in the ascending (Figures 8C,D) and the descending (Figures 8F,G) views. Analysis of Thermal Anomalies 2018–2019 Satellite Infrared Data Series The evolution of thermal anomalies over the Shiveluch dome area is derived from the S2 and L8 imagery in SWIR bands (Figure 10). Our analysis begins in December 2018, when intense degassing and fumarolic activity obscured the summit of the dome mainly over the central and northern sectors (Figure 10A). In the previous S2 MSI and L8 OLI images during October–December 2018 (see Supplementary Figure S4), signs of newly emplaced gray deposits (most likely related to PDCs, gravitational collapses, hot avalanches, and/or ash fall deposits), probably produced by sporadic explosive activity, are visible due to the contrast with the snow-covered ground. Moreover, some thermal anomalies are visible on the upper surface of the dome and become more apparent during December 2018; these anomalies, which appeared one and one-half months prior to the main explosion on December 30, 2018, were localized mainly in the central summit crater atop Shiveluch and its eastern sector (see Supplementary Figures S4, S5). The ﬁrst image to be collected after the strong explosion on December 30, 2018 was acquired one day later on December 31, 2018, revealing a glow emanating from the ﬂanks of the dome associated with extrusions and newly deposited, still- incandescent material (likely sourced by collapses, hot avalanches or PDCs) that is traceable up to 5.5 km from the summit towards the SW (Supplementary Figure S4). We were able to detect changes in the radar shadows located on the western (2005) and eastern (2010) collapse scars due to the accumulation and removal of materials. In the following, we refer to these materials as deposits, although we cannot distinguish the exact types of rock and deposition mechanisms (e.g., pyroclastics, collapse material, tephra). We applied the radar shadow method to the geocoded TSX amplitude images for the period from December 2018 to December 2019 to estimate the pointwise thicknesses and derive the deposit volumes by using Voronoi diagrams (see Data and Methods SAR Data and Monitoring the Material Redistribution (b)). The results are presented in Figure 9 and Table 2. Analysis of Thermal Anomalies 2018–2019 Satellite Infrared Data Series Based on the available SAR data, we can distinguish three main periods/episodes: the ﬁrst construction/deposition period that occurred between December 16, 2018 and August 26, 2019 estimated in relation to the December 16, 2018 image; the second destruction episode related to the August 29, 2019 explosion and partial dome collapse and described by one pre- eruption image (August 26, 2019) and one post-eruption image (September 17, 2019); and the third period of new deposition between September 17, 2019 and December 25, 2019 estimated in relation to the September 17, 2019 image. During the ﬁrst period, we observe a higher accumulation rate in the ﬁrst month with an average rate of 670,000 m3/day or 7.7 m3/s (December 16, 2018 – January 18, 2019) and a cumulative volume increase of 22 × 106 m3 (Figure 9A). After that, the accumulation rate slowed down to an average value of 106,000 m3/day or 1.2 m3/s (January 18 – March 25, 2019) and then to 39,000 m3/day or 0.4 m3/s (March 25 – August 26, 2019), reaching a cumulative volume increase of 29 × 106 m3 (Figure 9B) at the end of March and a cumulative volume increase of 35 × 106 m3 (Figure 9C) by the end of August. After the August 29, 2019 eruption and partial dome collapse, a signiﬁcant volume (65 × 106 m3) of material was removed (Figures 9D,G). Since the TSX acquisitions just before and after the collapse episode cover a time span of only 22 days, we cannot constrain the exact duration of the period during which material was removed from the scars. Subsequently, a new accumulation period, caused the reﬁlling of the scars, mainly the eastern collapse scar. The material accumulation rate was 790,000 m3/day or 9.1 m3/s from September 17 to October 20, 2019, reaching a cumulative volume increase of 26 × 106 m3 (Figure 9E), while the rate was 90,000 m3/day or 1 m3/s from October 20 to December 25, 2019, reaching a ﬁnal cumulative volume increase of 32 × 106 m3 (Figure 9F). During the construction episode deposits accumulated within During January 2019, we identify intense thermal activity due to an explosive event, hot ejecta, and rockfalls causing elongated thermal anomalies on the volcano ﬂanks (Figure 10B). Material Redistribution Observed by Satellite Radar Observations Due to the signiﬁcant surface changes, no further measurements could be made in descending orbit until December 2019, when a new extrusion occurred at the top of the dome with a steady-state pixel offset of approximately 0.50 px/day (Figure 8H). June 2021 \| Volume 9 \| Article 680051 Frontiers in Earth Science \| www.frontiersin.org 15 The 2018–2019 Shiveluch Volcano Eruption Shevchenko et al. Frontiers in Earth Science \| www.frontiersin.org Analysis of Thermal Anomalies 2018–2019 Satellite Infrared Data Series During this period of rockfalls and SW-directed lava extrusion, the uppermost summit area is only partially thermally expressed, with a few hotspots visible (Figures 10B–D), and persistent white plume emissions are observable from the northern sector of the dome (Figure 10D). g A series of cloud-free images from March 2019 allowed us to investigate the details of new features expressed near the summit of the Shiveluch dome (Figures 10E–H). Several glowing hotspots are present, representing a thermally active area in an elongated zone traversing the summit of the dome in a SW-NE direction. We ﬁnd that the eastern sector of this region is thermally more expressed than the western sector. Most of the thermal hotspots appear to change over time, but some remain stable and persist over several months (see also Supplementary Figures S5, S6). The SW-NE-elongated zone, well identiﬁed in the cloud-free thermal imagery (March–September 2019), is also morphologically expressed by a pronounced structural lineament and crater rows, as mentioned before (Figures 3A,D). Furthermore, small collapses of the lobe’s front are observed to trigger hot PDCs that mainly travel towards the south and east. Moreover, by September 2019, we recognize a thermal anomaly at a location where a new explosion crater will later develop in the northern part of the dome (Figure 10I). We note that the summit crater (the location of the August 29, 2019 explosion) appears cold in the thermal imagery (Figures 10I,J), whereas this new northern crater is hot (Figure 10J). However, the thermal activity inside this new northern crater wanes rapidly (Figure 10K). The thermal image taken in December 2019 (Figure 10L) reveals During the construction episode, deposits accumulated within the 2005 and 2010 collapse scars, reaching an average thickness of 15–20 m and a maximum thickness of 75 m in the upper part of the scars. After the August 29, 2019 destruction episode, the deposited material was concentrated mainly at the top section of the 2010 scar, reaching a thickness of 50 m. Hence, the August 2019 eruption and partial dome collapse caused the removal of material from the two scars (twice the volume of material that had accumulated in the ﬁrst period). Frontiers in Earth Science \| www.frontiersin.org June 2021 \| Volume 9 \| Article 680051 16 The 2018–2019 Shiveluch Volcano Eruption Shevchenko et al. Analysis of Thermal Anomalies 2018–2019 Satellite Infrared Data Series FIGURE 9 \| Time series of the minimum deposit thickness within the 2005 and 2010 collapse scars estimated using the radar shadow method for several SAR acquisitions between December 2018 and December 2019 (see Data and Methods SAR Data and Monitoring the Material Redistribution (b)). Reddish and bluish polygons show the Voronoi diagram approximations of the area of accumulation and removal of material, respectively (A–C) show the ﬁrst period of deposition starting from the dome reactivation in December 2018 up to just before the August 29, 2019 explosion estimated with respect to the December 16, 2018 image (D) shows the material removal episode that occurred after the explosion estimated in relation to the August 26, 2019 image (E,F) show the successive material accumulation episodes estimated in relation to the September 17, 2019 image. As the background images for (A–F), the dates of the compared geocoded TSX images are displayed; (G) presents the time series of the deposit volume change; all values are relative to the December 16, 2018 image. FIGURE 9 \| Time series of the minimum deposit thickness within the 2005 and 2010 collapse scars estimated using the radar shadow method for several SAR acquisitions between December 2018 and December 2019 (see Data and Methods SAR Data and Monitoring the Material Redistribution (b)). Reddish and bluish polygons show the Voronoi diagram approximations of the area of accumulation and removal of material, respectively (A–C) show the ﬁrst period of deposition starting from the dome reactivation in December 2018 up to just before the August 29, 2019 explosion estimated with respect to the December 16, 2018 image (D) shows the material removal episode that occurred after the explosion estimated in relation to the August 26, 2019 image (E,F) show the successive material accumulation episodes estimated in relation to the September 17, 2019 image. As the background images for (A–F), the dates of the compared geocoded TSX images are displayed; (G) presents the time series of the deposit volume change; all values are relative to the December 16, 2018 image. Frontiers in Earth Science \| www.frontiersin.org June 2021 \| Volume 9 \| Article 680051 17 The 2018–2019 Shiveluch Volcano Eruption Shevchenko et al. TABLE 2 \| Minimum volumetric accumulation of deposits and corresponding deposition rates in the western and eastern collapse scars combined. DISCUSSION The described eruptive activity during 2018–2019 was some of the strongest in the recent history of Young Shiveluch (since 1980) in terms of the eruption volume and morphological changes. A massive lava lobe developed at the dome during the extrusive period of December 2018 – April 2019, while a 430 m × 490 m SW–NE-elongated crater formed during the explosive eruption on August 29, 2019, which led to the signiﬁcant destruction of the NE sector and the collapse of the eastern ﬂank of the dome. The PDC generated from this eruption after the 2005 and 2010 ﬂows was the largest on record for this volcano (Dvigalo et al., 2011; Ramsey et al., 2012; Shevchenko and Svirid, 2014; Krippner et al., 2018). Such composite destructive events are common during large explosive eruptions at dome- building volcanoes and have been recently observed during major eruptions and erosional ravine-forming episodes, such as in 2015 at Volcan de Colima, Mexico (Reyes-Dávila et al., 2016), and in 2010 at Merapi volcano (Surono et al., 2012). Volumes before the August 29, 2019 eruption were estimated relative to the December 16, 2018 TSX acquisition, while those after the eruption were estimated relative to the September 17, 2019 TSX acquisition. thermal anomalies localized on the eastern ﬂank, mainly on the outer walls of the central crater, and only minor thermal activity on the western inner crater wall; in contrast, the new northern crater appears cold. Aerial Infrared Data Before and After the August 29, 2019 Eruption To further elaborate on the appearance of new explosion craters located close to the intersection of the newly identiﬁed SW–NE structure and the former amphitheater rim, we analyzed high-resolution aerial thermal infrared survey data. The data were acquired using handheld cameras from a helicopter on August 22, 2019 and October 22, 2019; thus, data were recorded both before and after the August 29, 2019 explosive event (Figure 11). The infrared data similarly reveal the locations of temperature anomalies throughout the dome, thereby highlighting the areas affected by ongoing extrusion, rockfalls, and degassing at a resolution not seen in the satellite imagery. The data taken a week before the event highlight four distinct areas with elevated temperatures at an approximate ground resolution of 3 m. First, we note the upper edge of the steep southern ﬂank, which regularly sources smaller rockfalls appearing as thin lines spreading radially outward from the dome towards the south. Similarly, on the eastern ﬂank, distributed smaller anomalies are observed, stemming mostly from fumaroles and cooling products. The central dome area is cold but displays a N–S-striking thermal anomaly of unknown origin (Figure 11A), which is located almost in the center of the future crater (marked by a yellow solid line in Figure 11B). We detect a patch of high-temperature anomalies on the northern edge of the dome that coincides with the strongest fumarole activity. Here, the highest measured temperatures exceed 270°C, although these temperatures may still be underestimated, as localized temperature anomalies may be smaller than the sensor resolution. Analysis of Thermal Anomalies 2018–2019 Satellite Infrared Data Series Date Volume (106 m3) Accumulation rate (106 m3/day)/(m3/sec) December 16, 2018 0 – December 27, 2018 7.00 0.64/7.37 January 07, 2019 18.00 1.00/11.6 January 18, 2019 22.00 0.36/4.21 January 29, 2019 24.00 0.18/2.10 February 20, 2019 26.00 0.09/1.05 March 25, 2019 29.00 0.09/1.05 May 08, 2019 31.00 0.05/0.53 June 21, 2019 33.00 0.05/0.53 July 24, 2019 34.00 0.03/0.35 August 26, 2019 35.00 0.03/0.35 September 17, 2019 0 – October 20, 2019 26.00 0.79/9.12 December 25, 2019 32.00 0.09/1.05 Volumes before the August 29, 2019 eruption were estimated relative to the December 16, 2018 TSX acquisition, while those after the eruption were estimated relative to the September 17, 2019 TSX acquisition. TABLE 2 \| Minimum volumetric accumulation of deposits and corresponding deposition rates in the western and eastern collapse scars combined. The post-eruption recording (Figure 11B) reveals a signiﬁcant redistribution of thermal activity. While the southern ﬂank remained intact, the high-temperature fumaroles were missing; in their place was a large central crater with low thermal emissions. The highest measured temperatures appear to the north of the dome within the fresh explosion crater. These observations perfectly coincide with the thermal satellite data (cf. Figure 10J), in which we detect a large anomaly north of the dome. Here, the highest recorded temperatures exceed 390°C, but as previously mentioned, these measurements may still underestimate the actual temperatures. Main Peculiarities of the 2018–2019 Eruptive Episode p One of the main distinctions between the 2019 and previous destructive events at Shiveluch is the preservation of the southern ﬂank of the dome. The southern ﬂank was partially destroyed during the 2005 eruption and completely destroyed during the 2010 collapse but appeared stable during the August 29, 2019 eruption. This preservation could be explained by the presence of the extensive lava lobe that formed in 2012, which remained stable on the ﬂank for more than 7 years. We speculate that this lobe could have acted like armor blanketing the ﬂank and preventing the collapse of unconsolidated material. For example, loading, gradual cooling, and degassing will close pore spaces and increase the compressive and tensile strengths of the lobe and dome rocks over time (Zorn et al., 2018). Additionally, hydrothermal alteration can signiﬁcantly change the strength of volcanic rocks depending on the type of alteration (Heap et al., 2021). In most studied cases, hydrothermal alteration weakened volcanic rocks (Wyering et al., 2014; Ball et al., 2015; Mordensky et al., 2019) or even induced explosive behavior (Heap et al., 2019), mainly through changes in the mineral content, porosity and permeability of the rocks. However, volcanic rocks also exhibit increased strength under certain June 2021 \| Volume 9 \| Article 680051 Frontiers in Earth Science \| www.frontiersin.org 18 Shevchenko et al. The 2018–2019 Shiveluch Volcano Eruption RE 10 \| Selected thermal imagery. S2 MSI RGB false-color composite bands 12-11-8a (SWIR) showing the evolution of the Shiveluch dome from Decemb o December 2019. Brownish and grayish colors display cold deposits and bedrock. Blue indicates snow cover. Reddish to yellowish gradients repres atic ejecta and materials (see the text for details). Images are cropped over the Shiveluch dome top within an area of 2 km × 2 km. White dashed lines indi s described in Morphological Changes Derived From a Comparison of the Optical DEMs. FIGURE 10 \| Selected thermal imagery. S2 MSI RGB false-color composite bands 12-11-8a (SWIR) showing the evolution of the Shiveluch dome from December 2018 to December 2019. Brownish and grayish colors display cold deposits and bedrock. Blue indicates snow cover. Reddish to yellowish gradients represent hot magmatic ejecta and materials (see the text for details). Images are cropped over the Shiveluch dome top within an area of 2 km × 2 km. Main Peculiarities of the 2018–2019 Eruptive Episode similarities with Bezymianny volcano, on which the slopes of the lava dome were protected with lava ﬂows that promoted stability and led to stratocone development (Shevchenko et al., 2020). Similarly, the western ﬂank of Shiveluch, covered by the 2019 lava lobe, remained intact during the August 29, 2019 explosive event and partial destruction of the dome, which may indicate that the stabilization effect produced by lava lobes is signiﬁcant for other episodes. Additionally, the locations of the new craters, either elongated along the SW–NE structural trend or along the former amphitheater rim (Figures 3C,D, 7H,I,K), argue for a structural control, as further discussed below. Craters that form during an explosion (Figures 3C, 7G) due to degassing or an interaction with meteoric water are typical on Shiveluch and have been previously recognized on lava lobes (Shevchenko et al., 2015) but never along or near the amphitheater rim. The central crater caused by the August 29, 2019 eruption is similar to the 2005 crater (∼750 m in diameter) but with smaller dimensions (430 m × 490 m), probably due to the smaller magnitude of the explosion. p Another notable distinction from the previous activity is the never-before-seen formation of multiple small craters along the northern and NE peripheries of the dome, either at or near the rim of the former amphitheater (Figures 3C,D). The ﬁrst of these craters appeared in mid-April 2019 (Figure 7H) at the northern rim of the amphitheater, which was covered by new material. The half-circular area of darkening in the region where this crater formed became noticeable in late January 2019, resembling the melting of perennial snow preserved by loose volcanic deposits coverage (see Chronology Derived From the TSX Amplitude Images and Figure 7E). The second peripheral crater appeared at the NE rim of the amphitheater after the August 29, 2019 eruption (Figure 7K) and was preceded by the appearance of a dark spot (Figure 7J). Furthermore, clusters of craters originated within the new 2019 collapse scar, probably due to the contact of the incandescent outcropped dome core with meteoric water. As these craters appeared suddenly and in association with only short eruptive/explosive activity, they may be considered monogenetic. Our careful analysis of available photogrammetric data does not reveal an extrusion or emission of large volumes of materials from these new crater sites. Main Peculiarities of the 2018–2019 Eruptive Episode White dashed lines indicate the features described in Morphological Changes Derived From a Comparison of the Optical DEMs. conditions through the deposition of minerals in hydrothermal areas (Heap et al., 2020). Here, it is not clear whether the dome or lobe were subject to either type of alteration, but since this lobe was relatively stationary and stable for several years, it would have likely experienced substantial cooling and degassing while also being subject to nearly constant hydrothermal alteration. Theoretically, these combined effects could have signiﬁcantly increased the rock strength of the lobe. We also note June 2021 \| Volume 9 \| Article 680051 Frontiers in Earth Science \| www.frontiersin.org 19 The 2018–2019 Shiveluch Volcano Eruption Shevchenko et al. FIGURE 11 \| Aerial FLIR data of the lava dome superimposed onto the DEMs of the pre-eruption survey on August 22, 2019 (A) and the post-eruption survey on October 22, 2019 (B). While the southern side of the dome remained mostly intact and unchanged, the eruption was followed by a signiﬁcant new anomaly in the northern part of the dome. The yellow lines in (B) outline new craters, and the dotted lines in (A) show the areas before these craters formed. The yellow dashed line outlines the 1964 amphitheater. The top of the dome is bisected by a N-S-striking anomaly shown with a solid yellow line in (A) in the area where the central crater was subsequently formed (B). The scale bar maximum is 200°C because only a few local pixels signiﬁcantly exceed this value. FIGURE 11 \| Aerial FLIR data of the lava dome superimposed onto the DEMs of the pre-eruption survey on August 22, 2019 (A) and the post-eruption survey on October 22, 2019 (B). While the southern side of the dome remained mostly intact and unchanged, the eruption was followed by a signiﬁcant new anomaly in the northern part of the dome. The yellow lines in (B) outline new craters, and the dotted lines in (A) show the areas before these craters formed. The yellow dashed line outlines the 1964 amphitheater. The top of the dome is bisected by a N-S-striking anomaly shown with a solid yellow line in (A) in the area where the central crater was subsequently formed (B). The scale bar maximum is 200°C because only a few local pixels signiﬁcantly exceed this value. thermal emissions, suggests that the origin of these craters could be phreatomagmatic. The SW–NE Lineament and Structural Implications vapor (Hashimoto et al., 2018); however, these craters did not show any strong visible steaming. As we observed cold crater ﬂoors in multiple S2 acquisitions and our helicopter overﬂight, we are conﬁdent that the low temperatures in these craters are real. Therefore, we can also assume that the extrusion of new blocky material observed in this crater that started in September shortly after the eruption could represent the extrusion of cold debris. Indeed, the crater that developed off-center and farther to the north exhibited one of the highest thermal anomalies in the study area during the period of observation (Figures 10J, 11B). The SW–NE lineament is inferred from 1) a pronounced linear kink in the slope during 2012, 2) structural features bisecting the peak-like remnant in 2018, 3) aligned explosion (monogenetic) craters that evolved during 2018–2019, and 4) thermal anisotropy. Moreover, we conjecture that the collapses (and especially the collapse directions) are largely governed by this structural feature. However, whether this feature has deeper roots and/or is the surface expression of a fault remains speculative. Accordingly, we hypothesize that this SW–NE lineament (Figure 12) could play an important role in the development of the dome and in its construction and destruction. The same SW–NE structural trend can be identiﬁed in optical and topographic data as early as 2012 (see Figures 2A, 3A) and was hypothesized in a previous study based on monogenetic cones located in the surrounding areas and lower apron of the Shiveluch ediﬁce (Koulakov et al., 2020). Nevertheless, this is the ﬁrst evidence of the presence and relevance of the structural architecture at the summit of Shiveluch volcano governed by a SW–NE-trending structure and of the alleged intersection between the curved plane and amphitheater of the 1964 sector collapse. g p g The growth rate calculated for the period from July 2018 to August 2019 was 365,000 m3/day (4.22 m3/s), which is signiﬁcantly higher than the long-term average growth rate over the 2001–2012 period (225,000 m3/day or 2.6 m3/s) but comparable to that over the 2001–2003 period (320,000 m3/day or 3.7 m3/s) (Shevchenko et al., 2015). These growth rates are also higher than the average growth rates at other dome-building volcanoes: 26,400 m3/day (0.31 m3/s) at Bezymianny in 1956–2017 (Shevchenko et al., 2020), 37,000 m3/day (0.43 m3/ s) at Santa Maria, Guatemala, in 1922–2000 (Ebmeer et al., 2012), and 173,000 m3/day (2 m3/s) at Mount St. Main Peculiarities of the 2018–2019 Eruptive Episode This ﬁnding, together with the observation of high We ﬁnd that some of the explosion craters are thermally well expressed, while others appear cold. Some craters, such as the central summit crater, are characterized by thermal anomalies around the rim only but not on the crater ﬂoor. A cold crater ﬂoor might have different explanations, both volcanological and observational: the extrusion of cold crystalline material (Mania et al., 2019), the surface crust of cool lava masking hotter material beneath (Sahetapy-Engel and Harris, 2008), the occurrence of a phreatic explosion not followed by the extrusion of lava (Barberi et al., 1992), low thermal activity due to hydrothermal sealing or the tectonic blocking of ﬂuid paths (Matthews et al., 1997; Laiolo et al., 2017), or even satellite techniques that are too inaccurate to detect anomalies with excessively inclined observation geometries or sensors that have an inadequate spatial resolution (Coppola et al., 2016; Blackett, 2017). One more reason that cannot be entirely excluded in this context is that the thermal infrared signal was partially covered and thus biased by the steam of whitish Frontiers in Earth Science \| www.frontiersin.org June 2021 \| Volume 9 \| Article 680051 20 The 2018–2019 Shiveluch Volcano Eruption Shevchenko et al. FIGURE 12 \| SW–NE lineament in relation to the Shiveluch dome’s features. The lineament is shown as the white dashed line. The yellow line outlines the collapse amphitheater, and the dotted lines outline the features (craters and collapse scars). The red arrows show the directions of partial dome collapses. The base images are aerial orthophotos. FIGURE 12 \| SW–NE lineament in relation to the Shiveluch dome’s features. The lineament is shown as the white dashed line. The yellow line outlines the collapse amphitheater, and the dotted lines outline the features (craters and collapse scars). The red arrows show the directions of partial dome collapses. The base images are aerial orthophotos FIGURE 12 \| SW–NE lineament in relation to the Shiveluch dome’s features. The lineament is shown as the white dashed line. The yellow line outlines the collapse amphitheater, and the dotted lines outline the features (craters and collapse scars). The red arrows show the directions of partial dome collapses. The base images are aerial orthophotos. June 2021 \| Volume 9 \| Article 680051 The SW–NE Lineament and Structural Implications Nevertheless, an assessment of the crater locations might provide evidence for an elongated zone or for several parallel alignments striking SW–NE, possibly associated with a fracture zone that is wide rather than focused along a single fault. The thermal anomaly locations (Figures 10E–H; Supplementary Figure S6) are also in agreement with this lineament. Moreover, this structure is parallel to the 1964 collapse direction, to the lineaments of the northern groups of volcanoes described in (Koulakov et al., 2020), to the regional tectonic structures (Kozhurin et al., 2006), and to the Kuril- Kamchatka volcanic arc in general. However, the recent partial dome collapse directions are approximately perpendicular (east- west) to the lineament in the upper sectors of the collapse scars and oblique (SE and SW) to the lineament in the lower sectors (Figure 12B). According to (Tibaldi et al., 2008), the tectonics of a volcano substrate inﬂuence the evolution of its ediﬁce, and normal and strike-slip faults propagating through a volcano induce its instability in the directions perpendicular and oblique to the fault strike, respectively. If we assume a SW–NE-trending, nearly vertical fracture zone to be present, then we may expect a curved intersection arc at the base of Young Shiveluch along the former décollement surface (Figure 13). We note, however, that although multiple lines of evidence support such a structural trend, a critical view regarding the emergence and locations of explosion vents is warranted. We note that many craters are consistent with this lineament, such as the crater on the upper surface of the 2012 lava lobe (Figures 3A, 12A), the summit crater in the center of the dome in 2018 (Figure 3B), and three craters (the central and northern craters and the side crater on the NE amphitheater rim) that originated during the period of study (Figures 3D, 12B), which is why we suggest that this structural feature can affect the formation of these craters. However, neither the radar observations nor the pixel offset method could conﬁrm whether the fracture slides as a strike-slip or dip-slip fault or is an opening fracture. The SW–NE Lineament and Structural Implications IGURE 13 \| Conceptual model of the 2018–2019 eruptive activity (A) The initial stage of increased eruptive activity and the deposition of the new lava lobe (B) The resence of the new lobe hindered the further extrusion of lava and induced endogenous swelling of the eastern ﬂank (C) The explosive eruption and collapse of the t d t Th SW NE li t i h ith th bl k d h d li j ti t th d ll t f FIGURE 13 \| Conceptual model of the 2018–2019 eruptive activity (A) The initial stage of increased eruptive activity and the deposition of the new lava lobe (B) The presence of the new lobe hindered the further extrusion of lava and induced endogenous swelling of the eastern ﬂank (C) The explosive eruption and collapse of the eastern dome sector. The SW–NE lineament is shown with the black dashed line as a projection onto the decollement surface. 2015). Nevertheless, an assessment of the crater locations might provide evidence for an elongated zone or for several parallel alignments striking SW–NE, possibly associated with a fracture zone that is wide rather than focused along a single fault. The thermal anomaly locations (Figures 10E–H; Supplementary Figure S6) are also in agreement with this lineament. Moreover, this structure is parallel to the 1964 collapse direction, to the lineaments of the northern groups of volcanoes described in (Koulakov et al., 2020), to the regional tectonic structures (Kozhurin et al., 2006), and to the Kuril- Kamchatka volcanic arc in general. However, the recent partial dome collapse directions are approximately perpendicular (east- west) to the lineament in the upper sectors of the collapse scars and oblique (SE and SW) to the lineament in the lower sectors (Figure 12B). According to (Tibaldi et al., 2008), the tectonics of a volcano substrate inﬂuence the evolution of its ediﬁce, and normal and strike-slip faults propagating through a volcano induce its instability in the directions perpendicular and oblique to the fault strike, respectively. Conceptual Model for the 2018–2019 Constructive and Destructive Events The complex interrelation and even bidirectional interaction of events responsible for constructing and destroying the volcano (Walter et al., 2015), supporting the idea that regional faults may indeed affect dome growth and collapse processes. 2015). The SW–NE Lineament and Structural Implications This lack of fault displacement evidence might indicate that the activity of this structure is below the detection threshold, that the fault kinematics simply did not activate over the observation period, that the lineament is a fracture zone without clear kinematics, or that it is possibly associated with shear fracturing, as identiﬁed in experimental studies of rock properties simulating shallow depths (Heap et al., The SW–NE Lineament and Structural Implications Helens in 2004–2005 (Schilling et al., 2008). At Shiveluch, we further identiﬁed particularly strong activity during the ﬁrst month of eruptive activity coincident with the extrusion of the lava lobe. While no discrete growth rates for the whole dome could be measured, only loose material accumulated within the scars during the ﬁrst month of eruptive activity, with the deposition rate reaching 670,000 m3/day (7.7 m3/s). Thus, the growth rates during the construction period signiﬁcantly exceed the average rates presented here, illustrating that this volcano is currently very productive compared to its own previous episodes and other volcanoes. The observed SW–NE-trending feature is identiﬁed on the Shiveluch dome only. We could not ﬁnd any evidence for its continuation farther north, such as crossing the amphitheater headwall, implying that this lineament is either a local volcano- tectonic feature or a structure that is ampliﬁed and strongly expressed on the Young Shiveluch ediﬁce only. Similar local tectonic features have been suggested to impact the structural architecture of lava domes at other volcanoes, such as Merapi June 2021 \| Volume 9 \| Article 680051 Frontiers in Earth Science \| www.frontiersin.org 21 The 2018–2019 Shiveluch Volcano Eruption Shevchenko et al. FIGURE 13 \| Conceptual model of the 2018–2019 eruptive activity (A) The initial stage of increased eruptive activity and the deposition of the new lava lobe (B) The presence of the new lobe hindered the further extrusion of lava and induced endogenous swelling of the eastern ﬂank (C) The explosive eruption and collapse of the eastern dome sector. The SW–NE lineament is shown with the black dashed line as a projection onto the decollement surface. Conceptual Model for the 2018–2019 Constructive and Destructive Events In contrast, the 2005 collapse was secondary and was caused by a large explosive eruption that formed a 750-m diameter crater, which opened into the large collapse scar (Ramsey et al., 2012; Shevchenko and Svirid, 2014; Shevchenko et al., 2015). The formation of the large crater on top of the dome during the August 29, 2019 eruption suggests that the sequence of events (primary explosion and secondary collapse) is similar to the sequence of the 2005 destruction episode, though the collapse direction and deposition of the main part of the PDC coincide with the 2010 collapse. The development of Shiveluch volcano is controlled through the interaction of constructive and destructive processes. Destruction follows construction when the dome height reaches a critical elevation and its ﬂanks become oversteepened or when massive extrusive bodies block the vent and endogenous growth destabilizes its structure. Then, construction continues after destruction when the vent opens again, allowing new material to freely extrude onto the surface. Further modeling might help to better understand the details of these relationships, which are governed by stress changes, growing and changing masses and slopes, and material heterogeneities, such as fracture lines and former decollement surfaces. We speculate that if construction processes prevail at Shiveluch, they might lead to the formation of a new stratocone, as happened at Bezymianny (Shevchenko et al., 2020). The prerequisite for this phenomenon has already occurred, i.e., the 2012 lava lobe that stabilized the southern ﬂank. Moreover, as was identiﬁed at Bezymianny, the establishment of a major and centralized summit crater, such as the Shiveluch central crater since 2018 (Figures 3B,D), might indicate the stabilization of a vent and the formation of a stratocone (Shevchenko et al., 2020). At Shiveluch, however, the relevance of the SW–NE structural trend and its intersection with the base of the ediﬁce are of major importance and may govern future episodes of construction and destruction. A previous study (Shevchenko et al., 2015) showed that the two major collapses at Shiveluch in 2005 and 2010 were preceded by periods of endogenous growth, while the dome remained stable during the extrusions of lava lobes due to the constant discharge of the new material. Conceptual Model for the 2018–2019 Constructive and Destructive Events The complex interrelation and even bidirectional interaction of events responsible for constructing and destroying the volcano June 2021 \| Volume 9 \| Article 680051 Frontiers in Earth Science \| www.frontiersin.org 22 The 2018–2019 Shiveluch Volcano Eruption Shevchenko et al. ediﬁce have been identiﬁed for volcanoes worldwide, both on land (Germa et al., 2015) and in the ocean (Sibrant et al., 2014). The relevant observations range from local-scale lava dome growth and collapse (Kelfoun et al., 2021) to large-scale ocean islands, where collapse-related unloading even affects deep crustal magma reservoirs (Manconi et al., 2009). The details of such growth and collapse, however, may be strongly dependent on the local site conditions. At Merapi, it was recently identiﬁed that the mechanism of collapse is controlled by the basement and dome slopes, which also control the directions of destruction episodes (Kelfoun et al., 2021). At Shiveluch, when considering the outlines and directions of the western (originating in 2005) and eastern (originating in 2010) collapse scars, we ﬁnd that these azimuthal directions have a certain longevity. These two scars serve as accumulation centers and erosion pathways; for example, they accumulate deposits from small explosive eruptions and gradual collapses or disintegration of the lava lobes and then repeatedly discharge materials during large destructive events (see Deposit Accumulation and Discharge Within the 2005 and 2010 Collapse Scars). These unconsolidated deposits are rather weak and unstable compared to the lava material composing the main dome ediﬁce; thus, the repeated discharge of loose material from these scars, which can occur due to gravitational processes or earthquakes, can be hazardous even if the dome ﬂanks remain stable. associated with rainfall and/or snowmelt (Figure 13B). Consequently, short-lived explosions formed craters aligned SW–NE. Further, the inability to discharge magma through extrusion led to a pressure buildup that could have caused the large August 29, 2019 explosive eruption and the formation of the central crater, which in turn led to the collapse of the eastern ﬂank; another side crater appeared along the fault lineament after the eruption (Figure 13C). The 2010 collapse was caused by dome instability due to gravitational processes and was followed by a relatively small decompression explosion. This is evidenced by the formation of the large and deep collapse scar that exposed the core of the dome, thereby revealing a small explosion crater at the top (Dvigalo et al., 2011; Shevchenko and Svirid, 2014; Shevchenko et al., 2015). Conceptual Model for the 2018–2019 Constructive and Destructive Events Collapses induced by intrusions and faulting activity at volcanoes have been debated based on experimental simulations (Acocella, 2005) and numerical modeling (Massaro et al., 2020), and evidence that intrusions such as plugs, dikes, and sills may ultimately cause complete or partial ﬂank collapse is growing (Giampiccolo et al., 2020). Conversely, ﬂank collapses may affect the locations and directions of intrusions (Maccaferri et al., 2017), so a complex interplay is likely (Lénat et al., 2012). This idea, together with the results of our remote sensing data analysis, led us to suggest the following conceptual model for the constructive and destructive events of Shiveluch (Figure 13). After intense extrusion (Figure 13A), a massive lava lobe covered the entire central part and SW ﬂank of the dome. The lateral expansion and deformation of the eastern ﬂank (see Lava Dome Deformations Revealed by PIV) started after the extrusion had slowed down, which could be evidence for blockage of the main vent by the massive solidiﬁed lava lobe on top of the dome that prevented the discharge of fresh eruptive material. As magma intruded the core of the dome, the ﬂanks deformed and steepened, and several events of local instability occurred (slumping, hot avalanches, etc.), exposing hot materials and generating thermal anomalies. We hypothesize that, during this stage, when magma did not reach the surface, it began to propagate along the SW-NE fracture system, along the base of Young Shiveluch, or along the intersection between the fracture system and the decollement. As magma approached the surface to the north, close to the amphitheater rim, it could come into contact with meteoric water DATA AVAILABILITY STATEMENT Data supporting the conclusions of this article are available through the GFZ Data Publishing Service at https://doi.org/10.5880/GFZ.2.1. 2021.002 AUTHOR CONTRIBUTIONS Photogrammetric analysis was performed by AVS and VND, radar analysis was performed by MSV (shadow method, TDX processing), EUZ (PIV), and AVS (amplitude image interpretation), and thermal analysis was performed by FM (satellite data) and EUZ (aerial data). TRW was involved in the preparation and realization of the analysis. The 2019 ﬁeld data were collected by AVS, EUZ, MSV, IYS, and SAC, with a ACKNOWLEDGMENTS We acknowledge Sergey Ushakov (IVS) for his assistance in organizing the ﬁeldwork; our colleagues from GFZ and IVS for their discussions; Diego Coppola and Marco Laiolo (MIROVA team, University of Torino) for providing the MODIS-MIROVA dataset; the German Aerospace Center (DLR) for providing the TSX data through proposal GEO1505 and the TDX data through proposal motagh_XTI_LAND6959; the Planet Team, 2017 for providing the Planet data via the Planet application program interface: In Space for Life on Earth. San Francisco, CA. https://api.planet.com the Kamchatka Volcanic Eruption Response Team (KVERT) for releasing eruption chronology data, and the Kamchatka Branch of the Uniﬁed Geophysical Service of the Russian Academy of Sciences for providing the seismic data obtained with large-scale research facilities: “Seismic infrasound array for monitoring Arctic cryolitozone and continuous seismic monitoring of the Russian Federation, neighboring territories and the world.” We also thank the three journal reviewers and the editor for their very detailed reviews and comments that allowed us to further improve this work. CONCLUSION In this article, we analyzed a broad remote sensing dataset to shed light on the growth and collapse of the Shiveluch volcano, Kamchatka. We were able to conduct helicopter overﬂights shortly before and after one of the main explosive eruptions. In this article, we analyzed a broad remote sensing dataset to shed light on the growth and collapse of the Shiveluch volcano, Kamchatka. We were able to conduct helicopter overﬂights shortly before and after one of the main explosive eruptions. June 2021 \| Volume 9 \| Article 680051 Frontiers in Earth Science \| www.frontiersin.org 23 The 2018–2019 Shiveluch Volcano Eruption Shevchenko et al. We compared the high-resolution aerial data with the Pleiades satellite photogrammetric data, derived DEMs of the volcano during the 2018–2019 activity, and complemented the study with satellite radar and thermal data. The likely cause for the explosion and partial dome collapse is a blockage of the vent caused by the massive 2018 lava lobe extrusion, inducing endogenous swelling on the eastern dome ﬂank. This gradually oversteepened the ﬂank and induced gravitational instability. We could identify the presence of an important lineament oriented SW–NE, expressed ﬁrst by a fracture bisecting the dome in 2012 and then by the formation of multiple side craters in the 2018–2019 period, which were not observed before. We discuss whether these craters evolved due to lateral migration of magmas along a basal decollement or along the newly identiﬁed SW–NE fracture system. The new craters all formed within the new deposits located at or close to the former amphitheater crater wall, arguing for a possible inﬂuence of structural reactivation and/ or water percolation involved in the explosion initiation. While most of the craters are cold, we identify thermal anomalies related to the SW–NE structural trend. We also found that the collapse scars originated in 2005 and 2010 retain their outlines very well, being periodically ﬁlled and discharged with unconsolidated material, proximally forming deeply incised valleys that eventually curve into the main amphitheater direction. contribution from AYO and assistance from VND, VAT, and IAB. AVS wrote the manuscript, and TRW, VND, EUZ, MSV, and FM contributed to the writing. FUNDING This is a contribution to VOLCAPSE, a research project funded by the European Research Council under the European Union’s H2020 Program/ERC consolidator grant ERC-CoG 646858. The ﬁeldwork was funded by GFZ (Project X-021-19-01) and by IVS. 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Inﬂuence of Porosity and Groundmass Crystallinity on Dome Rock Strength: a Case Study from Mt. Taranaki, New Zealand. Bull. Volcanol. 80, 35. doi:10.1007/s00445-018-1210-8 Walker, G. P. L. (1984). Downsag Calderas, Ring Faults, Caldera Sizes, and Incremental Caldera Growth. J. Geophys. Res. 89, 8407–8416. doi:10.1029/ JB089iB10p08407 Zorn, E. U., Walter, T. R., Johnson, J. B., and Mania, R. (2020). UAS-based Tracking of the Santiaguito Lava Dome, Guatemala. Sci. Rep. 10, 8644. doi:10. 1038/s41598-020-65386-2 Walter, T. R., Haghshenas Haghighi, M., Schneider, F. M., Coppola, D., Motagh, M., Saul, J., et al. (2019). Complex hazard cascade Culminating in the Anak Krakatau Sector Collapse. Nat. Commun. 10, 4339. doi:10.1038/s41467-019- 12284-5 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. Walter, T. R., Legrand, D., Granados, H. D., Reyes, G., and Arámbula, R. (2013a). Volcanic Eruption Monitoring by thermal Image Correlation: Pixel Offsets Show Episodic Dome Growth of the Colima Volcano. J. Geophys. Res. Solid Earth 118, 1408–1419. doi:10.1002/jgrb.50066 Copyright © 2021 Shevchenko, Dvigalo, Zorn, Vassileva, Massimetti, Walter, Svirid, Chirkov, Ozerov, Tsvetkov and Borisov. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). Frontiers in Earth Science \| www.frontiersin.org June 2021 \| Volume 9 \| Article 680051 REFERENCES 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. Walter, T. R., Ratdomopurbo, A., Subandriyo, N., Aisyah, K. S., Salzer, J., et al. (2013b). Dome Growth and Coulée Spreading Controlled by Surface Morphology, as Determined by Pixel Offsets in Photographs of the 2006 Merapi Eruption. J. Volcanology Geothermal Res. 261, 121–129. doi:10.1016/ j.jvolgeores.2013.02.004 June 2021 \| Volume 9 \| Article 680051 Frontiers in Earth Science \| www.frontiersin.org 27
https://openalex.org/W2063880783	https://open.uct.ac.za/bitstream/11427/13578/1/Matsumiya_myeloid-associated_gene_expression_2014.pdf	English	null	Inflammatory and myeloid-associated gene expression before and one day after infant vaccination with MVA85A correlates with induction of a T cell response	BMC infectious diseases	2,014	cc-by	9,715	Matsumiya et al. BMC Infectious Diseases 2014, 14:314 http://www.biomedcentral.com/1471-2334/14/314 Matsumiya et al. BMC Infectious Diseases 2014, 14:314 http://www.biomedcentral.com/1471-2334/14/314 Open Access Inflammatory and myeloid-associated gene expression before and one day after infant vaccination with MVA85A correlates with induction of a T cell response Magali Matsumiya1, Stephanie A Harris1, Iman Satti1, Lisa Stockdale1, Rachel Tanner1, Matthew K O’Shea1, Michelle Tameris2, Hassan Mahomed3,4, Mark Hatherill2, Thomas J Scriba2, Willem A Hanekom2, Helen McShane2 and Helen A Fletcher1,5 * Correspondence: magali.matsumiya@ndm.ox.ac.uk; helen.fletcher@lshtm.ac.uk 1Jenner Institute, University of Oxford, Old Road Campus Research Building, Oxford, UK 5Current affiliation: London School of Hygiene and Tropical Medicine, Keppel St, London WC1E 7HT, UK Full list of author information is available at the end of the article Background antigen presentation and the development of a stronger immune response [17]. Similar approaches have been used by other groups and are beginning to reveal some of the factors contributing to the variability of the hu- man immune response. The data show the importance of innate pathways in determining the magnitude of subsequent adaptive immune responses with a role for the stress response and gut microbiota in particular [18]. The cellular environment and its modification by vaccines and adjuvants are determined by many factors, underscoring the large variation seen in the immune re- sponse to vaccines across individuals and populations. Tuberculosis (TB) is a major global health problem with an estimated 8.6 million new cases and 1.3 million deaths in 2012 [1]. Effective vaccination is likely to be necessary for the long-term control of the TB epidemic however Bacille Calmette-Guerin (BCG), the only cur- rently licensed vaccine, provides variable protection against pulmonary disease [2]. Despite high BCG cover- age, the incidence of TB remains high in endemic coun- tries. Research efforts into new TB vaccines have focused largely on two strategies; either to modify BCG or replace it with an attenuated strain of Mycobacterium tuberculosis (M.tb), or to improve the protection provided by BCG through prime-boost regimes, often using viral vectors ex- pressing TB antigens to enhance the pool of circulating memory cells primed by vaccination with BCG [3]. Twelve novel TB vaccines are currently in clinical trials, including two in Phase 2b efficacy studies [1]. The results of the first efficacy trial of a novel vaccine, Modified Vaccinia virus Ankara expressing antigen 85A (MVA85A) were pub- lished in early 2013 [4]. Understanding the differences in immune responses in different groups is key to developing targeted approaches to vaccination. The immune system changes with age, with a decrease in response to vaccination often noted in the elderly [19], though a recent study found no de- crease in T cell responses in older adults following a novel MVA-vectored influenza vaccine [20]. Inflamma- tion, apoptosis and immune senescence have all been linked to lower responses to vaccination in this age group [16,21]. As the population of the developed world continues to age, understanding these factors will be im- portant in developing effective vaccination strategies. At the other end of the spectrum are the immature immune systems of infants, which also differ to those of adults [8,22]. Background Although children and infants, particularly in the developing world, are the target population for many vaccines against infectious diseases, the factors under- pinning the immune response to vaccination in this population remain poorly characterized. Understanding the immune response to vaccination in infants living in areas with a high burden of disease, and how this differs from the immune response of healthy, young adults liv- ing in areas of lower disease prevalence –but in whom early testing of vaccines is usually carried out- is there- fore a crucial component in the development and early selection of many of the vaccines in development. y Although the efficacy of boosting BCG with MVA85A was not superior to that of BCG alone, the study dem- onstrated that a trial of a novel TB vaccine is feasible in a high burden setting. Furthermore, the collection of blood samples from all infants during the trial will en- able research into the mechanisms of disease risk and response to vaccination in this setting. Previous studies with MVA85A have shown it to be safe and immuno- genic in several diverse populations including adults without or with latent TB infection in the UK; healthy, latently infected and HIV-infected adults in Africa and healthy adolescents, children and infants in Africa [5-8]. The vaccine shows a quantitatively lower immunogen- icity in African adults and in younger children and in- fants in Africa compared to UK adults [4]. In all trials to date, MVA85A induces antigen-specific Th1 and Th17 cells, believed to be important in protection against tuber- culosis [9-11]. If, as has been suggested [12], low T cell re- sponses to vaccination in this trial contributed to the lack of vaccine efficacy, understanding the mechanisms deter- mining the magnitude of the response to vaccination is important to the development of an improved vaccine. In this study, we have analysed gene expression signa- tures pre- and post-vaccination in infants from the MVA85A Phase 2b efficacy trial who did not develop TB disease during the trial (non-cases) and correlated these changes to the antigen-specific T cell response to vaccin- ation, as measured by IFN-γ ELISpot to Ag85A peptides, in an effort to understand the variability in response to vaccination in this setting. Finally, we compare these findings with previous work performed in UK adults re- ceiving the same vaccine [23], in an effort to characterise some of the differences between these populations. Background Several studies published in the last five years have demonstrated the power of genomics approaches in un- derstanding the molecular mechanisms of the immune response to vaccination [13-16]. Work using the yellow fever vaccine, YF-17D, identified a gene expression sig- nature in circulating leukocytes of vaccinated volunteers shortly after vaccination which could predict the magni- tude of the subsequent CD8+ T cell response [13]. Follow- up studies have yielded further mechanistic insight, showing that activation of the nutrient sensor GCN2 in dendritic cells following vaccination leads to increased Abstract BMC Infectious Diseases 2014, 14:314 http://www.biomedcentral.com/1471-2334/14/314 Matsumiya et al. BMC Infectious Diseases 2014, 14:314 http://www.biomedcentral.com/1471-2334/14/314 Page 2 of 14 Abstract Background: Tuberculosis (TB) remains a global health problem, with vaccination likely to be a necessary part of a successful control strategy. Results of the first Phase 2b efficacy trial of a candidate vaccine, MVA85A, evaluated in BCG-vaccinated infants were published last year. Although no improvement in efficacy above BCG alone was seen, cryopreserved samples from this trial provide an opportunity to study the immune response to vaccination in this population. Methods: We investigated blood samples taken before vaccination (baseline) and one and 28 days post-vaccination with MVA85A or placebo (Candin). The IFN-γ ELISpot assay was performed at baseline and on day 28 to quantify the adaptive response to Ag85A peptides. Gene expression analysis was performed at all three timepoints to identify early gene signatures predictive of the magnitude of the subsequent adaptive T cell response using the significance analysis of microarrays (SAM) statistical package and gene set enrichment analysis. Results: One day post-MVA85A, there is an induction of inflammatory pathways compared to placebo samples. Modules associated with myeloid cells and inflammation pre- and one day post-MVA85A correlate with a higher IFN-γ ELISpot response post-vaccination. By contrast, previous work done in UK adults shows early inflammation in this population is not associated with a strong T cell response but that induction of regulatory pathways inversely correlates with the magnitude of the T cell response. This may be indicative of important mechanistic differences in how T cell responses develop in these two populations following vaccination with MVA85A. Conclusion: The results suggest the capacity of MVA85A to induce a strong innate response is key to the initiation of an adaptive immune response in South African infants but induction of regulatory pathways may be more important in UK adults. Understanding differences in immune response to vaccination between populations is likely to be an important aspect of developing successful vaccines and vaccination strategies. Trial registration: ClinicalTrials.gov number NCT00953927 Keywords: Tuberculosis, Vaccine, Innate immunity, Transcriptomics, MVA85A Keywords: Tuberculosis, Vaccine, Innate immunity, Transcriptomics, MVA85A © 2014 Matsumiya 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. Matsumiya et al. Ex-vivo IFN-γ ELISpot assay Ex-vivo IFN-γ ELISpot assay γ p y The ex-vivo IFN-γ ELISpot assay was performed on thawed PBMC samples collected pre-vaccination and 28 days post- Table 2 Differentially expressed genes 1 day post- vaccination: MVA85A vs Candin PROBE_ID SYMBOL Fold change AveExpr adj.P.Val ILMN_1791759 CXCL10 3.2 7.18 0.04 ILMN_1799848 ANKRD22 2.61 6.65 0.04 ILMN_1656310 INDO 2.53 5.68 0.04 ILMN_2114568 GBP5 2.52 8.7 0.02 ILMN_2132599 ANKRD22 2.46 7.09 0.04 ILMN_3239965 IDO1 2.35 6.17 0.04 ILMN_3247506 FCGR1C 2.13 6.59 0.04 ILMN_1782487 LOC400759 1.96 5.38 0.01 ILMN_2066849 FAM26F 1.89 5.99 0.04 ILMN_1809086 XRN1 1.74 6.83 0.04 ILMN_2053527 PARP9 1.67 6.81 0.04 ILMN_1769520 UBE2L6 1.61 11.07 0.04 ILMN_1707979 CARD17 1.57 5.07 0.04 ILMN_2326509 CASP1 1.57 8.86 0.04 ILMN_1671452 MRPL44 1.5 6.05 0.04 ILMN_1700671 ETV7 1.49 4.83 0.04 ILMN_1678454 CASP4 1.48 9.83 0.04 ILMN_1715401 MT1G 1.43 4.78 0.01 ILMN_3238525 CARD17 1.35 4.6 0.04 ILMN_1693287 POMP 1.32 9.68 0.04 ILMN_1761159 ESYT1 −1.34 8.78 0.05 ILMN_1693410 BRI3BP −1.37 6.56 0.04 ILMN_1774828 VEZT −1.4 5.72 0.04 ILMN_3236036 LOC283663 −1.41 5.39 0.04 ILMN_3256478 LOC100129034 −1.41 5.38 0.04 ILMN_3240997 ARAP3 −1.43 5.7 0.04 ILMN_1767612 BBS2 −1.44 5.6 0.04 ILMN_1775542 FAIM3 −1.45 9.66 0.04 ILMN_3251155 PCBP2 −1.48 5.49 0.01 ILMN_1772876 ZNF395 −1.5 6.21 0.04 ILMN_3305938 SGK1 −1.56 6.59 0.04 ILMN_1731064 CABC1 −1.59 6.1 0.02 ILMN_3229324 SGK1 −1.6 6.3 0.04 Unstimulated whole blood genes identified using the R package limma Table 2 Differentially expressed genes 1 day post- vaccination: MVA85A vs Candin The samples used in this study were selected from a subset of 100 infants who had a small blood sample taken one day post-vaccination. The samples were se- lected to exclude cases (infants who went on to develop TB disease) and controls (selected to demographically match the cases), which will be used in a future study looking at correlates of risk of TB disease. Therefore none of the samples used in this study were taken from infants diagnosed with TB during the course of the trial. Cells were collected 0–7 days pre- and 28 days post- vaccination with MVA85A/placebo in cell preparation tubes with sodium heparin (CPT; Vacutainer; BD) and PBMC separated and cryopreserved. The cells were thawed and stimulated as detailed below. One day post-vaccination, 50-300 μL of whole blood was collected directly via heel prick into a tube filled with RLT buffer (RNeasy kit, Qiagen) containing 10 μL/mL β-mercaptoethanol using a BD Quikheel Lancet. The sample was immediately frozen and RNA extracted as detailed below. Unstimulated whole blood, genes identified using the R package limma. Origin of samples Samples used in these experiments were cryopreserved peripheral blood mononuclear cells (PBMC) or whole Page 3 of 14 Matsumiya et al. BMC Infectious Diseases 2014, 14:314 http://www.biomedcentral.com/1471-2334/14/314 Matsumiya et al. BMC Infectious Diseases 2014, 14:314 http://www.biomedcentral.com/1471-2334/14/314 blood in RNA lysis buffer from a double-blind, rando- mised, placebo-controlled Phase 2b efficacy trial of a candidate TB vaccine, MVA85A, in BCG-vaccinated, HIV- negative South African infants (South African National Clinical Trials Register DOH-27-0109-2654, Clinical- Trials.gov NCT00953927). Infants were randomized to receive either one dose of MVA85A (1 × 108 plaque forming units in 0.06 mL) or an equal volume of Candida skin test antigen (Candin, AllerMed, USA) as placebo at 4–6 months of age [4]. The trial was approved by the University of Cape Town Faculty of Health Sciences Human Research Ethics Committee, Oxford University Tropical Research Ethics Committee, and the Medicines Control Council of South Africa. Parents or legal guard- ians provided written, informed consent. Storage of sam- ples for exploratory immunological analyses was fully ethically approved. pelleted, supernatants discarded and resuspended in 10 mL R10 with 20 μL Benzonase (Merck Chemicals Ltd.) and rested overnight at 37°C, 5% CO2. PBMC were counted on a Casy Counter (Roche) and split into ap- propriate volumes for each assay. Not all assays were performed on all samples. Cell thawing Cryopreserved PBMC were rapidly thawed in a 37°C waterbath and transferred to a 15 mL Falcon tube con- taining 10 mL R10 (RPMI, 10% FCS, 1% L-glutamine, 1% Pen-Strep and 1% sodium pyruvate). PBMC were Table 1 Samples processed for each assay as part of this study Assay Samples processed No. of infants GEX: unstimulated PBMC days 0 and 28 60 30 GEX: whole blood day 1 82 82 GEX: Ag85A peptide-stimulated PBMC days 0 and 28 20 10 IFN-γ ELISpot (unstim, PHA, 85A) days 0 and 28 99 50 RNA-Sequencing: unstimulated PBMC days 0 and 28 12 6 GEX: gene expression analysis using Illumina Human HT-12 v4 microarray beadchips. Day 0: day of vaccination with MVA85A/Candin placebo. Table 1 Samples processed for each assay as part of this study Table 1 Samples processed for each assay as part of thi t d Page 4 of 14 Matsumiya et al. BMC Infectious Diseases 2014, 14:314 http://www.biomedcentral.com/1471-2334/14/314 vaccination as previously described [23]. 3 × 105 PBMC were stimulated in triplicate with a pool of Ag85A peptides, consisting of 66 15mers, overlapping by 10 amino acids (2 μg/ml) (Peptide Protein Research). vaccination as previously described [23]. 3 × 105 PBMC were stimulated in triplicate with a pool of Ag85A peptides, consisting of 66 15mers, overlapping by 10 amino acids (2 μg/ml) (Peptide Protein Research). Blood in RLT buffer was thawed and RNA extracted using the RNeasy kit (Qiagen) according to manufac- turer’s instructions, including the optional protocol for DNA digest (RNase-free DNase kit, Qiagen). The proto- col was modified in the following way for the heelprick samples due to the small volume of whole blood col- lected: 80% ethanol was added to precipitate the RNA (rather than the recommended 70%) and an extra wash with 350 μL RW1 buffer was performed prior to DNA digest. Gene expression assays PBMC from pre-vaccination and 28 days post-vaccination were incubated for 12 hours with either R10 media alone (unstimulated) or pooled Ag85A peptides as described for the ELISpot (2 μg/mL). After 12 hours supernatant was removed and the PBMC resuspended in 350uL RLT buffer (Qiagen) containing 10 μL/mL β-mercaptoethanol and frozen at -20C. Messenger RNA was amplified from the total RNA using the Illumina Totalprep kit (Ambion) according to manufacturer’s instructions. RNA quantity and quality was assessed using a Nanodrop ND-1000 Spectrophotometer and an Agilent Bioanalyser (Agilent RNA 6000 Nano Kit). Figure 1 Heatmap of differential gene expression 1 day post vaccination. Heatmap showing changes in gene expression one day post- vaccination with either MVA85A or a Candin placebo. Colors at the top show vaccination: MVA85A (blue) or Candin (red). Genes were selected on the basis of differential expression between the two groups (fdr < 0.05). Clustering using euclidean distance and average clustering methods. Red indicates up-regulated mRNA, blue indicates downregulated. Figure 1 Heatmap of differential gene expression 1 day post vaccination. Heatmap showing changes in gene expression one day post- vaccination with either MVA85A or a Candin placebo. Colors at the top show vaccination: MVA85A (blue) or Candin (red). Genes were selected on the basis of differential expression between the two groups (fdr < 0.05). Clustering using euclidean distance and average clustering methods. Red indicates up-regulated mRNA, blue indicates downregulated. Figure 1 Heatmap of differential gene expression 1 day post vaccination. Heatmap showing changes in gene expression one day post- vaccination with either MVA85A or a Candin placebo. Colors at the top show vaccination: MVA85A (blue) or Candin (red). Genes were selected on the basis of differential expression between the two groups (fdr < 0.05). Clustering using euclidean distance and average clustering methods. Red indicates up-regulated mRNA, blue indicates downregulated. Page 5 of 14 Matsumiya et al. BMC Infectious Diseases 2014, 14:314 http://www.biomedcentral.com/1471-2334/14/314 750 ng amplified cRNA was labeled and hybridized to Illumina Human HT-12 v4 beadchips as specified in the manufacturer’s instructions. Beadchips were scanned on an Illumina iScan machine and data extracted using the GenomeStudio software. used to perform background correction and normalization and the gene list was filtered using the gene filter pack- age to remove genes with an expression IQR < 0.3 (log2 transformed). Lists of differentially expressed genes were generated using limma (p-value cut-off of 0.05 after Benjamini-Hochberg correction [25-27]). Ex vivo IFN-γ ELISpot assay Phytohaemagglutinin (PHA) (Sigma) was used as a positive control and unstimulated wells were used as a measure of background IFN-γ production. Results are reported as spot forming cells (SFC) per million PBMC, calculated by sub- tracting the mean of the unstimulated wells from the mean of triplicate antigen wells and correcting for the number of PBMC in the well. An ELISpot response was deemed posi- tive if the average count in the positive control wells was at least twice that in the negative control wells and at least 5 spots more than the negative control wells. RNA sequencing G f h h Genes for which the reads per kilobase per million value (rpkm) was <1 in over 40% of samples were excluded. RPKM was calculated as (10^9C)/(NL) where C = number of reads uniquely mapped to transcript, N = total number of uniquely mapped reads in sample and L = maximum length of transcript. Raw counts were then analysed in the R package limma and converted to log2 transformed counts per million. These values were compared to the gene ex- pression values for the equivalent samples obtained by microarray analysis. Gene expression assays RNA-Seq was performed on pre- and post-vaccination PBMC from 6 infants (12 samples). Total RNA was sent to the Beijing Genomics Institute (BGI). Libraries were constructed using the TruSeq kit (Illumina) and se- quenced on a HiSeq2000 sequencer, using paired-end reads of 90 bp and 30 M sequencing depth. Quality con- trol was performed and the reads aligned to the genome hg19, downloaded from UCSC (http://genome.ucsc.edu/), using SOAPaligner2.21 with the following constraints: maximum number of mismatches allowed on one read is 5 bp, no gaps allowed, only repeat hits reported. The package Significance Analysis of Microarrays (SAM) was used to rank genes correlating with the IFN- γ ELISpot response according to the strength of the cor- relation [28-30]. The ranked gene list was then inputted into Broad Institute gene set enrichment analysis programme (GSEA) [31,32] as an externally supplied preranked list. The reference gene set used was the Blood Transcrip- tion Modules compiled by Li et al. [33]. The significance of module enrichment was assessed by permutation in the GSEA program. All heatmaps were generated in R, using Euclidean distance and average linkage as methods to calculate the distance matrix and hierarchical clustering respectively. Where correlations are shown, these use the Pearson product–moment correlation coefficient. Results and discussion Table 1 shows the number of samples used in each assay in this study. Accession codes associated with the immune response (differentially expressed genes are shown in Table 2 with genes asso- ciated with the immune response highlighted in bold). Hierarchical clustering showed that infants fall into three clusters with a mixed MVA85A/Candin cluster exhibiting a moderate level of expression of inflamma- tory genes (Figure 1). This analysis shows MVA85A- vaccinated infants exhibit a more inflammatory gene expression profile than those in the placebo group how- ever the range is large in both groups and there is an overlap between the two groups. This overlap could be due to the immunomodulatory properties of Candin, which induces inflammation and may lead to functional reprogramming of monocytes associated with protec- tion from subsequent infection [34]. Gene expression omnibus: GSE56559 (day 1 heelprick, South African infants), GSE56561 (PBMC, South African infants) GSE40719 (UK adults). Illumina microarray The R package arrayQualityMetrics [24] was used to as- sess sample quality. 82 heelprick samples and all PBMC samples passed quality control. The R package limma was 0 10 20 30 40 50 0 10 20 30 40 50 6 7 8 9 10 0 5 10 15 20 25 6.5 7.0 7.5 8.0 8.5 9.0 9.5 Figure 2 Stimulation of PBMC with Ag85A peptide pools. (a) IFN-γ ELISpot of PBMC taken pre- or post-vaccination in MVA85A or candin- vaccinated infants. Responses are significantly higher in the MVA85A group, 4 weeks post-vaccination (Wilcoxon test, p < 0.05). b,c. STAT1 expression in unstimulated (b) and 85A peptide-stimulated (c) PBMC. Squares = pre-vaccination, circles = 28 days post-vaccination; red = candin, blue = MVA85A. Dashed lines indicate a positive ELISpot response (x = 15) and elevated expression of STAT1 (y = 9.5). 0 10 20 30 40 50 6 7 8 9 10 0 5 10 15 20 25 6.5 7.0 7.5 8.0 8.5 9.0 9.5 0 10 20 30 40 50 Figure 2 Stimulation of PBMC with Ag85A peptide pools. (a) IFN-γ ELISpot of PBMC taken pre- or post-vaccination in MVA85A or candin- vaccinated infants. Responses are significantly higher in the MVA85A group, 4 weeks post-vaccination (Wilcoxon test, p < 0.05). b,c. STAT1 expression in unstimulated (b) and 85A peptide-stimulated (c) PBMC. Squares = pre-vaccination, circles = 28 days post-vaccination; red = candin, blue = MVA85A. Dashed lines indicate a positive ELISpot response (x = 15) and elevated expression of STAT1 (y = 9.5). Page 6 of 14 Matsumiya et al. BMC Infectious Diseases 2014, 14:314 http://www.biomedcentral.com/1471-2334/14/314 PBMC stimulated for 12 hours with Ag85A peptide pools, genes identified using the R package limma. Genes in italics have an adjusted p value 0.05 < p < 0.2). MC stimulated for 12 hours with Ag85A peptide pools, genes identified using the R package limma. Genes in italics have an adjusted p with Ag85A peptide pools, genes identified using the R package limma. Genes in italics have an adjusted p value 0.05 < p < 0.2). MVA85A induces an inflammatory signature one day post-vaccination Illumina microarray gene expression analysis of 82 whole blood samples taken one day post-vaccination (37 MVA85A, 45 Candin placebo) identified 32 differ- entially expressed genes. These genes were largely Table 3 Differentially expressed genes following Ag85A stimulation post-MVA85A vaccination Vaccinated infants: day 28- day-7 Day 28 samples: vaccinated- placebo SYMBOL logFC Fold change adj.P.Val SYMBOL logFC Fold change adj.P.Val LOC400759 2.04 4.11 0.0047 LOC400759 2.71 6.55 0.012 GBP5 2.16 4.46 0.0079 STAT1 2.69 6.46 0.012 LOC730249 1.93 3.82 0.0079 STAT1 2.31 4.96 0.012 WARS 1.37 2.58 0.0079 CXCL10 3.41 10.64 0.015 ANKRD22 1.67 3.19 0.018 CCL8 4.02 16.21 0.027 GBP4 1.4 2.63 0.018 GBP4 2.19 4.55 0.027 WARS 1.25 2.38 0.018 CXCL9 1.95 3.86 0.027 TAP1 0.79 1.73 0.0234 STAT1 1.78 3.44 0.027 AIM2 1.05 2.07 0.0251 WARS 1.69 3.23 0.027 PSME2 0.72 1.65 0.0251 IFI35 1.49 2.8 0.027 CXCL9 1.63 3.09 0.0259 WARS 1.73 3.31 0.03 GBP1 1.9 3.72 0.0349 GBP1 2.86 7.28 0.031 STAT1 1.15 2.22 0.0354 PARP9 1.56 2.94 0.031 STAT1 1.32 2.49 0.0476 FBXO6 1.84 3.57 0.037 STAT1 0.92 1.89 0.0746 PARP9 1.46 2.75 0.051 GBP1 1.87 3.66 0.0911 PARP14 1.24 2.36 0.051 CD38 0.8 1.74 0.0925 PSME2 1.12 2.17 0.051 CXCL10 2.3 4.92 0.1295 GBP5 2.65 6.3 0.058 LAP3 1.13 2.19 0.1295 EPSTI1 1.51 2.85 0.058 CEACAM1 0.96 1.95 0.1412 GBP1 3.01 8.05 0.062 PARP14 0.71 1.63 0.1412 IFNG 2.17 4.51 0.062 IFNG 1.67 3.19 0.1599 P2RX7 1.77 3.42 0.062 SAMD9L 1.37 2.58 0.062 PTER 1.18 2.27 0.062 UBE2L6 1.14 2.21 0.076 IFIT3 2.35 5.09 0.08 SNHG5 −1.17 −2.25 0.08 LOC730249 2.32 5 0.082 PLA2G4C 1.39 2.61 0.082 PBMC stimulated for 12 hours with Ag85A peptide pools, genes identified using the R package limma. Genes in italics have an adjusted p value 0.05 < p < 0.2). Table 3 Differentially expressed genes following Ag85A stimulation post-MVA85A vaccination Matsumiya et al. BMC Infectious Diseases 2014, 14:314 http://www.biomedcentral.com/1471-2334/14/314 Page 7 of 14 Page 7 of 14 Figure 3 Modular analysis of genes associated with a higher response to vaccination. Genes whose expression correlate with the IFNg ELISpot response were identified using the R package SAM (a) and ranked in order of their score. The ranked list was then analysed in GSEA using the Blood Transcription Modules compiled by Li et al. as the reference gene set [33]. MVA85A induces an inflammatory signature one day post-vaccination The results of the analysis for genes expressed in PBMC taken pre-vaccination or whole blood taken one day post-vaccination are shown (b). Length of the bar shows Normalised enrichment score for each module, number in the bars indicates genes in the test list present ineach reference set. Colour saturation indicates genes present as a percentage of total genes within the module (signal). Red modules are positively associated with ELISpot response, blue modules are negatively associated. Figure 3 Modular analysis of genes associated with a higher response to vaccination. Genes whose expression correlate with the IFNg ELISpot response were identified using the R package SAM (a) and ranked in order of their score. The ranked list was then analysed in GSEA using the Blood Transcription Modules compiled by Li et al. as the reference gene set [33]. The results of the analysis for genes expressed in PBMC taken pre-vaccination or whole blood taken one day post-vaccination are shown (b). Length of the bar shows Normalised enrichment score for each module, number in the bars indicates genes in the test list present ineach reference set. Colour saturation indicates genes present as a percentage of total genes within the module (signal). Red modules are positively associated with ELISpot response, blue modules are negatively associated. Matsumiya et al. BMC Infectious Diseases 2014, 14:314 http://www.biomedcentral.com/1471-2334/14/314 Page 8 of 14 IDO1 MAFB KYNU IFI30 ALDH2 FCER1G SIRPA SERPINA1 PLAUR LYN SERPINB2 TLR8 PILRA LILRA6 FES C5AR1 BST1 AQP9 APOB48R ANPEP TLR6 MYD88 KCNJ2 ITGAX VNN1 TNFRSF1B TMEM176B PTGS2 PGD LYZ HHEX EMR1 EMILIN2 DPYD DOK3 DOCK5 C1orf162 C19orf59 BCL6 VNN3 VNN2 NLRP12 NFE2 FPR2 FPR1 FCGR2A ST6GALNAC2 SLC22A4 PROK2 NCF4 KCNJ15 EMR3 BASP1 G0S2 TNFAIP6 TNFAIP3 SLAMF7 NFKBIA IL1B DUSP1 CXCL2 CXCL1 CCL20 IL8 CXCL6 CXCL5 CXCL13 CCL7 CCL23 CCL2 CCL19 UBASH3A PTPRCAP IL32 KLRB1 GZMB GNLY CCL5 STAT4 KLRF1 KLRD1 IL2RB GPR56 TMOD1 SEMA4A RXRA PLXNB2 KLF1 GATA1 FES EPB42 TLR8 TLR5 P2RY13 LILRB3 LILRB2 FPR1 DYSF CSF3R FES PILRA LILRA6 FGR ALOX5 BST1 APOB48R TLR6 MYD88 KCNJ2 HSPA6 AQP9 RSAD2 OAS3 IRF7 DDX58 CXCL10 C1QB BCL3 ANXA3 SERPING1 HERC5 MX2 LAMP3 IFIT3 IFIT2 HLX CCL8 ATF3 a b Figure 4 Heatmaps of BTMs associated with a higher response to vaccination. Heatmaps show expression of genes for MVA85A infants in unstimulated PBMC taken pre-vaccination (a) or whole blood taken one day post-vaccination (b). MVA85A induces an inflammatory signature one day post-vaccination The colour coding along t the heatmap shows responder (blue) and non-responder (black) infants as measured by IFN-γ ELISpot. Genes are arranged by module IDO1 MAFB KYNU IFI30 ALDH2 FCER1G SIRPA SERPINA1 PLAUR LYN SERPINB2 TLR8 PILRA LILRA6 FES C5AR1 BST1 AQP9 APOB48R ANPEP TLR6 MYD88 KCNJ2 ITGAX VNN1 TNFRSF1B TMEM176B PTGS2 PGD LYZ HHEX EMR1 EMILIN2 DPYD DOK3 DOCK5 C1orf162 C19orf59 BCL6 VNN3 VNN2 NLRP12 NFE2 FPR2 FPR1 FCGR2A ST6GALNAC2 SLC22A4 PROK2 NCF4 KCNJ15 EMR3 BASP1 G0S2 TNFAIP6 TNFAIP3 SLAMF7 NFKBIA IL1B DUSP1 CXCL2 CXCL1 CCL20 IL8 CXCL6 CXCL5 CXCL13 CCL7 CCL23 CCL2 CCL19 a UBASH3A PTPRCAP IL32 KLRB1 GZMB GNLY CCL5 STAT4 KLRF1 KLRD1 IL2RB GPR56 TMOD1 SEMA4A RXRA PLXNB2 KLF1 GATA1 FES EPB42 TLR8 TLR5 P2RY13 LILRB3 LILRB2 FPR1 DYSF CSF3R FES PILRA LILRA6 FGR ALOX5 BST1 APOB48R TLR6 MYD88 KCNJ2 HSPA6 AQP9 RSAD2 OAS3 IRF7 DDX58 CXCL10 C1QB BCL3 ANXA3 SERPING1 HERC5 MX2 LAMP3 IFIT3 IFIT2 HLX CCL8 ATF3 b Figure 4 Heatmaps of BTMs associated with a higher response to vaccination. Heatmaps show expression of genes for MVA85A-vaccinated infants in unstimulated PBMC taken pre-vaccination (a) or whole blood taken one day post-vaccination (b). The colour coding along the top of the heatmap shows responder (blue) and non-responder (black) infants as measured by IFN-γ ELISpot. Genes are arranged by module (right). b Figure 4 Heatmaps of BTMs associated with a higher response to vaccination. Heatmaps show expression of genes for MVA85A-vaccinated infants in unstimulated PBMC taken pre-vaccination (a) or whole blood taken one day post-vaccination (b). The colour coding along the top of the heatmap shows responder (blue) and non-responder (black) infants as measured by IFN-γ ELISpot. Genes are arranged by module (right). Figure 4 Heatmaps of BTMs associated with a higher response to vaccination. Heatmaps show expression of genes for MVA85A-vaccinated infants in unstimulated PBMC taken pre-vaccination (a) or whole blood taken one day post-vaccination (b). The colour coding along the top of the heatmap shows responder (blue) and non-responder (black) infants as measured by IFN-γ ELISpot. Genes are arranged by module (right). Page 9 of 14 Matsumiya et al. BMC Infectious Diseases 2014, 14:314 http://www.biomedcentral.com/1471-2334/14/314 The immune response to stimulation with Ag85A peptides The antigen-specific immune response to Ag85A was assessed by IFN-γ ELISpot and Illumina microarray gene expression analysis. Infants vaccinated with MVA85A had a significantly higher post-vaccination Ag85A-specific ELISpot response than the Candin group (Figure 2a). MVA85A induces an inflammatory signature one day post-vaccination Differentially expressed genes between the vaccine and placebo groups, and pre- and post-vaccination time points, are shown in Table 3. The genes induced follow- ing Ag85A peptide stimulation are all associated with the STAT1 pathway and exhibit a highly correlated pat- tern of expression. Furthermore, upregulation of this pathway occurred in infants who also had a detectable Ag85A-specific T cell response by IFN-γ ELISpot assay but not in unstimulated cells or infants who received the candin placebo (Figure 2b,c). This observation suggests that, in this population, gene expression analysis does not add substantial information to that measured by the IFN- γ ELISpot assay in capturing the response to Ag85A pep- tide stimulation following MVA85A vaccination. suggested that low or absent responses to MVA85A may be one explanation for the lack of efficacy observed in the trial [12]. Further analysis of cases and controls is under way to address this question however, in this smaller study, we next investigated some of the mecha- nisms underlying the magnitude of the adaptive immune response which develops following MVA85A vaccination. Gene expression signatures correlating with immunogenicity differ in South African infants and UK adults We have previously described changes in gene expression in unstimulated PBMC from UK adults vaccinated with the same regime: a BCG prime followed by an MVA85A boost [23,42]. We therefore wanted to compare the obser- vations made in these two different populations. In the adults, there was no placebo and PBMC were collected for gene expression analysis pre-, two and seven days post-MVA85A. Comparing gene expression to the magni- tude of the induced T cell response showed a positive Table 4 Comparison of differentially expressed genes in South African infants and UK adults post-MVA85A Fold change SYMBOL RSA infants UK adults CXCL10 3.2 6.5 ANKRD22 2.6 2.0 INDO 2.5 1.3 GBP5 2.5 2.2 ANKRD22 2.5 1.7 IDO1 2.4 1.4 FCGR1C 2.1 1.7 LOC400759 2.0 2.1 FAM26F 1.9 1.8 XRN1 1.7 1.0 PARP9 1.7 1.8 UBE2L6 1.6 1.6 SGK1 −1.6 1.1 CABC1 −1.6 −1.3 CARD17 1.6 1.2 CASP1 1.6 1.7 SGK1 −1.6 1.1 MRPL44 1.5 1.1 ZNF395 −1.5 −1.3 ETV7 1.5 1.0 CASP4 1.5 1.2 PCBP2 −1.5 −1.2 FAIM3 −1.5 −1.3 South African infants: unstimulated whole blood taken 1 day post-vaccination. UK adults: unstimulated PBMC taken 2 days post-vaccination. All genes are differentially expressed between MVA85A and candin vaccinated infants (fdr < 0.05); genes in bold are also significantly different between day 0 and day 2 in UK adults vaccinated with MVA85A. Table 4 Comparison of differentially expressed genes in South African infants and UK adults post-MVA85A The ratio of myeloid to lymphoid cells has previously been associated with differences in TB disease risk in HIV-infected South African adults and susceptibility to malaria and influenza in other cohorts [37-40] and this may be another example of an outcome associated with this ratio. The gene expression profiles associated with responder infants are present pre-vaccination and show a strong overlap pre- and one day post vaccination. This suggests the baseline inflammatory profile of the infant, including the differing proportion of circulating leukocytes, is key to determining the response to vac- cination. This may be influenced by genetic influences on innate immunity or environmental exposure preced- ing vaccination, including the response to BCG, which all infants received at birth. As MVA preferentially in- fects myeloid cells [41], a higher proportion of myeloid cells may lead to overall increased viral expression of Ag85A protein in infants with higher frequencies of myeloid cells. Matsumiya et al. BMC Infectious Diseases 2014, 14:314 http://www.biomedcentral.com/1471-2334/14/314 Matsumiya et al. BMC Infectious Diseases 2014, 14:314 http://www.biomedcentral.com/1471-2334/14/314 Matsumiya et al. BMC Infectious Diseases 2014, 14:314 http://www.biomedcentral.com/1471-2334/14/314 Page 10 of 14 subsequently defined infants as responders or non- responders. An ELISpot response was deemed positive if the average count in the positive control wells was at least twice that in the negative control wells and at least 5 spots more than the negative control wells [36]. Sets of correlating genes pre-vaccination and one day post- vaccination were generated and ranked according to their significance score (Figure 3a). This ranked list of genes was then analysed using the Broad Institute Gene Set Enrichment Analysis PreRanked function, using the Blood Transcription Modules defined by Li et al. as a reference gene set [33]. Pre-vaccination, responder in- fants have an over-representation of genes enriched in monocytes, activated dendritic cells and neutrophils as well chemokines and inflammatory pathways (Figure 3b). One day post-vaccination, there is a negative association between lymphoid cells and the subsequent development of an ELISpot response (Figure 3c). Moreover, there is positive enrichment of gene sets associated with myeloid cells, inflammation and an antiviral response. The expres- sion pattern of these clusters and their relationship to the ELISpot response is shown in heatmaps in Figure 4. Higher expression of genes associated myeloid cells and inflammation pre- and 1 day post-vaccination are both as- sociated with the development of an antigen-specific T cell response to vaccination with MVA85A, suggesting the ability of MVA to induce a strong innate response is key to its function as a vaccine vector in this population. Add- itionally, higher expression of genes associated with activa- tion of lymphoid cells such as NK cells and cytotoxic T cells one day post-vaccination is associated with an absent ELISpot response 28 days later. In a subset of samples, the RNA extracted from the PBMC collected pre- and 28 days post-MVA85A was also measured by RNA Sequencing, as part of a pilot project for future studies. Gene expression values as measured in- dependently by these two methods are highly correlated (Figure 5), providing a technical validation for this result. Myeloid cells and inflammation are associated with a higher ELISpot response The following analyses were performed using only sam- ples from infants vaccinated with MVA85A, to further investigate the mechanisms underlying the immune re- sponse to this vaccine. The R package Significance Ana- lysis of Microarrays (SAMR) was used to identify genes whose expression correlated with the Ag85A-specific T cell frequencies measured 28 days post-vaccination by IFN-γ ELISpot assay. SAM identifies genes significantly correlat- ing with a continuous response variable, in this case the IFN-γ ELISpot, and outputs a positive and negative set of genes based on the strength of the correlation of each gene with higher (positive) or lower (negative) values of the response phenotype [29,35]. Since the IFN-γ ELISpot responses in this study were very low, we have A proportion of infants did not respond to antigenic stimulation with Ag85A peptides following MVA85A vaccination. This lack of response was observed both by IFN-γ ELISpot and gene expression analysis. It has been 4 6 8 10 12 14 0 2 4 6 8 10 12 Microarray RNA-Seq Number of XY pairs=74 Pearson's r=0.77 95% confidence interval=0.66-0.85 P-value (two-tailed)=4.4e-16 Figure 5 Comparison of gene expression values measured by beadchip microarray and RNA sequencing. Plot of median values from the RNA- Seq and microarray data of 12 unstimulated PBMC samples for 74 genes used in the modular analysis. Significance assessed using Pearson’s correlation. Number of XY pairs=74 Pearson's r=0.77 95% confidence interval=0.66-0.85 P-value (two-tailed)=4.4e-16 Figure 5 Comparison of gene expression values measured by beadchip microarray and RNA sequencing. Plot of me d d f l d l f d h d l l f d Figure 5 Comparison of gene expression values measured by beadchip microarray and RNA sequencing. Plot of median values from the RNA- Seq and microarray data of 12 unstimulated PBMC samples for 74 genes used in the modular analysis. Significance assessed using Pearson’s correlation. South African infants: unstimulated whole blood taken 1 day post-vaccination. UK adults: unstimulated PBMC taken 2 days post-vaccination. All genes are differentially expressed between MVA85A and candin vaccinated infants (fdr < 0.05); genes in bold are also significantly different between day 0 and day 2 in UK adults vaccinated with MVA85A. Gene expression signatures correlating with immunogenicity differ in South African infants and UK adults UK adults have a stronger innate response to the vac- cine, however the magnitude of this response does not predict the magnitude of the antigen-specific response. By contrast, in the South African infants the magnitude of inflammatory gene expression one day post-MVA85A correlated with the ELISpot response at week 4. Further investigation of these mechanisms and how they differ between the different populations in which vaccines are tested and deployed may prove important in future vac- cine development strategies. association with TLR1 expression at baseline and a nega- tive correlation with regulatory genes including STAT5B and CTLA4 at day 2 [42]. Table 4 shows the genes differ- entially expressed in infants one day post-MVA85A (com- pared to a placebo group at the equivalent timepoint). Genes in bold were also differentially expressed in UK adults two days post-MVA85A compared to baseline and fold changes are shown for both comparisons. In both populations, CXCL10 had the highest fold change at the innate timepoint (one or two days post-MVA85A). One interesting difference is the gene encoding indoleamine 2,3-dioxygenase (probes INDO and IDO1) which is highly differentially expressed in the infants but not the adults. two populations and in the pathways that determine the adaptive immune response. However, it is impossible to determine whether the differences observed are attribut- able to age or to genetic or environmental differences be- tween the populations. Furthermore, the post-vaccination timepoint differed in both cell type (whole blood in the in- fants, PBMC in adults) and time taken (one or two days post-MVA85A). PBMC were used in the pre-vaccination timepoint in both studies. Despite this, the similarities in the list of differentially expressed genes suggest these com- parisons are meaningful and warrant further investigation. UK adults have a stronger innate response to the vac- cine, however the magnitude of this response does not predict the magnitude of the antigen-specific response. By contrast, in the South African infants the magnitude of inflammatory gene expression one day post-MVA85A correlated with the ELISpot response at week 4. Further investigation of these mechanisms and how they differ between the different populations in which vaccines are tested and deployed may prove important in future vac- cine development strategies. Further to this, the relationships between these genes show interesting differences between the two popula- tions (Figure 6). Gene expression signatures correlating with immunogenicity differ in South African infants and UK adults In the South African infants, expression of both CXCL10 and IDO1 one day post-vaccination correlates with the IFN-γ ELISpot response and with each other. In the UK adults however, expression of these genes correlates neither with each other nor with the IFN-γ ELISpot response. In the UK adults, we have previously described a negative association between ex- pression of CTLA4 post-vaccination and the IFN-γ ELI- Spot response. However, this association was not found in South African infants. Furthermore, CTLA4 corre- lated positively with IDO1 in the UK adults but nega- tively in the South African infants. Conclusions This study has shown an association between high levels of inflammation and myeloid signatures both pre- and post-vaccination and the development of the antigen- specific T cell response to MVA85A in BCG-vaccinated South African infants. In this population, the capacity of the vaccine to induce a strong innate response appears key to its ability to initiate an adaptive immune re- sponse. Furthermore, we describe differences in the re- sponse to vaccination with MVA85A in UK adults and South African infants, suggesting different immune pathways may determine immune responses in these two very different cohorts. This study has focused on in- vestigating the mechanisms underlying vaccine immuno- genicity, not vaccine-induced protection. Ultimately, an understanding of both these aspects of vaccination and how they differ across individuals and populations is likely to be necessary in achieving protection in the di- verse groups and areas of the world still plagued by TB. y Indoleamine 2,3-dioxygenase (IDO1) is an enzyme catalyzing the first and rate-limiting step in tryptophan catabolism. This enzyme has multiple physiological ef- fects, including immunosuppression and regulation of T cells. In UK adults, expression of IDO1 in PBMC cor- relates with that of the inhibitory co-receptor CTLA4. A recent study showing IDO1 is a critical resistance mechanism in antitumor T cell immunotherapy target- ing CTLA-4 in a mouse model of melanoma suggests the interaction between these two genes is an important component to consider when inducing an immune re- sponse for therapeutic purposes [43]. In the South African infants however, we did not observe a correlation between expression of IDO1 and CTLA4 but IDO1 correlated with CXCL10 instead, suggesting either a different role for this enzyme or that inflammatory and regulatory responses are closely coupled in this population, perhaps to protect against excessive immune responses. Competing interests The authors declare that they have no competing interests. Author contributions MM, SAH, IS, LS, RT and MO carried out the experiments; MT, HMcS, MH, TJS, WAH and HAF were involved in study design and sample collection; MM Gene expression signatures correlating with immunogenicity differ in South African infants and UK adults Conversely, killing of infected myeloid cells by cytotoxic T cells and NK cells may decrease antigen expression, inhibiting the development of a re- sponse to Ag85A. South African infants: unstimulated whole blood taken 1 day post-vaccination. UK adults: unstimulated PBMC taken 2 days post-vaccination. All genes are differentially expressed between MVA85A and candin vaccinated infants (fdr < 0.05); genes in bold are also significantly different between day 0 and day 2 in UK adults vaccinated with MVA85A. South African infants: unstimulated whole blood taken 1 day post-vaccination. UK adults: unstimulated PBMC taken 2 days post-vaccination. All genes are differentially expressed between MVA85A and candin vaccinated infants (fdr < 0.05); genes in bold are also significantly different between day 0 and day 2 in UK adults vaccinated with MVA85A. Matsumiya et al. BMC Infectious Diseases 2014, 14:314 http://www.biomedcentral.com/1471-2334/14/314 Page 11 of 14 Figure 6 (See legend on next page.) Figure 6 (See legend on next page.) Figure 6 (See legend on next page.) Matsumiya et al. BMC Infectious Diseases 2014, 14:314 http://www.biomedcentral.com/1471-2334/14/314 Matsumiya et al. BMC Infectious Diseases 2014, 14:314 http://www.biomedcentral.com/1471-2334/14/314 Page 12 of 14 ( g p p g ) Figure 6 Differences in inflammation and regulation: RSA infants and UK adults. a. Expression in unstimulated whole blood 1 day post- MVA85A of CXCL10 and IDO1 but not CTLA4 correlates with ELISpot response in RSA infants. CTLA4 expression in unstimulated PBMC 2 days post-MVA85A is inversely correlated with ELISpot response in UK adults but IDO1 and CXCL10 show no significant association. b. In RSA infants, IDO1 expression correlates positively with CXCL10 and negatively with CTLA4 expression. In UK adults, CTLA4 and IDO1 expression show a positive association. All tests are Pearson’s correlations. two populations and in the pathways that determine the adaptive immune response. However, it is impossible to determine whether the differences observed are attribut- able to age or to genetic or environmental differences be- tween the populations. Furthermore, the post-vaccination timepoint differed in both cell type (whole blood in the in- fants, PBMC in adults) and time taken (one or two days post-MVA85A). PBMC were used in the pre-vaccination timepoint in both studies. Despite this, the similarities in the list of differentially expressed genes suggest these com- parisons are meaningful and warrant further investigation. 3. Ottenhoff THM, Kaufmann SHE: Vaccines against tuberculosis: where are we and where do we need to go? PLoS Pathog 2012, 8:e1002607. 19. Goronzy JJ, Weyand CM: Understanding immunosenescence to improve responses to vaccines. Nat Immunol 2013, 14:428–436. 4. Tameris MD, Hatherill M, Landry BS, Scriba TJ, Snowden MA, Lockhart S, Shea JE, McClain JB, Hussey GD, Hanekom WA, Mahomed H, McShane H: Safety and efficacy of MVA85A, a new tuberculosis vaccine, in infants previously vaccinated with BCG: a randomised, placebo-controlled phase 2b trial. Lancet 2013, 6736:1–8. 20. 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Author details 1 1Jenner Institute, University of Oxford, Old Road Campus Research Building, Oxford, UK. 2South African Tuberculosis Vaccine Initiative, Institute of Infectious Disease and Molecular Medicine and School of Child and Adolescent Health, University of Cape Town, Cape Town, South Africa. 3Division of Community Health, Stellenbosch University, Stellenbosch, South Africa. 4Metropolitan District Health Services, Western Cape, Government Health, Cape Town, South Africa. 5Current affiliation: London School of Hygiene and Tropical Medicine, Keppel St, London WC1E 7HT, UK. 15. Nakaya HI, Wrammert J, Lee EK, Racioppi L, Marie-Kunze S, Haining WN, Means AR, Kasturi SP, Khan N, Li G-M, McCausland M, Kanchan V, Kokko KE, Li S, Elbein R, Mehta AK, Aderem A, Subbarao K, Ahmed R, Pulendran B: Systems biology of vaccination for seasonal influenza in humans. Nat Immunol 2011, 12:786–795. Received: 8 April 2014 Accepted: 28 May 2014 Published: 9 June 2014 Received: 8 April 2014 Accepted: 28 May 2014 Published: 9 June 2014 16. Furman D, Jojic V, Kidd B, Shen-Orr S, Price J, Jarrell J, Tse T, Huang H, Lund P, Maecker HT, Utz PJ, Dekker CL, Koller D, Davis MM: Apoptosis and other immune biomarkers predict influenza vaccine responsiveness. Mol Syst Biol 2013, 9:659. Author contributions These disparities suggest there may be important dif- ferences in the immune response to vaccination in these Page 13 of 14 Matsumiya et al. BMC Infectious Diseases 2014, 14:314 http://www.biomedcentral.com/1471-2334/14/314 analysed the data and drafted the manuscript. All authors reviewed and approved the final manuscript. 11. De Cassan SC, Pathan AA, Sander CR, Minassian A, Rowland R, Hill AVS, McShane H, Fletcher HA: Investigating the induction of vaccine-induced Th17 and regulatory T cells in healthy, Mycobacterium bovis BCG- immunized adults vaccinated with a new tuberculosis vaccine, MVA85A. Clin Vaccine Immunol 2010, 17:1066–1073. Funding Thi k g This work was funded by Aeras, the Wellcome Trust and the Oxford Emergent Tuberculosis Consortium and NEWTBVAC (EC FP7); HMcS is a Wellcome Trust Senior Clinical Reseach Fellow. 14. Gaucher D, Therrien R, Kettaf N, Angermann BR, Boucher G, Filali-Mouhim A, Moser JM, Mehta RS, Drake DR, Castro E, Akondy R, Rinfret A, Yassine-Diab B, Said EA, Chouikh Y, Cameron MJ, Clum R, Kelvin D, Somogyi R, Greller LD, Balderas RS, Wilkinson P, Pantaleo G, Tartaglia J, Haddad EK, Sékaly R-P: Yellow fever vaccine induces integrated multilineage and polyfunctional immune responses. J Exp Med 2008, 205:3119–3131. Acknowledgements The authors would like to thank the trial participants and their families and the MVA85A 020 Trial Study team. 12. Dye C, Fine PE: A major event for new tuberculosis vaccines. Lancet 2013, 6736:3–5. 13. Querec TD, Akondy RS, Lee EK, Cao W, Nakaya HI, Teuwen D, Pirani A, Gernert K, Deng J, Marzolf B, Kennedy K, Wu H, Bennouna S, Oluoch H, Miller J, Vencio RZ, Mulligan M, Aderem A, Ahmed R, Pulendran B: Systems biology approach predicts immunogenicity of the yellow fever vaccine in humans. Nat Immunol 2009, 10:116–125. References ld 17. Ravindran R, Khan N, Nakaya HI, Li S, Loebbermann J, Maddur MS, Park Y, Jones DP, Chappert P, Davoust J, Weiss DS, Virgin HW, Ron D, Pulendran B: Vaccine activation of the nutrient sensor GCN2 in dendritic cells enhances antigen presentation. Science 2013, 2(December):1–8. 1. World Health Organisation: Global Tuberculosis Report 2013. 2013. Available at: http://www.who.int/tb/publications/global_report/en/. 1. 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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: doi:10.1186/1471-2334-14-314 Cite this article as: Matsumiya et al.: Inflammatory and myeloid-associated gene expression before and one day after infant vaccination with MVA85A correlates with induction of a T cell response. 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https://openalex.org/W2943770061	https://www.jotse.org/index.php/jotse/article/download/659/399	English	null	Methodological approach as a strategy for professional employees’ training in small and medium sized enterprises in Russia	Journal of technology and science education	2,019	cc-by	3,359	Abstract In the era of rapid development and the emergence of new methods and ideas for the employees’ training in small and medium sized enterprises in Russia, managers need to think about the role that the methodological organization of the educational process, which refers, not only with respect to vocational training, but also in providing the methodological foundation for teaching certain disciplines in small and medium sized enterprises in Russia. The article describes the integrative-praxicological approach in the professional training of employees, it describes the principles and requirements as a methodological basis for the educational process. Keywords – Small and medium sized enterprises, Vocational Training, Principles, Integration, Praxicology, Employees. To cite this article: Ivanova, N.L., & Ippolitova, N.V. (2019). Methodological approach as a strategy for professional employees’ training in small and medium sized enterprises in Russia. Journal of Technology and Science Education, 9(3), 239-244. https://doi.org/10.3926/jotse.659 ---------- Journal of Technology and Science Education JOTSE, 2019 – 9(3): 239-244 – Online ISSN: 2013-6374 – Print ISSN: 2014-5349 https://doi.org/10.3926/jotse.659 METHODOLOGICAL APPROACH AS A STRATEGY FOR PROFESSIONAL EMPLOYEES’ TRAINING IN SMALL AND MEDIUM SIZED ENTERPRISES IN RUSSIA Natalia L. Ivanova1 , Natalia V. Ippolitova2 , Irina N. Odarich3 1Tyumen Industrial University (Russia) 2Shadrinsk State Pedagogical University (Russia) 3Togliatti State University (Russia) NatalieIvanova@yandex.ru, inv_@mail.ru, azshar2017@mail.ru NatalieIvanova@yandex.ru, inv_@mail.ru, azshar2017@mail.ru NatalieIvanova@yandex.ru, inv_@mail.ru, azshar2017@mail.ru Received April 2018 Accepted February 2019 3. Results The consideration of the philosophical aspects of the integrative-praxicological approach and the determination of its belonging to one of the levels of methodology requires an additional analysis of the approaches affecting the problems of integration and activity. The most general interpretation of integration involves “the uniting of any parts, elements” (Flynn, McKevitt & Davis, 2015). In philosophy, “integration means the side of the development process associated with the unification of previously heterogeneous parts and elements. Integration processes can take place both within the existing system - in this case they lead to an increase in the level of its integrity and organization, and when a new system emerges from previously unrelated elements. Individual parts of an integrated whole may have varying degrees of autonomy. In the course of integration processes, the volume and intensity of interrelations and interactions between elements increase in the system” (Sloman, 2017). “Integrity as a generalized characteristic of objects possessing a complex internal structure expresses the integration, self-sufficiency, autonomy of these objects, and their opposition to the environment, related to their internal activity. The relationship between a part and a whole is characterized by the presence of definite, fixed, multilateral interactions between parts in the whole, having the form of connections. The emergence of links in the formation of the whole of the parts is considered as the primary necessary condition for integration, i.e. the emergence of a new integrity” (Zhou, 2016). From these positions, integration expresses the dynamics of a complex object, and its integrity is the result of movement. Thus, the study of various phenomena and processes from the standpoint of an integrative approach presupposes a support for the principle of integrativity that characterizes the phenomenon under consideration as an integrity achieved by integrating its constituent elements. The second component of the integrative-praxicological approach is related to the concept of activity (Johnson, 2017). The analysis of human activity as a complex system from the philosophical point of view reveals the interconnection between the concepts of activity and practice and shows that practice performs an integrative function to the whole system of human activity in all the diversity of its forms and varieties. Important in this case is that all activities have an outlet to practice, enrich its capabilities. 1. Introduction When studying the problems of higher professional education, and in connection with the transition of small and medium sized enterprises to a “competence” paradigm, it requires a revision of the methodological approaches that determine the strategy for studying and organizing the professional training of future specialists. The methodological tools of modern small and medium sized enterprises include a set of ways to gain knowledge about pedagogical approaches. “The methodological approach is a set of ideas that determine the general scientific outlook of the scientist, the principles that form the basis of the research strategy, as well as the methods, techniques, procedures that ensure the implementation of the chosen strategy in practice”. An effective-methodological approach, which corresponds to the main trends in the development of vocational education (integration, globalization), is the integrative-praxicological approach. -239- -239- Journal of Technology and Science Education – https://doi.org/10.3926/jotse.659 2. Methodology To organize and conduct the study, we selected the appropriate tools, presented in the form of two groups of methods. The first group consisted of theoretical methods of research: an analysis that enabled detailed elaboration of the content of various sources of information; synthesis, necessary to obtain the main features and provisions, which subsequently formed the basis for the basic definitions and characteristics of the study. The second group included empirical methods of pedagogical research - the study of literature, which made it possible to find out which aspects of the problem we are studying have been well researched sufficiently; generalization of experience, through which we could logically come to the formulation of concepts. 3. Results Proceeding from this, we refer to the integrative-praxicological approach to the third level - the concrete scientific methodology, because, firstly, it relies on a set of approaches and cannot be qualified within one of them when discarding others, and second, presupposes a set of methods and research principles used in psycho-pedagogical, philosophical studies related to pedagogical activity and training of stakeholders for its implementation (Bruhn, Karlan & Schoar, 2018). So, the integrative-praxicological approach regards phenomena and processes as complex objects whose integrity is achieved on the basis of the integration of the constituent elements, which are in interrelation and interaction. Characterization of activities and preparation for it from the standpoint of this approach implies the consideration of the first as the main means of achieving the goal of the second. At the same time, preparation for activity is viewed as a system with a certain structure and the existence of interrelations between components. When creating a certain set of conditions, the implementation of this system ensures the achievement of the intended result, increases the efficiency of the functioning of this system (Organization, 2013). This approach is based on a set of principles that reflect the features of professional training of future specialists as a pedagogical system. The idea of integrativity, as the basis of this approach, reflects the leading trends of the society as a whole and the higher school in particular. The cardinal changes taking place in modern society are manifested in the integration of various branches of production, deepening the production ties of state and interstate character. The tasks of increasing the efficiency of the production sphere put forward new, higher requirements for specialists and, consequently, the level and quality of their preparation in the process of training at the university. Hence the need to integrate small and medium sized enterprises institutions with other institutions of society. The training of a specialist is objectively conditioned by the needs of modern society. Socio-economic, scientific-technical, production factors have an increasing influence on pedagogical science. The convergence of science and production in modern society, its transformation into a real productive force, is the basis for the intensification of modern production. The search for new approaches to professional training of university employees is objectively conditioned by the requirements of the developing society. 3. Results Extrapolating this provision to the psychological and pedagogical aspect of the problem in question, it should be noted that it is in the process of activity that the personality develops, the development of human capabilities, and a necessary condition for this is to include it in a system of interrelated activities. Analysis of professional human activity involves finding ways to improve its effectiveness, which is the subject of research such a science as praxicology. The founder of praxicology as the theory of effective human activity is the Polish scientist Sonnenstuhl and Trice (2018), who set the task of finding “the general laws of all human activity and deriving the most general rules of such activity”. Bell (2016) understands praxicology as a science on the rationality and effectiveness of actions. According to these theories, the structure of activity is linear or networked, the activity process is aimed at achieving the result and must be structured in accordance with the conditions and opportunities to obtain the result (Albrecht, Bakker, Gruman, Macey & Saks, 2015). -240- -240- Journal of Technology and Science Education – https://doi.org/10.3926/jotse.659 In view of these provisions, we apply a combination of integrative and praxicological approaches for a holistic analysis of the preparation of employees for professional work (Zuber-Skerritt, 2013). In view of these provisions, we apply a combination of integrative and praxicological approaches for a holistic analysis of the preparation of employees for professional work (Zuber-Skerritt, 2013). We define the place of the integrative-praxicological approach in a number of levels of methodological analysis. This approach cannot be attributed to the level of philosophical methodology as it is not directly connected either with the development of world outlook problems or with the performance of the functions of philosophical criticism of the forms and principles of scientific cognition. This approach cannot be attributed to the second level of general scientific principles and forms of research, tk. it does not extend simultaneously to the aggregate of fundamental scientific disciplines and is not connected with the solution of a wide range of methodological problems. It is wrong to consider the integrative-praxicological approach as belonging to the level of methodology and technique of research, it is not a specialized set of procedures ensuring the receipt of a uniform and reliable empirical material and its primary processing (Ogunyomi & Bruning, 2016). 3. Results The use of the results of pedagogical research in this area contributes to the intensification of the training of future specialists and the improvement of the quality and effectiveness of the pedagogical process in specific educational institutions. The development of modern science, and pedagogical science in particular, calls for the integration of interdisciplinary scientific knowledge, the implementation of scientific research on the basis of related sciences, attempts to create concepts and theories reflecting the development of the underlying categories used in related sciences. Formation of holistic ideas about activity, personality, social processes, etc. based on the interpenetration of philosophical, psychological, pedagogical, sociological sciences. The interdisciplinary -241- Journal of Technology and Science Education – https://doi.org/10.3926/jotse.659 integration of scientific knowledge within the framework of pedagogical science presupposes the assimilation of these related sciences, taking into account the specifics of the objects under study. The implementation of the praxicological approach in the preparation of future specialists for professional activities presupposes the support of a diverse in the forms and content of the activities of employees, which acts as the main means of achieving the goal of preparation. Since the activity of employees in the process of professional training is aimed at mastering professional pedagogical activity, it is necessary to consider it from the position of analysis of the pedagogical activity itself. The psychological structure of activity, according to Dreher (2014), is a kind of reflection of the activities of pedagogical systems and contains constructive, organizational, communicative and gnostic components. Accordingly, the activities of employees should be aimed at the development of these components. It is also important to note that the activity of employees in the process of professional training presupposes a combination of educational, cognitive, educational, practical and independent practical activities. At the same time, educational and cognitive activity acts as a process of solving learning problems aimed at understanding the laws, principles, ways of organizing the learning process and mastering the basics of professional skills. Educational and practical activity is a process of solving practical problems aimed at fulfilling practical tasks in theoretical disciplines that require the application of theoretical knowledge in practice. Independent practical activity presupposes the solution of practical problems in organizing educational work with children on the basis of self-constructing the content and methods of pedagogical activity, understanding their purpose and principles, analyzing and evaluating its results. 4. Conclusions Thus, the implementation of the praxicological aspect of the integrative-praxicological approach in the preparation of employees for professional activity presupposes: taking into account the components of the psychological structure of this activity (constructive, organizational, communicative, gnostic) when it is mastered by employees; realization in interrelation of various types of educational activity of employees in the process of training (educational-cognitive, educational-practical, independent practical), contributing to mastering the dialectical unity of theoretical knowledge and practical skills and skills necessary for any professional activity. Taking into account all the above, it is possible to single out the requirements for an integrative-praxicological approach: A. Ensuring the integration of the various components of professional training of future specialists. B. Ensuring the unity of the assimilation of theoretical knowledge and the formation of practical skills and skills by employees on the basis of their inclusion in a variety of types and content of activities. C. Ensuring that employees master the algorithms of pedagogical activity in the process of their professional training. Concluding the analysis of the essence of integrative-praxicological approach to the organization of professional training of future specialists, it is necessary to note the following. This approach means that the process of professional training of employees is seen as a holistic phenomenon, consisting of elements that are in a relationship of interdependence and interdependence, which ensures their integration into a single whole one. A variety of employees’ activities is the main means of achieving its goal in the process of professional training. 3. Results The latter, in turn, are subdivided into stochastic (probabilistic) and heuristic algorithms. As the author notes, any activity can be really algorithmized only to the extent that the standards are realized by the subject of activity, are explicated, socially approved and fixed in the subject’s knowledge”. algorithms are distinguished. The latter, in turn, are subdivided into stochastic (probabilistic) and heuristic algorithms. As the author notes, any activity can be really algorithmized only to the extent that the standards are realized by the subject of activity, are explicated, socially approved and fixed in the subject’s knowledge”. For professional activities, mechanical, rigid stochastic and heuristic algorithms may be characteristic, reflecting the creative nature of the activity, the uniqueness and sometimes unpredictability of each particular pedagogical situation. In the professional training of future specialists in the oil and gas industry, for example, the use of algorithms is fully justified, since it teaches them to analyze and take into account the various conditions for choosing methods and methods of action in each specific situation. Important in this case is not only awareness of the impossibility of using rigid algorithms in professional activity, but also mastering them of stochastic and heuristic algorithms in the process of teaching, cognitive, educational and self- practice. Algorithmicity in mastering the knowledge, skills and skills necessary for professional activities, contributes to improving employees’ readiness to implement all the functions of a specialist in conditions of independent professional activity. Declaration of Conflicting Interests The author declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Funding he author received no financial support for the research, authorship, and/or publication of this artic author received no financial support for the research, authorship, and/or publication of this article. rences 3. Results The interconnection of these activities is conditioned by the fact that during the training sessions employees acquire a fundamental knowledge of the fundamentals of the theory of general professional disciplines and the basics of professional skills, and in the process of professional practice, actualization, the application of theoretical knowledge in specific conditions, the formation and development of practical skills and skills. Educational and practical activities synthesize cognitive and practical activities of employees, as a result, an interconnected system of knowledge and skills is formed, and their correlation is achieved. The dialectical connection of these activities is achieved through their parallel organization, when, acquiring knowledge, employees use them in solving practical problems, and in practice observe the manifestation of the laws of the pedagogical process, comprehend and generalize knowledge. In addition to the principles of integrativity and praxicology, other principles are also based on the integrative-praxicological approach. The principle of adequacy of the content of vocational training of future specialists to the requirements of modern society, meaning the need to determine the content of professional training of employees, taking into account modern methodological approaches, new concepts of vocational training and upbringing, changes in the technology of the pedagogical process. The principle of helicity, which combines the elements of linear and concentric construction of curricula and programs for professional employees’ training, involves the mastering of knowledge and the formation of skills and skills not only in a certain sequence, but also mastering their complex with the complexity of the system of knowledge and specific skills at various levels of training. The principle of variability reflects, on the one hand, the need for continuous development, improvement of the foundations of professional pedagogical training on the basis of taking into account changes occurring in society, the system of small and medium sized enterprises in Russia, and on the other hand, it presupposes a multiplicity of ways of preparation depending on the contingent of employees, the level of vocational training, local conditions and etc. The principle of algorithmization means the implementation of the training of employees for professional activity on the basis of mastering various algorithms of pedagogical activity corresponding to various situations. “An algorithm is a rule or an action program for obtaining the desired result under given conditions. By the degree of accuracy of the task, mechanical and non-mechanical (variational, flexible) -242- Journal of Technology and Science Education – https://doi.org/10.3926/jotse.659 algorithms are distinguished. 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Professional development in higher education: A theoretical framework for action research. Routledge. Published by OmniaScience (www.omniascience.com) Article’s contents are provided on an Attribution-Non Commercial 4.0 Creative commons International Lice p Readers are allowed to copy, distribute and communicate article’s contents, provided the author’s and JOTSE journal’s names are included. It must not be used for commercial purposes. To see the complete licence contents, please visit https://creativecommons.org/licenses/by-nc/4.0/. -244-
https://openalex.org/W4313365272	https://cellandbioscience.biomedcentral.com/counter/pdf/10.1186/s13578-022-00950-z	English	null	SPOP inhibits BRAF-dependent tumorigenesis through promoting non-degradative ubiquitination of BRAF	Cell & bioscience	2,022	cc-by	11,139	© 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/. 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 gene encoding the E3 ubiquitin ligase substrate-binding adapter Speckle-type BTB/POZ protein (SPOP) is frequently mutated in prostate cancer (PCa) and endometrial cancer (EC); however, the molecular mecha- nisms underlying the contribution of SPOP mutations to tumorigenesis remain poorly understood. Methods: BRAF harbors a potential SPOP-binding consensus motif (SBC) motif. Co-immunoprecipitation assays dem- onstrated that BRAF interacts with SPOP. A series of functional analyses in cell lines were performed to investigate the biological significance of MAPK/ERK activation caused by SPOP mutations. Results: Cytoplasmic SPOP binds to and induces non-degradative ubiquitination of BRAF, thereby reducing the interaction between BRAF and other core components of the MAPK/ERK pathway. SPOP ablation increased MAPK/ERK activation. EC- or PCa-associated SPOP mutants showed a reduced capacity to bind and ubiquitinate BRAF. Moreover, cancer-associated BRAF mutations disrupted the BRAF-SPOP interaction and allowed BRAF to evade SPOP-mediated ubiquitination, thereby upregulating MAPK/ERK signaling and enhancing the neoplastic phenotypes of cancer cells. Conclusions: Our findings provide new insights into the molecular link between SPOP mutation-driven tumorigen- esis and aberrant BRAF-dependent activation of the MAPK/ERK pathway. Keywords: SPOP, BRAF, Mutation, Ubiquitination, MAPK/ERK †Kai Feng and Qing Shi are contributed equally to this work Correspondence: pzzhang@fudan.edu.cn; yaoli@fudan.edu.cn; chenjiwang@fudan.edu.cn Correspondence: pzzhang@fudan.edu.cn; yaoli@fudan.edu.cn; chenjiwang@fudan.edu.cn 1 Shanghai Stomatological Hospital & School of Stomatology, State Key Laboratory of Genetic Engineering, MOE Engineering Research Center of Gene Technology, Shanghai Engineering Research Center of Industrial Microorganisms, School of Life Sciences, Fudan University, Shanghai 200438, China 4 Department of Pathology, School of Basic Medical Sciences, Fudan University Shanghai Cancer Center, Fudan University, Shanghai 200032, China Full list of author information is available at the end of the article Introductionh The MAPK/ERK pathway, also known as the RAS-RAF- MEK-ERK pathway, transmits extracellular proliferative signals from the ligand-mediated activation of receptor tyrosine kinases to the cell nucleus [1, 2]. BRAF is a mem- ber of the rapidly accelerated fibrosarcoma (RAF) kinase family, which includes ARAF and CRAF (RAF1) [3]. RAFs transduce signals downstream of the RAS via the MEK-ERK cascade. Among the three RAF kinases, BRAF binds most avidly to RAS and is the most active in phos- phorylating MEK1/2 [4]. Oncogenic BRAF mutations have been detected in approximately 6% of human can- cers, with most occurring in hairy cell leukemia (> 97%), melanoma (40–50%), thyroid cancer (30–50%), colorectal SPOP inhibits BRAF‑dependent tumorigenesis through promoting non‑degradative ubiquitination of BRAF Kai Feng1,2†, Qing Shi1†, Dongyue Jiao1, Yingji Chen1, Wanqi Yang1, Ke Su2, Yalan Wang3, Yan Huang1, Pingzhao Zhang4, Yao Li1 and Chenji Wang1* Feng et al. Cell & Bioscience (2022) 12:211 https://doi.org/10.1186/s13578-022-00950-z Feng et al. Cell & Bioscience (2022) 12:211 https://doi.org/10.1186/s13578-022-00950-z Cell & Bioscience Open Access Plasmid constructions Expression vectors for SPOP-WT and its mutants have been described previously [14]. BRAF cDNA was obtained from Genecopia and subcloned into the pCMV vector. BRAF mutants were generated using the KOD- Plus-Mutagenesis Kit (TOYOBO) following the manu- facturer’s instructions. Colony formation assays 3 Ishikawa cells (1 × 103) were seeded in triplicate in 6-well plates containing each well. After incubation for 2 weeks, the cells were fixed with 4% paraformaldehyde for 15 min and stained with Giemsa dye (Solarbio) for 20 min. The cells were then washed with water by gentle dropping and air dried at room temperature. The number of colo- nies was quantified using a Nikon digital camera and the ImageJ software. In this study, we identified BRAF as a bona fide substrate of the CRL3-SPOP E3 ubiquitin ligase complex. The CRL3-SPOP complex mediates non-deg- radative ubiquitination of BRAF, which leads to attenu- ated MAPK/ERK activation. Moreover, mutations in the SPOP-BRAF-binding interface disrupt the SPOP-BRAF CRISPR‑Cas9 mediated gene knock out stable cell generationh The pX459 plasmid was used to clone guide oligonu- cleotides targeting BRAF and SPOP genes. Ishikawa and SPEC-2 cells were plated and transfected overnight with pX459. 24 h after transfection, 1 μg/ml puromycin was used to screen cells for 3 days. Living cells were seeded in 96 well plate at a limited dilution to isolate the mono- clonal cell lines. KO cell clones were screened by West- ern blot (WB) and validated by Sanger sequencing. The sequences of gene-specific sgRNAs are listed in Addi- tional file 1: Table S1. Protein half‑life assays To measure protein half-life, cycloheximide (50 μg/ml) was added to the media of the parental and SPOP-KO Ishikawa cells. At the indicated time points, cell lysates were prepared and analyzed by WB using the indicated antibodies. © 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/. 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. Feng et al. Cell & Bioscience (2022) 12:211 Page 2 of 15 regulatory pattern and promote aberrant MAPK/ERK activation and malignant transformation in cancer cells. regulatory pattern and promote aberrant MAPK/ERK activation and malignant transformation in cancer cells. cancer (10%), and non-small cell lung cancer (3–5%) [5]. In contrast, mutations in ARAF and CRAF are relatively rare in human cancers. Oncogenic BRAF mutations lead to the constitutive activation of the MAPK/ERK pathway, resulting in uncontrolled proliferation, survival, invasive- ness, and drug resistance [1]. BRAF inhibitors such as vemurafenib, dabrafenib, and encorafenib are currently used to treat patients with BRAF-mutant cancers [6, 7]. Methods Cell culture 293 T, Ishikawa, KLE, SPEC-2, and HeLa cells were main- tained in DMEM supplemented with 10% (v/v) fetal bovine serum (FBS). SPEC-2 cells were maintained in F12 medium supplemented with 10% (v/v) FBS. All cells were grown at 37 °C with 5% CO2. Cullin-RING E3 ubiquitin ligases (CRLs) are a super- family of modular, multi-subunit E3 ubiquitin ligases that mediate the ubiquitination and degradation of various cellular proteins involved in a plethora of physiological and pathological processes. Human cells express seven different Cullins (CUL1, 2, 3, 4A, 4B, 5, and 7), each of which nucleates a multi-subunit complex [8]. The Cullin3-RING E3 ubiquitin ligase complex subfamily employs a BTB domain-containing protein for substrate- binding. Speckle-type BTB/POZ protein (SPOP) is a substrate-binding adaptor of CRL3s [9]. Investigation of the pathophysiological functions of SPOP has attracted extensive attention in recent years because of its high mutation frequency in prostate cancer (PCa) (5–15%) and endometrial cancer (EC) (8–10%), two hormone- related cancers [10, 11]. Recurrent SPOP mutations have also been detected in other cancer types, although at a relatively low frequency. SPOP mutations occur as het- erozygous missense mutations with dominant-negative, selective loss-of-function effects on the remaining wild- type allele [12]. SPOP selectively recruits substrates via its N-terminal MATH domain, whereas its BTB and BACK domains mediate oligomerization and interaction with CUL3 [9]. The vast majority of EC- or PCa-associated SPOP mutations occur in the substrate-binding MATH domain. To date, dozens of SPOP substrates (AR, ERα, SRC-3, BET, GLP, etc.) have been identified through dif- ferent biochemical methods and functionally investigated in PCa or EC settings, and the list of SPOP substrates is still growing [13–17]. Research has shown that cancer- associated SPOP mutants generally display an impaired capacity to bind substrates, leading to reduced ubiquit- ination of substrates. Accumulating evidence from can- cer cell lines and animal models has demonstrated that SPOP mutations promote the initiation and progression of EC and PCa, potentially owing to the dysregulation of SPOP-mediated ubiquitination of its substrates [15, 18]. However, the cellular pathways affected by SPOP muta- tions are not yet fully understood. Sphere formation assays 3 Ishikawa cells (1 × 103 cells/well) were mixed with Matrigel (BD Biosciences) and plated in 24-well ultra- low-attachment plates (Corning) in DMEM containing 10% FBS. Fresh medium was added every 3 days. Floating spheres that grew in 1–2 weeks were quantified using a Nikon digital photo camera and the ImageJ software. Cell proliferation assaysh The proliferation rate of Ishikawa cells was determined using the Cell Counting Kit-8 (CCK-8) Kit (Beyotime) according to the manufacturer’s instructions. Briefly, the cells were seeded onto 96-well plates at a density of 1 × 103 cells/well in triplicate. During the 2-to 6 day cul- ture period, 10 μl of CCK-8 solution was added to the cell culture and incubated for 2 h. The absorbance of the sam- ples was measured at 450 nm wavelength using a micro- plate absorbance reader. mmunofluorescence and confocal microscopyl In vitro ubiquitination assays were performed accord- ing to a protocol reported previously [12]. Briefly, 2 µg APP-BP1/Uba3, 2 µg His-UBE2M, and 5 µg NEDD8 were incubated at 30 °C for 2 h in the presence of ATP. The thioester-loaded His-UBE2M–NEDD8 was further incu- bated with 3 µg His-DCNL2 and 6 µg CUL3–RBX1 at 4 °C for 2 h to obtain neddylated CUL3–RBX1. The ned- dylated CUL3–RBX1, 5 µg of GST-SPOP, 5 µg of ubiqui- tin, 500 ng of E1 enzyme, 750 ng of E2 enzyme (UbcH5a and UbcH5b), and 5 µg of GST-BRAF were incubated with 0.6 µl of 100 mM ATP, 1.5 µl of 20 µM ubiquitin aldehyde, 3 µL of 10 × ubiquitin reaction buffer (500 mM Tris–HCl (pH 7.5), 50 mM KCl, 50 mM NaF, 50 mM MgCl2, and 5 mM DTT), 3 µL of 10 × energy regenera- tion mix (200 mM creatine phosphate and 2 µg/µL cre- atine phosphokinase), and 3 µL of 10 × protease inhibitor cocktail at 30 °C for 2 h, followed by WB analysis. Recom- binant proteins Information is presented in Additional file 1: Table S2. For immunofluorescence, cells were plated on chamber slides and fixed with 4% paraformaldehyde at room tem- perature for 30 min. After washing with PBS, cells were permeabilized with 0.1% Triton X-100 in PBS for 15 min. The cells were then washed with PBS, blocked with 0.5% BSA in PBS for 1 h, and incubated with primary antibod- ies in PBS at 4 °C overnight. After washing with PBS, flu- orescence-labeled secondary antibodies were applied and DAPI was counterstained for 1 h at room temperature. The cells were visualized and imaged using a confocal microscope (LSM710, Zeiss). Statistical analysis Statistical analysis was performed using GraphPad Prism (GraphPad Software, Inc.), and all data are shown as mean values ± standard deviation (SD) for experiments performed with at least three replicates. The differences between the two groups were analyzed using one-way or two-way ANOVA test unless otherwise specified. * represents p < 0.05, represents p < 0.01, * repre- sents p < 0.001, **** represents p < 0.0001, n.s. represents non-significant. Identification of BRAF as a novel SPOP interactori Identification of BRAF as a novel SPOP interactori Identification of BRAF as a novel SPOP interactor Previously, we and others have identified multiple onco- proteins as degradative or non-degradative substrates of the CRL-SPOP complex using the yeast two-hybrid method or affinity purification coupled with mass spec- trometry. Substrate-binding consensus (SBC) motifs (Φ-π-S/T-S/T-S/T, where Φ is a nonpolar residue and π is a polar residue) have been well-characterized in the majority, if not all, of these substrates [9]. To expand the SPOP interactome to a proteome-wide level, we performed an SBC motif search against the human pro- teome sequence using the webtool ScanProsite (https:// prosite.expasy.org/scanprosite/). Among the hundreds of potential candidates identified by this search, we noticed that BRAF harbors a perfectly matched SBC motif (120- VTSSS-124 aa), which is very similar to those present in In vivo ubiquitination assays 293 T cells were transfected with HA-Ubiquitin and other indicated constructs. 36 h after transfection, cells were lysed in 1% SDS buffer (Tris [pH 7.5], 0.5 mM Feng et al. Cell & Bioscience (2022) 12:211 Page 3 of 15 different fields. Matrigel invasion assays were performed using Transwell inserts (Costar) coated with Matrigel/ fibronectin (BD Biosciences). EDTA, 1 mM DTT) and boiled for 10 min. For co-immu- noprecipitation (Co-IP), cell lysates were diluted tenfold in Tris–HCl buffer and incubated with anti-FLAG M2 agarose beads (Sigma) for 4 h at 4 °C. The bound beads were then washed four times with BC100 buffer (20 mM Tris–Cl, pH 7.9, 100 mM NaCl, 0.2 mM EDTA, 20% glycerol) containing 0.2% Triton X-100. Proteins were eluted with 3X FLAG peptide for 2 h at 4 °C. The ubiqui- tinated form of BRAF was detected by WB with anti-HA antibody. EDTA, 1 mM DTT) and boiled for 10 min. For co-immu- noprecipitation (Co-IP), cell lysates were diluted tenfold in Tris–HCl buffer and incubated with anti-FLAG M2 agarose beads (Sigma) for 4 h at 4 °C. The bound beads were then washed four times with BC100 buffer (20 mM Tris–Cl, pH 7.9, 100 mM NaCl, 0.2 mM EDTA, 20% glycerol) containing 0.2% Triton X-100. Proteins were eluted with 3X FLAG peptide for 2 h at 4 °C. The ubiqui- tinated form of BRAF was detected by WB with anti-HA antibody. SPOP promotes non‑degradative ubiquitination of BRAF SPOP promotes non‑degradative ubiquitination of BRAF Next, we investigated whether BRAF protein stability was controlled by the ubiquitin–proteasome pathway. Treatment of Ishikawa cells with the proteasome inhibi- tor MG132 had no obvious effect on BRAF protein lev- els, indicating that BRAF has a relative long half-life, at least in Ishikawa cells. MLN4924, a small-molecule Fig. 1 SPOP interacts with BRAF in cells. a Amino acid sequence alignment of the SBC motif in SPOP substrates. ERG, ATF2, Caprin1, and DAXX are reported SPOP substrates containing the well-characterized SBC motif (Φ-π-S–S/T-S/T; Φ: nonpolar residues, π: polar residues). b Diagram showing the RAF protein family, putative SBC motif, and two cancer-associated BRAF mutants. c–e Western blot of indicated proteins in whole-cell lysates (WCL) and co-IP samples of anti-FLAG antibody obtained from 293 T cells transfected with indicated plasmids. f Western blot of indicated proteins in WCL and co-IP samples of anti-FLAG antibody obtained from 293 T cells transfected with indicated plasmids. g Western blot of indicated proteins in WCL and co-IP samples of IgG or anti-SPOP antibody obtained from the cell extracts of Ishikawa cells. h Western blot of indicated proteins in WCL and co-IP samples of IgG or anti-BRAF antibody obtained from the cell extracts of Ishikawa cells. i Schematic representation of SPOP deletion mutants. The binding capacity of SPOP to BRAF is indicated with the symbol. j Western blot of indicated proteins in WCL and co-IP samples of anti-FLAG antibody obtained from 293 T cells transfected with indicated plasmids Fig. 1 SPOP interacts with BRAF in cells. a Amino acid sequence alignment of the SBC motif in SPOP substrates. ERG, ATF2, Caprin1, and DAXX are reported SPOP substrates containing the well-characterized SBC motif (Φ-π-S–S/T-S/T; Φ: nonpolar residues, π: polar residues). b Diagram showing the RAF protein family, putative SBC motif, and two cancer-associated BRAF mutants. c–e Western blot of indicated proteins in whole-cell lysates (WCL) and co-IP samples of anti-FLAG antibody obtained from 293 T cells transfected with indicated plasmids. f Western blot of indicated proteins in WCL and co-IP samples of anti-FLAG antibody obtained from 293 T cells transfected with indicated plasmids. g Western blot of indicated proteins in WCL and co-IP samples of IgG or anti-SPOP antibody obtained from the cell extracts of Ishikawa cells. h Western blot of indicated proteins in WCL and co-IP samples of IgG or anti-BRAF antibody obtained from the cell extracts of Ishikawa cells. Migration and invasion assays Cell migration and invasion were determined using Transwell (Costar) migration and invasion assays. Ishi- kawa cells were precultured in serum-free medium for 48 h. For the migration assay, 3 × 104 cells were seeded in serum-free medium in the upper chamber, and the lower chamber was filled with DMEM containing 5% FBS. After 48 h, the non-migrating cells in the upper chambers were carefully removed with a cotton swab and migrated cells underside of the filter were stained and counted in nine Feng et al. Cell & Bioscience (2022) 12:211 Page 4 of 15 several known SPOP substrates, including ERG, ATF2, Caprin1, and DAXX (Fig. 1a). Notably, this motif was absent in BRAF paralogs of ARAF and CRAF (Fig. 1b). Given that BRAF is frequently hyperactivated in vari- ous human cancers, we explored whether BRAF is an authentic substrate of the CRL3-SPOP complex, and whether BRAF activity is dysregulated in SPOP-mutated cancers. Using reciprocal co-IP assays, we demonstrated that ectopically expressed BRAF interacted with SPOP (Fig. 1c, d). In contrast, ectopically expressed ARAF or CRAF did not interact with SPOP (Fig. 1e). Only SPOP, but none of the other CRL3-based BTB domain-contain- ing adaptors examined, interacted with BRAF (Fig. 1f). Moreover, a specific endogenous interaction between SPOP and BRAF was observed in Ishikawa endometrial several known SPOP substrates, including ERG, ATF2, Caprin1, and DAXX (Fig. 1a). Notably, this motif was absent in BRAF paralogs of ARAF and CRAF (Fig. 1b). Given that BRAF is frequently hyperactivated in vari- ous human cancers, we explored whether BRAF is an authentic substrate of the CRL3-SPOP complex, and whether BRAF activity is dysregulated in SPOP-mutated cancers. Using reciprocal co-IP assays, we demonstrated that ectopically expressed BRAF interacted with SPOP (Fig. 1c, d). In contrast, ectopically expressed ARAF or CRAF did not interact with SPOP (Fig. 1e). Only SPOP, but none of the other CRL3-based BTB domain-contain- ing adaptors examined, interacted with BRAF (Fig. 1f). Moreover, a specific endogenous interaction between SPOP and BRAF was observed in Ishikawa endometrial cancer cells (Fig. 1g, h). In accordance with a previ- ous study showing that the MATH domain of SPOP is responsible for recruiting substrates [9], deletion of the MATH domain, but not the CUL3-binding BTB domain, completely abolished the interaction between SPOP and BRAF (Fig. 1i, j). Taken together, our data indicate that SPOP interacts with BRAF in cells. SPOP promotes non‑degradative ubiquitination of BRAF i Schematic representation of SPOP deletion mutants. The binding capacity of SPOP to BRAF is indicated with the symbol. j Western blot of indicated proteins in WCL and co-IP samples of anti-FLAG antibody obtained from 293 T cells transfected with indicated plasmids Fig. 1 SPOP interacts with BRAF in cells. a Amino acid sequence alignment of the SBC motif in SPOP substrates. ERG, ATF2, Caprin1, and DAXX are reported SPOP substrates containing the well-characterized SBC motif (Φ-π-S–S/T-S/T; Φ: nonpolar residues, π: polar residues). b Diagram showing the RAF protein family, putative SBC motif, and two cancer-associated BRAF mutants. c–e Western blot of indicated proteins in whole-cell lysates (WCL) and co-IP samples of anti-FLAG antibody obtained from 293 T cells transfected with indicated plasmids. f Western blot of indicated proteins in WCL and co-IP samples of anti-FLAG antibody obtained from 293 T cells transfected with indicated plasmids. g Western blot of indicated proteins in WCL and co-IP samples of IgG or anti-SPOP antibody obtained from the cell extracts of Ishikawa cells. h Western blot of indicated proteins in WCL and co-IP samples of IgG or anti-BRAF antibody obtained from the cell extracts of Ishikawa cells. i Schematic representation of SPOP deletion mutants. The binding capacity of SPOP to BRAF is indicated with the symbol. j Western blot of indicated proteins in WCL and co-IP samples of anti-FLAG antibody obtained from 293 T cells transfected with indicated plasmids Feng et al. Cell & Bioscience (2022) 12:211 Page 5 of 15 co-expressed BRAF (Fig. 2b). To characterize the effect of SPOP on endogenous BRAF, we generated Tet-on- inducible Ishikawa cells. Induction of FLAG-SPOP by doxycycline treatment did not affect BRAF protein lev- els, whereas BRD4 was destabilized in a time-dependent manner (Fig. 2c). Depletion of SPOP by shRNA-mediated knockdown or CRISPR/Cas9-mediated KO in EC cell inhibitor of the NEDD8-activating enzyme required for the activation of the CRL complex, did not alter BRAF protein levels. In contrast, MG132 or MLN4924 treat- ment caused a marked increase in the protein levels of BRD4 and Caprin1, which are two reported substrates of SPOP (Fig. 2a). Ectopic overexpression of SPOP-WT or its domain deletion mutants did not alter the levels of Fig. 2 SPOP promotes non-degradative ubiquitination of BRAF. a Western blot of indicated proteins in WCL from Ishikawa cells treated with DMSO, MLN4924 (100 nM), or with MG132 (20 μM) for 8 h. SPOP promotes non‑degradative ubiquitination of BRAF b Western blot of WCL from 293 T cells transfected with FLAG-BRAF and increasing doses of Myc-tagged SPOP-WT or deletion mutants. c Western blot of indicated proteins in WCL from Tet-on-inducible Ishikawa cells treated with doxycycline (DOX). d Western blot of the indicated proteins in WCL from Ishikawa or SPEC-2 cells with SPOP knockout through CRISPR/Cas9 methods. e, f Western blot of indicated proteins in WCL from 293 T cells treated with 50 µg/ml cycloheximide (CHX) and harvested at different time points. Data were shown as mean ± SD (n = 3) f. The p-values were calculated using the Two-way ANOVA test. n.s., non-significant. g, i 293 T cells were transfected with the indicated plasmids. 24 h after transfection, the WCLs were prepared and the in vivo ubiquitination assays were performed. The polyubiquitinated forms of BRAF were detected by Western blot with anti-HA antibody. h 293 T cells were infected with lentivirus expressing SPOP-specific shRNA or negative control. The stable cell lines were transfection with HA-Ub and the cell lysates were prepared and immunoprecipitation was performed. The polyubiquitinated forms of BRAF were detected by Western blot with anti-HA antibody Fig. 2 SPOP promotes non-degradative ubiquitination of BRAF. a Western blot of indicated proteins in WCL from Ishikawa cells treated with DMSO, MLN4924 (100 nM), or with MG132 (20 μM) for 8 h. b Western blot of WCL from 293 T cells transfected with FLAG-BRAF and increasing doses of Myc-tagged SPOP-WT or deletion mutants. c Western blot of indicated proteins in WCL from Tet-on-inducible Ishikawa cells treated with doxycycline (DOX). d Western blot of the indicated proteins in WCL from Ishikawa or SPEC-2 cells with SPOP knockout through CRISPR/Cas9 methods. e, f Western blot of indicated proteins in WCL from 293 T cells treated with 50 µg/ml cycloheximide (CHX) and harvested at different time points. Data were shown as mean ± SD (n = 3) f. The p-values were calculated using the Two-way ANOVA test. n.s., non-significant. g, i 293 T cells were transfected with the indicated plasmids. 24 h after transfection, the WCLs were prepared and the in vivo ubiquitination assays were performed. The polyubiquitinated forms of BRAF were detected by Western blot with anti-HA antibody. h 293 T cells were infected with lentivirus expressing SPOP-specific shRNA or negative control. The stable cell lines were transfection with HA-Ub and the cell lysates were prepared and immunoprecipitation was performed. SPOP promotes non‑degradative ubiquitination of BRAF The polyubiquitinated forms of BRAF were detected by Western blot with anti-HA antibody Feng et al. Cell & Bioscience (2022) 12:211 Page 6 of 15 Feng et al. Cell & Bioscience (2022) 12:211 Feng et al. Cell & Bioscience (2022) 12:211 ubiquitination (Fig. 2i), indicating that SPOP may cata- lyze the synthesis of mixed-linkage polyUb chains on BRAF. We also utilized linkage-specific K27/K29/48/63- Ub antibodies to demonstrate that ubiquitinated BRAF contains K27-, K29-, K48-, and K63-Ub linkages (Addi- tional file 1: Fig. S1e). Taken together, our data indicate that SPOP promotes non-degradative ubiquitination of BRAF. lines elevated the protein levels of BRD4 and Caprin1, but not BRAF (Fig. 2d, Additional file 1: Fig. S1a–c). Moreo- ver, the half-life of BRAF was comparable between paren- tal and SPOP KO cells (Fig. 2e, f). Although BRAF was not degraded by SPOP, it was robustly polyubiquitinated in response to the co-expression of SPOP-WT but not by the SPOP-ΔBTB or -ΔMATH mutant (Fig. 2g). We fur- ther demonstrated that the SPOP–CUL3–RBX1 E3 ubiq- uitin ligase complex catalyzed BRAF polyubiquitination in vitro (Additional file 1: Fig. S1d). Accordingly, SPOP depletion decreased the ubiquitination levels of endog- enous BRAF (Fig. 2h). These results indicate that SPOP induces non-degradative polyubiquitination of BRAF. The SBC motif in BRAF is required for SPOP‑mediated BRAF ubiquitination To examine whether the potential SBC motif is required for the SPOP-BRAF interaction, we generated a BRAF mutant in which the SBC motif was deleted. Co-IP assay results showed that SPOP only interacted with wild- type BRAF but did not bind to the BRAF-ΔSBC mutant (Fig. 3a, Additional file 1: Fig. S2a). The deletion of the SBC motif in BRAF abrogated SPOP-mediated BRAF ubiquitination (Fig. 3b). Mutations in amino acids in the SBC motif considerably reduced the SPOP-BRAF inter- action (Fig. 3c) and SPOP-mediated BRAF ubiquitination (Fig. 3d). Given that SPOP-mediated BRAF ubiquitination is non-degradative, we examined the polyUb chain-linkage specificity of BRAF. We performed in vivo ubiquitina- tion assays using a panel of ubiquitin mutants containing single KR mutations at each of the seven lysine residues. Co-expression of either Ub-KR mutant did not obvi- ously alter SPOP-mediated BRAF ubiquitination. We also used a reciprocal series of Ub-KO mutants that contain only one lysine residue, with the other six lysine residues mutated to arginine. The expression of either Ub-KO mutant nearly abolished SPOP-mediated BRAF Given that BRAF interacts with SPOP in an SBC motif-dependent manner, we further explored whether Fig. 3 The SBC motif in BRAF is recognized by SPOP. a, c, e Western blot of WCL and co-IP samples of anti-FLAG antibody obtained from 293 T cells transfected with BRAF-WT or deletion mutants. b, d, f 293 T cells were transfected with the indicated plasmids. 24 h after transfection, the WCLs were prepared and the in vivo ubiquitination assays were performed. The polyubiquitinated forms of BRAF were detected by Western blot with anti-HA antibody Fig. 3 The SBC motif in BRAF is recognized by SPOP. a, c, e Western blot of WCL and co-IP samples of anti-FLAG antibody obtained from 293 T cells transfected with BRAF-WT or deletion mutants. b, d, f 293 T cells were transfected with the indicated plasmids. 24 h after transfection, the WCLs were prepared and the in vivo ubiquitination assays were performed. The polyubiquitinated forms of BRAF were detected by Western blot with anti-HA antibody Fig. 3 The SBC motif in BRAF is recognized by SPOP. a, c, e Western blot of WCL and co-IP samples of anti-FLAG antibody obtained from 293 T cells transfected with BRAF-WT or deletion mutants. b, d, f 293 T cells were transfected with the indicated plasmids. SPOP suppresses MAPK/ERK pathway activation SPOP suppresses MAPK/ERK pathway activation BRAF plays a critical role in the regulation of MAPK/ ERK pathway activation [3]. We examined MAPK/ERK activity in SPOP KO and SPOP overexpressed cells. The induction of FLAG-SPOP by doxycycline treatment led to a marked decrease in the phosphorylation levels of MEK1/2, ERK1/2, and MSK1, indicating that MAPK/ ERK activation was attenuated (Fig. 6a). Conversely, stronger MAPK/ERK activation was observed when SPOP was ablated (Fig. 6b). Moreover, SPOP-ΔNLS overexpression attenuated the MAPK/ERK pathway acti- vation in EGF-stimulated cells (Fig. 6c, d). Conversely, SPOP KO enhanced MAPK/ERK pathway activation in EGF-stimulated cells (Fig. 6e, f). Stronger MAPK/ ERK activation was observed in SPOP KO Ishikawa cells reconstituted with the SPOP-S80R mutant than in those reconstituted with the SPOP-Y87C mutant, which retains a similar binding capacity to BRAF as SPOP-WT (Addi- tional file 1: Fig. S3a, b). The SBC motif in BRAF is required for SPOP‑mediated BRAF ubiquitination 24 h after transfection, the WCLs were prepared and the in vivo ubiquitination assays were performed. The polyubiquitinated forms of BRAF were detected by Western blot with anti-HA antibody Feng et al. Cell & Bioscience (2022) 12:211 Page 7 of 15 Page 7 of 15 cancer-associated BRAF mutations that occur in the SBC motif would attenuate the SPOP-BRAF interaction and permit BRAF to evade SPOP-mediated ubiquitination. To this end, we examined the cancer sequencing data deposited in cancer gene mutation databases (cBioPortal and COSMIC). We identified a BRAF-T121I mutation in glioblastoma and BRAF-S122Y mutation in osteosar- coma (Fig. 1b, Additional file 1: Table. S3). We found that the SPOP-binding capacity of these BRAF mutants was markedly reduced (Fig. 3e, Additional file 1: Fig. S2b), and SPOP-mediated ubiquitination of these mutants was also markedly attenuated (Fig. 3f). Taken together, our data indicate that cancer-derived BRAF mutations occur- ring in the SBC motif allow BRAF to evade SPOP-medi- ated ubiquitination. sequence (SPOP-ΔNLS) accumulated exclusively in the cytoplasm in punctate patterns and colocalized perfectly with cytoplasmic BRAF. Moreover, the deletion of the SBC motif in BRAF did not alter its diffuse cytoplasmic localization, and the SPOP-induced speckle pattern of BRAF was not observed (Fig. 5a). Next, we investigated the effect of cancer-associated SPOP mutations on BRAF localization. We focused on three hotspot mutants (E47K, S80R, and W131G). Three cancer-associated SPOP mutants lacking the NLS sequence (ΔNLS-E47K, -S80R, and -W131G) accumu- lated exclusively in the cytoplasm in punctate patterns similar to those of SPOP-ΔNLS, but these mutants did not colocalize with BRAF. Similarly, the cancer-associ- ated BRAF-T121I mutant did not co-localize with SPOP (Fig. 5b). Our previous studies showed that the SPOP- ΔNLS mutant was more effective in promoting the ubiq- uitination of its cytoplasmic substrates (INF2, MyD88, Caprin1, and HIPK2) than SPOP-WT, but it was una- ble to ubiquitinate nuclear substrates [19–22]. Indeed, we found that SPOP-ΔNLS immunoprecipitated more BRAF than SPOP-WT (Fig. 5c) and SPOP-ΔNLS was more effective in promoting BRAF ubiquitination than SPOP-WT (Fig. 5d). Taken together, our data indicate that SPOP can recruit BRAF into cytoplasmic speckles and that this activity strictly depends on its cytoplasmic localization. EC‑ and PCa‑associated SPOP mutants are defective in promoting BRAF ubiquitination To date, the vast majority of SPOP mutations associated with EC or PCa occur within the MATH domain, which is responsible for selective substrate binding (Fig. 4a, b) [10, 11]. We postulated that EC- or PCa-associated SPOP mutants may be defective in mediating BRAF ubiquitina- tion. The BRAF-binding ability of EC-associated SPOP mutants was moderately or severely impaired as com- pared to that of SPOP-WT (Fig. 4c). SPOP-mediated BRAF ubiquitination was moderately or severely attenu- ated in these mutants (Fig. 4d). Similar effects were observed when PCa-associated SPOP mutants were tested (Fig. 4e, f). In accordance with a previous study showing that cancer-associated SPOP mutants function as dominant-negative variants to deregulate their sub- strates [12], we found that co-expression of the SPOP mutant markedly reduced the interaction between SPOP- WT and BRAF (Fig. 4g), resulting in the suppression of wild-type SPOP-mediated BRAF ubiquitination (Fig. 4h). Taken together, our data indicate that EC or PCa-associ- ated SPOP mutants exert dominant-negative effects by downregulating BRAF ubiquitination due to impaired BRAF binding capacity. Cytoplasmic but not nuclear SPOP promotes BRAF ubiquitination BRAF is a cytoplasmic protein kinase. Our previous study showed that SPOP is localized exclusively in the nucleus as speckles or in both the cytoplasm and nucleus [19]. We investigated the subcellular localization of the SPOP-BRAF interaction. BRAF was diffusely localized in the cytoplasm. When SPOP was localized in both the cytoplasm and nucleus, BRAF was recruited into SPOP speckles in the cytoplasm. When SPOP was localized exclusively in the nucleus, it did not co-localize with cytoplasmic BRAF. We found that SPOP lacking the NLS i To determine whether the escape of SPOP-medi- ated-BRAF ubiquitination has any impact on MAPK/ ERK pathway activation, we reconstituted BRAF- KO Ishikawa cells (Additional file 1: Fig. S3c–e) with BRAF-WT or the -T121I mutant, which is incapable of binding to SPOP. As shown in Fig. 6g, h, EGF-induced Feng et al. Cell & Bioscience (2022) 12:211 Page 8 of 15 Fig. 4 EC- or PCa-associated SPOP mutants are defective in promoting BRAF ubiquitination. a, b Distribution of the recurrent mutation sites on the SPOP found in EC(a) or PCa b. c, e, g Western blot of WCL and co-IP samples of anti-FLAG antibody obtained from (293 T cells transfected with the indicated plasmids. d, f, h 293 T cells were transfected with the indicated plasmids. The WCLs were prepared and the in vivo ubiquitination assays were performed. The polyubiquitinated forms of BRAF were detected by Western blot with anti-HA antibody BRAF ubiquitination. a, b Distribution of the recurrent mutation sites on the ples of anti-FLAG antibody obtained from (293 T cells transfected with the d plasmids. The WCLs were prepared and the in vivo ubiquitination assays BRAF ubiquitination. a, b Distribution of the recurrent mutation sites on the ples of anti-FLAG antibody obtained from (293 T cells transfected with the d plasmids. The WCLs were prepared and the in vivo ubiquitination assays y Western blot with anti-HA antibody Fig. 4 EC- or PCa-associated SPOP mutants are defective in promoting BRAF ubiquitination. a, b Distribution of the recurrent mutation sites on the SPOP found in EC(a) or PCa b. c, e, g Western blot of WCL and co-IP samples of anti-FLAG antibody obtained from (293 T cells transfected with the indicated plasmids. d, f, h 293 T cells were transfected with the indicated plasmids. The WCLs were prepared and the in vivo ubiquitination assays were performed. Cytoplasmic but not nuclear SPOP promotes BRAF ubiquitination The polyubiquitinated forms of BRAF were detected by Western blot with anti-HA antibody MAPK/ERK activation was stronger in BRAF-KO Ishi- kawa cells reconstituted with the BRAF-T121 mutant than in those reconstituted with BRAF-WT. We found that stable overexpression of SPOP-WT or ΔNLS mutant reduced the interaction between BRAF and its upstream regulator, H-RAS, and downstream effec- tors MEK1/2 and ERK1/2 (Fig. 6i). Conversely, stable overexpression of SPOP-S80R or -F125V, increased the interactions between BRAF and MAPK/ERK path- way components. Surprisingly, stable overexpression of the SPOP-Y87C mutant even moderately reduced the interactions between BRAF and MAPK/ERK path- way components (Fig. 6j). Moreover, the interactions between BRAF and MAPK/ERK pathway components were moderately increased in BRAF-KO Ishikawa cells reconstituted with the BRAF-T121 mutant as com- pared to those reconstituted with BRAF-WT (Fig. 6k). Taken together, our data indicate that SPOP suppresses MAPK/ERK pathway activation. Page 9 of 15 Feng et al. Cell & Bioscience (2022) 12:211 Feng et al. Cell & Bioscience Fig. 5 The SPOP-BRAF interaction occurs in the cytoplasm. a, b Representative images of HeLa cells transfected with indicated plasmids, and hen immunostained with Myc (SPOP), FLAG (BRAF), and DAPI. Scale bar, 10 μm. Enlargements of selected areas were shown. c Western blot of the ndicated proteins in WCL and samples from co-IP with anti-FLAG antibody in 293 T cells transfected with the indicated plasmids. d 293 T cells were ransfected with the indicated plasmids. The WCLs were prepared and the in vivo ubiquitination assays were performed. The polyubiquitinated orms of BRAF were detected by Western blot with anti-HA antibody Fig. 5 The SPOP-BRAF interaction occurs in the cytoplasm. a, b Representative images of HeLa cells transfected with indicated plasmids, and then immunostained with Myc (SPOP), FLAG (BRAF), and DAPI. Scale bar, 10 μm. Enlargements of selected areas were shown. c Western blot of the indicated proteins in WCL and samples from co-IP with anti-FLAG antibody in 293 T cells transfected with the indicated plasmids. d 293 T cells were transfected with the indicated plasmids. The WCLs were prepared and the in vivo ubiquitination assays were performed. The polyubiquitinated forms of BRAF were detected by Western blot with anti-HA antibody Fig. 5 The SPOP-BRAF interaction occurs in the cytoplasm. a, b Representative images of HeLa cells transfected with indicated plasmids, and then immunostained with Myc (SPOP), FLAG (BRAF), and DAPI. Scale bar, 10 μm. Enlargements of selected areas were shown. Cytoplasmic but not nuclear SPOP promotes BRAF ubiquitination Similarly, Transwell assay results showed that BRAF-T121I mutant expression resulted in increased cell migration and inva- sion compared to BRAF-WT (Fig. 7c, d). Moreover, the 3D sphere formation assay results showed that the num- ber and size of spheres in BRAF KO cells reconstituted with the BRAF-T121I mutant were markedly increased compared to those reconstituted with BRAF-WT, sug- gesting that the cancer-derived BRAF mutant increased anchorage-independent cell growth (Fig. 7e, f). Taken together, our data indicate that loss of SPOP-mediated BRAF ubiquitination promotes cell malignancy. at a markedly reduced growth rate, as demonstrated by colony formation and CCK-8 assays. Reconstitution with BRAF-WT restored BRAF-KO cell growth in vitro, and more importantly, BRAF-KO cells reconstituted with the BRAF-T121I mutant showed a higher growth rate than those reconstituted with BRAF-WT (Fig. 7a, b). Similarly, Transwell assay results showed that BRAF-T121I mutant expression resulted in increased cell migration and inva- sion compared to BRAF-WT (Fig. 7c, d). Moreover, the 3D sphere formation assay results showed that the num- ber and size of spheres in BRAF KO cells reconstituted with the BRAF-T121I mutant were markedly increased compared to those reconstituted with BRAF-WT, sug- gesting that the cancer-derived BRAF mutant increased anchorage-independent cell growth (Fig. 7e, f). Taken together, our data indicate that loss of SPOP-mediated BRAF ubiquitination promotes cell malignancy. BRAF and the MAPK/ERK pathway components were only moderately increased in BRAF-T121 reconstituted cells to BRAF-WT-reconstituted cells. It would be inter- esting to investigate whether this effect is more obvious in growth factor-treated conditions. Taken together, our data raise the possibility that disruption of the SPOP- BRAF regulatory axis may promote malignant transfor- mation of cancer cells. BRAF and the MAPK/ERK pathway components were only moderately increased in BRAF-T121 reconstituted cells to BRAF-WT-reconstituted cells. It would be inter- esting to investigate whether this effect is more obvious in growth factor-treated conditions. Taken together, our data raise the possibility that disruption of the SPOP- BRAF regulatory axis may promote malignant transfor- mation of cancer cells. Proteins can be tagged with mono-ubiquitin or modi- fied by polyUb chains that can vary in length and link- age specificity, and these variations influence how ubiquitination signals are interpreted [25]. Accumulat- ing evidence suggests that both degradative and non- degradative ubiquitination play vital roles in modulating BRAF protein stability and function. RNF149, FBXW7, and TRAF2 have been reported as E3 ubiquitin ligases of BRAF [26–28]. (See figure on next page.) Fig. 6 SPOP suppresses the activation of MAPK/ERK cascade. a Western blot of indicated proteins in WCLs from Tet-on-inducible Ishikawa cells treated with DOX. b Western blot of the indicated proteins in WCLs from parental and SPOP-KO Ishikawa cells. c, d EV and SPOP-ΔNLS overexpressed Ishikawa cells were serum-starved for 48 h and then treated with EGF (100 nM) for the corresponding times. The WCLs were prepared for WB analysis. At each time point, the intensity of p-ERK1/2, p-MEK1/2, p-MSK1 was normalized to the intensity of ERK1/2, MEK1/2, MSK1 respectively and then to the value at 0 min d. e, f Parental and SPOP-KO Ishikawa cells were serum-starved for 48 h and then treated with EGF (100 nM) for the corresponding times. The WCLs were prepared for WB analysis. At each time point, the intensity of p-ERK1/2, p-MEK1/2, p-MSK1 was normalized to the intensity of ERK1/2, MEK1/2, MSK1 respectively and then to the value at 0 min f. g, h SPOP-KO Ishikawa cells stably overexpressing BRAF-WT, or T121I mutant were serum-starved for 48 h and then treated with EGF (100 nM) for the corresponding times. The WCLs were prepared for WB analysis. At each time point, the intensity of p-ERK1/2, p-MEK1/2, p-MSK1 was normalized to the intensity of ERK1/2, MEK1/2, MSK1 respectively and then to the value at 0 min h. i, j The WCLs from SPOP-KO Ishikawa cells stably overexpressing EV, SPOP-WT, SPOP- ΔNLS i and mutants j were prepared and subjected to co-IP with anti-BRAF antibody. The immunoprecipitates were analyzed by WB with the indicated antibodies. k The WCLs from BRAF-KO Ishikawa cells stably overexpressing BRAF-WT or T121I mutant were prepared and subjected to co-IP with anti-BRAF antibody. The immunoprecipitates were analyzed by WB with the indicated antibodies. The band intensity of the indicated proteins was first normalized to loading input l and the value was further normalized to the one in cells. All data shown are mean values ± SD (n = 3). The p-values were calculated using the Two-way ANOVA test in c, e, g. p < 0.05, p < 0.01, p < 0.001, *p < 0.0001 Cytoplasmic but not nuclear SPOP promotes BRAF ubiquitination USP28, a deubiquitinating enzyme, causes BRAF degradation by stabilizing FBXW7 [29]. In melanoma cells treated with proinflammatory cytokines, the HECT domain-containing E3 ligase ITCH catalyzes the non-degradative K27-linked ubiquitination of BRAF, which leads to PP2A recruitment, 14-3-3 dissociation, and sustained BRAF/MEK/ERK pathway activation [30]. This effect is distinct from our finding that SPOP-medi- ated BRAF ubiquitination inactivates BRAF. One possible explanation for this discrepancy could be that the differ- ent linkage types of polyUb chains catalyzed by CRL3- SPOP or ITCH determine the distinct fates of BRAF. Whether PolyUb chains on BRAF catalyzed by CRL3- SPOP directly hinder BRAF complex assembly or recruit other ubiquitin-binding proteins should be investigated in future studies. Cytoplasmic but not nuclear SPOP promotes BRAF ubiquitination c Western blot of the indicated proteins in WCL and samples from co-IP with anti-FLAG antibody in 293 T cells transfected with the indicated plasmids. d 293 T cells were transfected with the indicated plasmids. The WCLs were prepared and the in vivo ubiquitination assays were performed. The polyubiquitinated forms of BRAF were detected by Western blot with anti-HA antibody Fig. 5 The SPOP-BRAF interaction occurs in the cytoplasm. a, b Representative images of HeLa cells transfected with indicated plasmids, and then immunostained with Myc (SPOP), FLAG (BRAF), and DAPI. Scale bar, 10 μm. Enlargements of selected areas were shown. c Western blot of the indicated proteins in WCL and samples from co-IP with anti-FLAG antibody in 293 T cells transfected with the indicated plasmids. d 293 T cells were transfected with the indicated plasmids. The WCLs were prepared and the in vivo ubiquitination assays were performed. The polyubiquitinated forms of BRAF were detected by Western blot with anti-HA antibody BRAF‑T121I mutant is more effective to promote cell growth, migration, and invasion than BRAF‑WT We found that stable overexpression of SPOP-WT reduced the growth of Ishikawa, KLE, and SPEC-2 cells as compared to parental cells. In contrast, BRAF‑T121I mutant is more effective to promote cell growth, migration, and invasion than BRAF‑WT We found that stable overexpression of SPOP-WT reduced the growth of Ishikawa, KLE, and SPEC-2 cells as compared to parental cells. In contrast, BRAF‑T121I mutant is more effective to promote cell growth, migration, and invasion than BRAF‑WT stable overexpression of EC-associated SPOP mutants increased the growth of KLE, SPEC-2, and Ishikawa cells as compared to parental cells (Additional file 1: Fig. S4a–d). Consistent with the substantial roles of BRAF in cellular malignancy [5], BRAF KO Ishikawa cells grew We found that stable overexpression of SPOP-WT reduced the growth of Ishikawa, KLE, and SPEC-2 cells as compared to parental cells. In contrast, Page 10 of 15 Feng et al. Cell & Bioscience (2022) 12:211 Page 10 of 15 Feng et al. Cell & Bioscience (2022) 12:211 at a markedly reduced growth rate, as demonstrated by colony formation and CCK-8 assays. Reconstitution with BRAF-WT restored BRAF-KO cell growth in vitro, and more importantly, BRAF-KO cells reconstituted with the BRAF-T121I mutant showed a higher growth rate than those reconstituted with BRAF-WT (Fig. 7a, b). Discussion Aberrant activation of the MAPK/ERK cascade is com- mon in human cancer. BRAF mutations are frequently observed in melanoma, thyroid cancer, and others. In contrast, oncogenic BRAF mutations are uncommon in EC and PCa, with prevalence rates ranging from 2 to 5%, depending on the specific case series [23, 24]. Whether specific genetic alterations drive aberrant regulation of wild-type BRAF activity in EC and PCa remains poorly understood. In the current study, we showed that the CRL3-SPOP complex mediates the non-degradative ubiquitination of BRAF, which reduces the interaction between BRAF and other MAPK/ERK pathway com- ponents, resulting in subsequent MAPK/ERK inactiva- tion (Fig. 8). Importantly, EC- or PCa-associated SPOP mutants showed reduced capacity to ubiquitinate BRAF. Moreover, some cancer-associated BRAF mutants (T121I and S122Y) evaded SPOP-mediated regulation and were stronger activators of the MAPK/ERK cascade than wild-type BRAF. We noted that the interactions between The CRL3-SPOP complex catalyzes both degradative and non-degradative ubiquitination depending on the substrate. For example, CRL3-SPOP catalyzes K27-linked non-degradative ubiquitination of Geminin, which pre- vents DNA replication over-firing by indirectly blocking the association of Cdt1 with the MCM protein complex Page 11 of 15 Feng et al. Cell & Bioscience (2022) 12:211 Fig. 6 (See legend on previous page.) Fig. 6 (See legend on previous page.) Feng et al. Cell & Bioscience (2022) 12:211 Page 12 of 15 Fig. 7 Deficiency in SPOP-mediated BRAF ubiquitination confers oncogenicity. a Clone formation assays in parental, BRAF-KO Ishikawa cells, or BRAF-KO Ishikawa cells stably overexpressing BRAF-WT or BRAF-T121I mutant. Quantitative data are shown in the right panel. Data are shown as the means ± SD (n = 4). b CCK-8 assays in parental, BRAF-KO Ishikawa cells, or BRAF-KO Ishikawa cells stably overexpressing BRAF-WT or BRAF-T121I mutant. Data are shown as the means ± SD (n = 4). c Transwell migration assays in parental, BRAF-KO Ishikawa cells, or BRAF-KO Ishikawa cells stably overexpressing BRAF-WT or BRAF-T121I mutant. Quantitative data are shown in the right panel. Data are shown as the means ± SD (n = 4). d Transwell invasion assays in parental, BRAF-KO Ishikawa cells, or BRAF-KO Ishikawa cells stably overexpressing BRAF-WT or BRAF-T121I mutant. Quantitative data are shown in the right panel. Data are shown as the means ± SD (n = 4). e Representative pictures of 3D sphere formation assays in parental, BRAF-KO Ishikawa cells, or BRAF-KO Ishikawa cells stably overexpressing BRAF-WT or BRAF-T121I mutant. Discussion Scale bars, 10 × image: 400 μm, 40 × image: 50 μm. f Quantitative data of parental, BRAF KO and rescued with BRAF or BRAF mutation in BRAF-knock-out Ishikawa cells after 10 days in three-dimensional culture. Numbers and average sizes per sphere. Data are shown as means ± SD (n = 4 in left, n = 10 in right). The p-values were calculated using the One-way ANOVA test in a, c, d, f and the Two-way ANOVA test in b. p < 0.05, p < 0.01, p < 0.001, **p < 0.0001 Fig. 7 Deficiency in SPOP-mediated BRAF ubiquitination confers oncogenicity. a Clone formation assays in parental, BRAF-KO Ishikawa cells, or BRAF-KO Ishikawa cells stably overexpressing BRAF-WT or BRAF-T121I mutant. Quantitative data are shown in the right panel. Data are shown as the means ± SD (n = 4). b CCK-8 assays in parental, BRAF-KO Ishikawa cells, or BRAF-KO Ishikawa cells stably overexpressing BRAF-WT or BRAF-T121I mutant. Data are shown as the means ± SD (n = 4). c Transwell migration assays in parental, BRAF-KO Ishikawa cells, or BRAF-KO Ishikawa cells stably overexpressing BRAF-WT or BRAF-T121I mutant. Quantitative data are shown in the right panel. Data are shown as the means ± SD (n = 4). d Transwell invasion assays in parental, BRAF-KO Ishikawa cells, or BRAF-KO Ishikawa cells stably overexpressing BRAF-WT or BRAF-T121I mutant. Quantitative data are shown in the right panel. Data are shown as the means ± SD (n = 4). e Representative pictures of 3D sphere formation assays in parental, BRAF-KO Ishikawa cells, or BRAF-KO Ishikawa cells stably overexpressing BRAF-WT or BRAF-T121I mutant. Scale bars, 10 × image: 400 μm, 40 × image: 50 μm. f Quantitative data of parental, BRAF KO and rescued with BRAF or BRAF mutation in BRAF-knock-out Ishikawa cells after 10 days in three-dimensional culture. Numbers and average sizes per sphere. Data are shown as means ± SD (n = 4 in left, n = 10 in right). The p-values were calculated using the One-way ANOVA test in a, c, d, f and the Two-way ANOVA test in b. p < 0.05, p < 0.01, p < 0.001, **p < 0.0001 Fig. 7 Deficiency in SPOP-mediated BRAF ubiquitination confers oncogenicity. a Clone formation assays in parental, BRAF-KO Ishikawa cells, or BRAF-KO Ishikawa cells stably overexpressing BRAF-WT or BRAF-T121I mutant. Quantitative data are shown in the right panel. Discussion In the current study, we found that SPOP-mediated BRAF ubiquitination had no predominant ubiquitination linkage specificity, in sharp contrast to other SPOP substrates reported using Ub linkage mutants. However, the mechanisms underlying this phenomenon are currently unknown. Given that Ub linkage mutants have been artificially generated, non- physiological effects may occur. Alternatively, we used a panel of linkage-specific Ub antibodies to demonstrate that ubiquitinated BRAF at least contains K27-, K29-, K48-, and K63-Ub linkages. Further studies are needed to thoroughly clarify the polyUb-linkage composition using mass spectrometry-based proteomics, such as the selected reaction monitoring (Ub-AQUA-SRM) method [33]. [31]. CRL3-SPOP catalyzed mixed K6-, K27-, and K29- linked non-degradative ubiquitination of p62, which suppresses p62-dependent autophagy [32]. CRL3-SPOP catalyzes mixed K27-, K29-, K33-, K48-, and K63-linked non-degradative ubiquitination of INF2 and HIPK2 to regulate mitochondrial division and DNA damage response, respectively [19, 22]. In the current study, we found that SPOP-mediated BRAF ubiquitination had no predominant ubiquitination linkage specificity, in sharp contrast to other SPOP substrates reported using Ub linkage mutants. However, the mechanisms underlying this phenomenon are currently unknown. Given that Ub linkage mutants have been artificially generated, non- physiological effects may occur. Alternatively, we used a panel of linkage-specific Ub antibodies to demonstrate that ubiquitinated BRAF at least contains K27-, K29-, K48-, and K63-Ub linkages. Further studies are needed to thoroughly clarify the polyUb-linkage composition using mass spectrometry-based proteomics, such as the selected reaction monitoring (Ub-AQUA-SRM) method [33]. Conclusionsi Our findings show that SPOP and BRAF are biochemi- cally linked. SPOP promoted non-degradative ubiquit- ination of BRAF, which resulted in subsequent MAPK/ ERK inactivation. Importantly, PCa- or EC-associated SPOP mutations lead to abnormal activation of the MAPK/ERK pathway in a BRAF-dependent man- ner. Our data raise the possibility that disruption of the SPOP-BRAF regulatory axis may promote the malignant transformation of cancer cells (Fig. 8). Given that BRAF activity is elevated in SPOP-mutated cancers, elucidation of the SPOP-BRAF regulatory axis in further studies will be helpful in designing therapeutic strategies for SPOP- mutated cancers. Targeted treatment with BRAF and MEK inhibition has demonstrated clinical efficacy in melanomas harboring the BRAF-V600E/K mutation, and the combination of dabrafenib and trametinib, vemurafenib, and cobimetinib represent effective therapeutic options for these patients [5]. However, vemurafenib and dabrafenib caused a para- doxical induction of MAPK/ERK pathway activation and induced proliferation via a RAS-dependent mechanism Discussion Data are shown as the means ± SD (n = 4). b CCK-8 assays in parental, BRAF-KO Ishikawa cells, or BRAF-KO Ishikawa cells stably overexpressing BRAF-WT or BRAF-T121I mutant. Data are shown as the means ± SD (n = 4). c Transwell migration assays in parental, BRAF-KO Ishikawa cells, or BRAF-KO Ishikawa cells stably overexpressing BRAF-WT or BRAF-T121I mutant. Quantitative data are shown in the right panel. Data are shown as the means ± SD (n = 4). d Transwell invasion assays in parental, BRAF-KO Ishikawa cells, or BRAF-KO Ishikawa cells stably overexpressing BRAF-WT or BRAF-T121I mutant. Quantitative data are shown in the right panel. Data are shown as the means ± SD (n = 4). e Representative pictures of 3D sphere formation assays in parental, BRAF-KO Ishikawa cells, or BRAF-KO Ishikawa cells stably overexpressing BRAF-WT or BRAF-T121I mutant. Scale bars, 10 × image: 400 μm, 40 × image: 50 μm. f Quantitative data of parental, BRAF KO and rescued with BRAF or BRAF mutation in BRAF-knock-out Ishikawa cells after 10 days in three-dimensional culture. Numbers and average sizes per sphere. Data are shown as means ± SD (n = 4 in left, n = 10 in right). The p-values were calculated using the One-way ANOVA test in a, c, d, f and the Two-way ANOVA test in b. p < 0.05, p < 0.01, p < 0.001, **p < 0.0001 Feng et al. Cell & Bioscience (2022) 12:211 Page 13 of 15 Fig. 8 A model was proposed according to the findings of the present study. The schematic diagram depicts a model that SPOP regulates the MAPK/ERK pathway through promoting BRAF ubiquitination Fig. 8 A model was proposed according to the findings of the present study. The schematic diagram depicts a model that SPOP regulates the MAPK/ERK pathway through promoting BRAF ubiquitination in wild-type BRAF cells [34]. AZ304, a potent dual BRAF inhibitor of both wild-type and BRAF-V600E mutant BRAF, exerts anti-tumor effects in colorectal cancer inde- pendent of BRAF genetic status [35]. Given that BRAF activity is elevated in SPOP-mutated cancers, the efficacy of AZ304 in these cancers should be investigated. [31]. CRL3-SPOP catalyzed mixed K6-, K27-, and K29- linked non-degradative ubiquitination of p62, which suppresses p62-dependent autophagy [32]. CRL3-SPOP catalyzes mixed K27-, K29-, K33-, K48-, and K63-linked non-degradative ubiquitination of INF2 and HIPK2 to regulate mitochondrial division and DNA damage response, respectively [19, 22]. Author details Cas9-mediated knockout of SPOP by sgRNA#1 or sgRNA#2 in Ishikawa cells. (c) Sanger sequencing confirming that the SPOP gene was edited by sgRNA#1 or sgRNA#2 in Ishikawa cells. (d) Western blot of the products of in vitro ubiquitination assays performed by incubating the reconstituted SPOP–CUL3–RBX1 E3 ligase complex with E1 and E2 enzymes, ubiquitin and GST-BRAF at 30 °C for 2 h. (e) Western blot of the products of in vivo ubiquitination assays from 293T cells transfected with the indicated plasmids. Figure S2. The SBC motif in BRAF is recognized by SPOP (related to Figure 3). (a, b) Western blot of WCL and co-IP samples of anti-FLAG antibody obtained from 293T cells transfected with the indicated plas- mids. Figure S3. SPOP suppresses the activation of MAPK/ERK cascade (related to Figure 6). (a, b) SPOP-KO Ishikawa cells stably overexpress- ing SPOP-Y87C, or S80R mutant were serum-starved for 48 hr and then treated with EGF (100 nM) for the corresponding times. The WCLs were prepared for WB analysis. At each time point, the intensity of p-ERK1/2, p-MEK1/2, p-MSK1 was normalized to the intensity of ERK1/2, MEK1/2, MSK1 respectively and then to the value at 0 min (b). (c) Schematic of CRISPR/Cas9-mediated knockout of BRAF by sgRNA in Ishikawa cells. (d) Sanger sequencing confirming that the BRAF gene was edited by sgRNA in Ishikawa cells. (e) Western blot of the indicated proteins in WCLs from parental and BRAF-KO Ishikawa cells. All data shown are mean values ± SD(n=3). The p-values were calculated using the Two-way ANOVA test in (b). p<0.05, p<0.01, p<0.001, **p<0.0001. Figure S4. The impact of SPOP-WT and EC-associated SPOP mutants on the growth of three EC cell lines. (a-c) CCK-8 assays in KLE (a), SPEC-2 (b), and Ishikawa (c) cells stably overexpressing EV, SPOP-WT or EC-associated SPOP mutants. Data are shown as the means ± SD (n=4). The p-values were calculated using the Two-way ANOVA test in (a-c). p<0.05, p<0.01, p<0.001, ***p<0.0001. (d) Western blot of the indicated proteins in WCL from KLE, SPEC-2, and Ishikawa cells stably overexpressing EV, SPOP-WT, or EC-associated SPOP mutants. Table S1. Sequence information. Table S2. Antibody and recombinant protein information. Table S3. Cell cultures, chemicals and Kit. 1 Shanghai Stomatological Hospital & School of Stomatology, State Key Laboratory of Genetic Engineering, MOE Engineering Research Center of Gene Technology, Shanghai Engineering Research Center of Industrial Microorganisms, School of Life Sciences, Fudan University, Shanghai 200438, China. References 1. Malumbres M, Barbacid M. RAS oncogenes: the first 30 years. Nat Rev Cancer. 2003;3:459–65. 2. Roberts PJ, Der CJ. Targeting the Raf-MEK-ERK mitogen-activated protein kinase cascade for the treatment of cancer. Oncogene. 2007;26:3291–310 3. Wellbrock C, Karasarides M, Marais R. The RAF proteins take centre stage. Nat Rev Mol Cell Biol. 2004;5:875–85. 4. Wan PT, Garnett MJ, Roe SM, Lee S, Niculescu-Duvaz D, Good VM, et al. Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF. Cell. 2004;116:855–67. 5. Dankner M, Rose AAN, Rajkumar S, Siegel PM, Watson IR. 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Dab- rafenib plus trametinib in patients with previously treated BRAF(V600E)- mutant metastatic non-small cell lung cancer: an open-label, multicentre phase 2 trial. Lancet Oncol. 2016;17:984–93. Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s13578-022-00950-z. The online version contains supplementary material available at https://doi. org/10.1186/s13578-022-00950-z. Additional file 1: Figure S1. SPOP promotes non-degradative ubiquitina- tion of BRAF (related to Figure 2). (a) Western blot of the indicated proteins in WCL from Ishikawa and KLE cells infected with lentivirus expressing SPOP-specific shRNAs or negative control. (b) Schematic of CRISPR/ Page 14 of 15 Page 14 of 15 Feng et al. Cell & Bioscience (2022) 12:211 Feng et al. Cell & Bioscience (2022) 12:211 Author details 2 State Key Laboratory of Genetic Engineering, Human Phenome Institute, School of Life Sciences, Fudan University, Shanghai 200438, China. 3 Obstetrics and Gynecology Hospital, NHC Key Laboratory of Reproduction Regulation, Shanghai Institute of Planned Parenthood Research, State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai 200438, China. 4 Department of Pathology, School of Basic Medical Sciences, Fudan University Shanghai Cancer Center, Fudan University, Shang- hai 200032, China. Received: 20 May 2022 Accepted: 27 December 2022 Funding Thi k This work was supported in part by the National Natural Science Foundation of China (No. 91957125, 81672558, and 81972396 to C.W.; No. 81773024 to Y.H., No. 81872260, 82172938 to PZZ). the Shanghai Science and Technology Devel- opment Foundation (No. 22ZR1406600 to C.W.; 20ZR1404500 to Y.L.) Science and Technology Research Program of Shanghai (No. 9DZ2282100). p g 11. Barbieri CE, Baca SC, Lawrence MS, Demichelis F, Blattner M, Theurillat JP, et al. Exome sequencing identifies recurrent SPOP, FOXA1 and MED12 mutations in prostate cancer. Nat Genet. 2012;44:685–9. 12. Theurillat JP, Udeshi ND, Errington WJ, Svinkina T, Baca SC, Pop M, et al. Prostate cancer. ubiquitylome analysis identifies dysregulation of effector substrates in SPOP-mutant prostate cancer. Science. 2014;346:85–9. Availability of data and materials All data generated or analyzed in this study are included in this published article. All the data supporting the findings of this study are available from the corresponding author upon request. 13. Li C, Ao J, Fu J, Lee DF, Xu J, Lonard D, et al. Tumor-suppressor role for the SPOP ubiquitin ligase in signal-dependent proteolysis of the oncogenic co-activator SRC-3/AIB1. Oncogene. 2011;30:4350–64. Consent for publication Not applicable. 16. Geng C, Rajapakshe K, Shah SS, Shou J, Eedunuri VK, Foley C, et al. Andro- gen receptor is the key transcriptional mediator of the tumor suppressor SPOP in prostate cancer. Cancer Res. 2014;74:5631–43. Author contributions 8. Lydeard JR, Schulman BA, Harper JW. Building and remodelling cullin- RING E3 ubiquitin ligases. EMBO Rep. 2013;14:1050–61. CJW conceived the study. KF, QS, DYJ, YJC, WQY, KS, and YLW performed experiments and data analyses. KF, HY, PZZ, YL, and CJW analyzed and inter- preted the data. CJW wrote and revised the manuscript. All authors read and approved the final manuscript. 9. Zhuang M, Calabrese MF, Liu J, Waddell MB, Nourse A, Hammel M, et al. Structures of SPOP-substrate complexes: insights into molecular architec- tures of BTB-Cul3 ubiquitin ligases. Mol Cell. 2009;36:39–50. 10. Le Gallo M, O’Hara AJ, Rudd ML, Urick ME, Hansen NF, O’Neil NJ, et al. Exome sequencing of serous endometrial tumors identifies recurrent somatic mutations in chromatin-remodeling and ubiquitin ligase com- plex genes. Nat Genet. 2012;44:1310–5. Declarations 14. An J, Wang C, Deng Y, Yu L, Huang H. Destruction of full-length androgen receptor by wild-type SPOP, but not prostate-cancer-associated mutants. Cell Rep. 2014;6:657–69. Ethics approval and consent to participate Not applicable. Ethics approval and consent to participate Not applicable. 15. Zhang P, Gao K, Jin X, Ma J, Peng J, Wumaier R, et al. Endometrial cancer-associated mutants of SPOP are defective in regulating estrogen receptor-alpha protein turnover. Cell Death Dis. 2015;6:e1687. Competing interests 17. Zhang J, Gao K, Xie H, Wang D, Zhang P, Wei T, et al. SPOP mutation induces DNA methylation via stabilizing GLP/G9a. Nat Commun. 2021;12:5716. p g The authors declare no competing interests. Page 15 of 15 Feng et al. Cell & Bioscience (2022) 12:211 Feng et al. Cell & Bioscience (2022) 12:211 Feng et al. Cell & Bioscience (2022) 12:211 18. Blattner M, Liu D, Robinson BD, Huang D, Poliakov A, Gao D, et al. SPOP mutation drives prostate tumorigenesis in vivo through coordinate regulation of PI3K/mTOR and AR signaling. Cancer Cell. 2017;31:436–51. 19. Jin X, Wang J, Gao K, Zhang P, Yao L, Tang Y, et al. Dysregulation of INF2- mediated mitochondrial fission in SPOP-mutated prostate cancer. PLoS Genet. 2017;13:e1006748. 20. Jin X, Shi Q, Li Q, Zhou L, Wang J, Jiang L, et al. CRL3-SPOP ubiquitin ligase complex suppresses the growth of diffuse large B-cell lymphoma by negatively regulating the MyD88/NF-kappaB signaling. Leukemia. 2020;34:1305–14. 21. Shi Q, Zhu Y, Ma J, Chang K, Ding D, Bai Y, et al. Prostate cancer-associated SPOP mutations enhance cancer cell survival and docetaxel resistance by upregulating caprin1-dependent stress granule assembly. Mol Cancer. 2019;18:170. 22. Jin X, Qing S, Li Q, Zhuang H, Shen L, Li J, et al. Prostate cancer-associated SPOP mutations lead to genomic instability through disruption of the SPOP-HIPK2 axis. Nucleic Acids Res. 2021;49:6788–803. 23. Moreno-Bueno G, Sanchez-Estevez C, Palacios J, Hardisson D, Shiozawa T. Low frequency of BRAF mutations in endometrial and in cervical carcino- mas. Clin Cancer Res. 2006;12(3865):3865–6. q y mas. Clin Cancer Res. 2006;12(3865):3865–6. 24. Cancer Genome Atlas Research N. The molecular taxonomy of primary prostate cancer. Cell. 2015;163:1011–25. 25. Brzovic PS, Klevit RE. Ubiquitin transfer from the E2 pe 25. Brzovic PS, Klevit RE. Ubiquitin transfer from the E2 pers UbcH5 so promiscuous? Cell Cycle. 2006;5:2867–73. 26. Hong SW, Jin DH, Shin JS, Moon JH, Na YS, Jung KA, et al. Ring fin- ger protein 149 is an E3 ubiquitin ligase active on wild-type v-Raf murine sarcoma viral oncogene homolog B1 (BRAF). J Biol Chem. 2012;287:24017–25. 27. Wang Q, Gao G, Zhang T, Yao K, Chen H, Park MH, et al. TRAF1 is critical for regulating the BRAF/MEK/ERK pathway in non-small cell lung carcino- genesis. Cancer Res. 2018;78:3982–94. 28. Yeh CH, Bellon M, Wang F, Zhang H, Fu L, Nicot C. Competing interests 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: Competing interests Loss of FBXW7-medi- ated degradation of BRAF elicits resistance to BET inhibitors in adult T cell leukemia cells. Mol Cancer. 2020;19:139. 29. Saei A, Palafox M, Benoukraf T, Kumari N, Jaynes PW, Iyengar PV, et al. Loss of USP28-mediated BRAF degradation drives resistance to RAF cancer therapies. J Exp Med. 2018;215:1913–28. 30. Yin Q, Han T, Fang B, Zhang G, Zhang C, Roberts ER, et al. K27-linked ubiquitination of BRAF by ITCH engages cytokine response to maintain MEK-ERK signaling. Nat Commun. 2019;10:1870. 31. Ma J, Shi Q, Cui G, Sheng H, Botuyan MV, Zhou Y, et al. SPOP mutation induces replication over-firing by impairing geminin ubiquitination and triggers replication catastrophe upon ATR inhibition. Nat Commun. 2021;12:5779. 32. Shi Q, Jin X, Zhang P, Li Q, Lv Z, Ding Y, et al. SPOP mutations promote p62/SQSTM1-dependent autophagy and Nrf2 activation in prostate can- cer. Cell Death Differ. 2022. https://doi.org/10.1038/s41418-021-00913-w. f 33. Heunis T, Lamoliatte F, Marin-Rubio JL, Dannoura A, Trost M. Technical report: targeted proteomic analysis reveals enrichment of atypical ubiqui- tin chains in contractile murine tissues. J Proteomics. 2020;229:103963. 34. Karoulia Z, Wu Y, Ahmed TA, Xin Q, Bollard J, Krepler C, et al. An integrated model of RAF inhibitor action predicts inhibitor activity against onco- genic BRAF signaling. Cancer Cell. 2016;30:501–3. 35. Ma R, Xu L, Qu X, Che X, Zhang Y, Fan Y, et al. AZ304, a novel dual BRAF inhibitor, exerts anti-tumour effects in colorectal cancer independently of BRAF genetic status. 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https://openalex.org/W2418573307	https://europepmc.org/articles/pmc5111464?pdf=render	English	null	Retrospective Study of Incidence and Prognostic Significance of Eosinophilia after Allogeneic Hematopoietic Stem Cell Transplantation: Influence of Corticosteroid Therapy	Turkish journal of hematology	2,016	cc-by	3,910	Abstract Amaç: Allojenik hematopoetik kök hücre nakli sonrası eozinofilinin önemi tartışmalıdır. Bu çalışma kortikosteroid tedavisinin etkisini hesaba katarak, eozinofilinin allojenik hematopoetik kök hücre naklinin sonuçları üzerine etkisini geriye dönük değerlendirmeyi amaçladık. Objective: The clinical significance of eosinophilia after allogeneic hematopoietic stem cell transplantation is controversial. This study aimed to retrospectively study the impact of eosinophilia on the outcome of allogeneic hematopoietic stem cell transplantation by taking into account the influence of corticosteroid therapy. Materials and Methods: We retrospectively studied 204 patients with acute myeloid leukemia, acute lymphoblastic leukemia, and myelodysplastic syndrome who underwent allogeneic hematopoietic stem cell transplantation from January 2001 to December 2010. Gereç ve Yöntemler: Ocak 2001’den Aralık 2010’a kadar akut myeloid lösemi, akut lenfoblastik lösemi ve myelodisplastik sendrom tanısıyla allojenik hematopoetik kök hücre nakli olan 204 hastayı geriye dönük değerlendirdik. Results: The median age was 43 years (minimum-maximum: 17- 65 years). Myeloablative conditioning was used in 153 patients and reduced intensity conditioning was employed in 51 patients. Donor cells were from bone marrow in 132 patients, peripheral blood in 34, and cord blood in 38. Eosinophilia was detected in 71 patients and there was no significant predictor of eosinophilia by multivariate analysis. There was no relationship between occurrence of eosinophilia and the incidence or grade of acute graft-versus-host disease when the patients were stratified according to corticosteroid treatment. Although eosinophilia was a prognostic factor for 5-year overall survival by univariate analysis, it was not a significant indicator by multivariate analysis. Bulgular: Ortanca yaş 43 (aralık: 17-65 yaş) idi. Yüz elli üç hastada miyeloablatif, 51 hastada azaltılmış yoğunluklu hazırlama rejimi uygulandı. Kök hücre kaynağı 132 hastada kemik iliği, 34 hastada periferik kan ve 38 hastada kordon kanıydı. Yetmiş bir hastada eozinofili saptandı ve çoklu değişken analizinde eozinofiliyi anlamlı olarak öngörecek bir belirteç saptanmadı. Hastalar kortikosteroid tedavisine göre gruplandığında eozinofili gelişimi ile akut graft- verus-host hastalığı sıklığı ya da derecesi arasında bağlantı yoktu. Tek değişkenli analizde eozinofili 5 yıllık genel sağkalım açısından prognostik bir faktör olmasına karşın, çok değişkenli analizde anlamlı bir belirteç değildi. Sonuç: Bu sonuçlar allojenik hematopoetik kök hücre nakli olan hastalarda eozinofilinin klinik öneminin, sistemik kortikosteroid uygulamasını dikkate alarak değerlendirilmesi gerektiğinin düşündürmektedir. Conclusion: These results suggest that the clinical significance of eosinophilia in patients receiving allogeneic hematopoietic stem cell transplantation should be assessed with consideration of systemic corticosteroid administration. Received/Geliş tarihi: January 23, 2015 Accepted/Kabul tarihi: November 18, 2015 Address for Correspondence/Yazışma Adresi: Wataru YAMAMOTO, M.D., Kanagawa Cancer Center, Department of Hematology, Yokohama, Japan Phone : 81-45-391-5761 E-mail : watadesu@yahoo.co.jp Allojenik Hematopoetik Kök Hücre Nakli Sonrası Eozinofilinin Sıklığı ve Prognostik Öneminin Değerlendirildiği Geriye Dönük Çalışma: Kortikosteroid Tedavisinin Etkisi Wataru Yamamoto1, Eriko Ogusa1, Kenji Matsumoto1, Atsuo Maruta1, Yoshiaki Ishigatsubo2, Heiwa Kana 1Kanagawa Cancer Center, Department of Hematology, Yokohama, Japan 2Yokohama City University Graduate Faculty of Medicine, Department of Internal Medicine and Clinical Immunology, Yokohama, Japan Results A total of 204 patients received allo-HSCT for AML, ALL, or MDS. The median follow-up period was 5.7 years and patients’ clinical characteristics are shown in Table 1. The median age was 43 years (minimum-maximum: 17-65 years) and there were 102 patients of each sex. The underlying disease was AML in 126 patients, ALL in 63, and MDS in 15. Myeloablative conditioning was used in 153 patients and reduced intensity conditioning was employed in 51 patients. Donor cells were from bone marrow in 132 patients, peripheral blood in 34, and cord blood in 38. Proliferation of eosinophils is induced by stimulation with cytokines [1] and eosinophilia occurs in various clinical settings. Eosinophilia is often found in patients receiving allogeneic hematopoietic stem cell transplantation (allo-HSCT) and a relationship between eosinophilia and the outcome and/or graft-versus-host disease (GVHD) has been reported [2,3,4,5,6,7,8,9,10]. However, the role of corticosteroid (CS) therapy should be taken into consideration with regard to evaluation of eosinophilia after allo-HSCT, because it is known that eosinophilia is influenced by such drugs [11,12]. Therefore, we retrospectively studied the impact of eosinophilia on the outcome of allo-HSCT by taking into account the influence of CS therapy. Eosinophilia was detected in 71 patients (34.8%). Its appearance was associated with total body irradiation (TBI), unrelated donor, and CS administration within 100 days after transplantation by univariate analysis. However, no significant predictors of eosinophilia were identified by multivariate analysis (Table 1). Among the 204 patients, 90 patients (44%) received systemic CS therapy. The reason for CS treatment was acute GVHD in 76 patients, engraftment syndrome in 4, interstitial pneumonia in 4, organizing pneumonia in 3, disease relapse in 1, diffuse alveolar hemorrhage in 1, and vasculitis in 1. The incidence of eosinophilia within 100 days after transplantation was higher in patients without CS (47/114 patients, 41.2%) than in patients with CS (24/90, 26.7%) (p=0.038). Among the 90 patients with CS, 11 were first given CS therapy after the appearance of eosinophilia. The frequency of eosinophilia was higher among patients who were not given CS therapy before eosinophilia appeared than among patients who were already receiving CS therapy (58/125 patients, 46.4% vs. 13/79, 16.5%, respectively; p<0.001). Materials and Methods Patients who underwent allo-HSCT for hematologic malignancies from January 2001 to December 2010 at the Kanagawa Cancer Center were retrospectively investigated. We defined eosinophilia as a peripheral blood eosinophil count of >500 µL on more than one occasion, while systemic steroid therapy meant CS administration at more than 0.5 mg/kg/day within 100 days after allo-HSCT. Standard-risk disease was defined as acute myeloid leukemia (AML)/acute lymphoblastic leukemia (ALL) in the first or second remission and myelodysplastic syndrome (MDS) without leukemic transformation, while high- risk disease was defined as all others. Grading of acute GVHD was done according to established criteria [13]. Abstract Anahtar Sözcükler: Eozinofili, Allojenik hematopoetik kök hücre nakli, Kortikosteroid tedavisi, Prognostik faktör, Graft-versus-host hastalığı Keywords: Eosinophilia, Allogeneic hematopoietic stem cell transplantation, Corticosteroid therapy, Prognostic factor, Graft- versus-host disease Address for Correspondence/Yazışma Adresi: Wataru YAMAMOTO, M.D., Kanagawa Cancer Center, Department of Hematology, Yokohama, Japan Phone : 81-45-391-5761 E-mail : watadesu@yahoo.co.jp 196 Yamamoto W, et al: Eosinophilia after Allogeneic Hematopoietic Stem Cell Transplantation Turk J Hematol 2016;33:196-201 Discussion In the present study, we investigated the incidence and clinical implications of eosinophilia occurring within 100 days after allo- HSCT, and we also analyzed the prognostic value of eosinophilia in relation to the influence of CS administration. The incidence of eosinophilia (34.8%) in our patient cohort was comparable with that in previous reports, although the definition of eosinophilia varies among studies. It is well known that a decrease of eosinophils is caused by CS administration [10,11]; hence, we assessed the clinical implications of eosinophilia in relation to systemic CS administration. The incidence of eosinophilia was significantly lower in patients receiving CS treatment compared with that for those without CS treatment in the present study, and the same result for patients with acute GVHD has already been described [2]. In our study, MDS, use of TBI, and transplantation from an unrelated donor were also associated with a lower incidence of eosinophilia, but the reasons for these associations are unknown. Figure 1. Outcome stratified according to the presence/absence of eosinophilia. A) Overall survival, B) cumulative incidence of relapse, C) non-relapse mortality. The relationship between eosinophilia and acute GVHD after allo- HSCT remains controversial. We could not find any association between eosinophilia and acute GVHD among patients with or without CS therapy in this study. Some previous reports suggested that eosinophilia is significantly related to the onset of acute GVHD [2,9,10]. However, Aisa et al. reported that the onset of eosinophilia within 100 days after allo-HSCT is associated with a lower rate of grade II-IV acute GVHD (43% vs. 98%; p<0.001) [3]. They speculated that this association between eosinophilia and a lower incidence of severe acute GVHD may reflect the immunosuppressive effect of Th2 cytokines, which induce eosinophilia. Figure 1. Outcome stratified according to the presence/absence of eosinophilia. A) Overall survival, B) cumulative incidence of relapse, C) non-relapse mortality. A relationship between the occurrence of eosinophilia after allo- HSCT and a good prognosis [2,3,4,5,6] has been reported previously. Figure 3. Outcomes stratified according to eosinophilia and corticosteroid therapy. A) Overall survival, B) cumulative incidence of relapse, C) non-relapse mortality. Figure 2. Outcomes stratified according to corticosteroid administration. A) Overall survival, B) cumulative incidence of relapse, C) non-relapse mortality. Figure 3. Outcomes stratified according to eosinophilia and corticosteroid therapy. A) Overall survival, B) cumulative incidence of relapse, C) non-relapse mortality. Figure 2. Outcomes stratified according to corticosteroid administration. Statistical Analysis Furthermore, the cumulative incidence of relapse showed no significant association with eosinophilia (Figures 1B, 2B, and 3B). However, non-relapse mortality was significantly higher in patients receiving CS therapy (Figures 2C and 3C). According to univariate analysis, eosinophilia was a good prognostic indicator for 5-year OS (hazard ratio: 0.6, 95% CI: 0.4-0.9; p=0.017), but it was not an independent prognostic indicator by multivariate analysis (hazard ratio: 0.8, 95% CI: 0.5-1.3; p=0.385). Finally, high-risk disease, unrelated donor, and CS therapy were adverse prognostic indicators for 5-year OS according to multivariate analysis (Table 3). OS between patients with and without eosinophilia among those not receiving CS therapy (66.3%, 95% CI: 53.6-82.1 vs. 57.4%, 95% CI: 46.5-70.8, respectively; p=0.139) (Figure 3A). Furthermore, the cumulative incidence of relapse showed no significant association with eosinophilia (Figures 1B, 2B, and 3B). However, non-relapse mortality was significantly higher in patients receiving CS therapy (Figures 2C and 3C). According to univariate analysis, eosinophilia was a good prognostic indicator for 5-year OS (hazard ratio: 0.6, 95% CI: 0.4-0.9; p=0.017), but it was not an independent prognostic indicator by multivariate analysis (hazard ratio: 0.8, 95% CI: 0.5-1.3; p=0.385). Finally, high-risk disease, unrelated donor, and CS therapy were adverse prognostic indicators for 5-year OS according to multivariate analysis (Table 3). Statistical Analysis The cumulative incidence of grade II-IV and grade III-IV acute GVHD was 45.1% and 18.6%, respectively. Table 2 shows the patients stratified according to CS treatment, GVHD grade, and occurrence of eosinophilia. There was no significant relationship between the grade of acute GVHD and occurrence of eosinophilia when patients were stratified by CS administration. Statistical analyses were performed with R software (version 2.11.1; R Development Core Team). Differences between groups were analyzed by the Wilcoxon rank sum test or Fisher’s exact test, as was appropriate for univariate analysis and logistic regression analysis for multivariate analysis. Overall survival (OS) was calculated from the date of transplantation to the date of death from any cause or the date of last follow-up. Non-relapse mortality was defined as death without disease relapse or resistance. Time-to-event curves were drawn according to the Kaplan-Meier method and the statistical significance of differences in survival was assessed by the log-rank test. Prognostic factors included age, sex mismatch, disease risk, conditioning regimen, GVHD prophylaxis, donor type, cytomegalovirus infection, CS therapy, and eosinophilia. Either the Cox proportional hazard model or the Fine-Gray proportional hazard model was used for analysis. Death without relapse was considered to be a competing risk for relapse, relapse was a competing risk for non-relapse mortality, and relapse and death without GVHD were competing risks for GVHD. The OS, cumulative incidence of relapse, and non-relapse mortality rate are stratified according to eosinophilia and systemic CS therapy in Figures 1 and 2, respectively. Patients with eosinophilia had a higher 5-year OS compared to those without eosinophilia (59.8%, 95% confidence interval [CI]: 48.9-73.1 vs. 45.4%, 95% CI: 37.5-55.0; p=0.016) (Figure 1A). In contrast, patients receiving CS therapy had a lower 5-year OS compared to those without CS therapy (38.9%, 95% CI: 29.9-50.7 vs. 60.9%, 95% CI: 52.3-70.9; p<0.001) (Figure 2A). However, there was no significant difference in 5-year OS between patients with or without eosinophilia among those who received CS therapy (47.3%, 95% CI: 30.3- 73.9 vs. 33.7%, 95% CI: 23.9-47.7, respectively; p=0.200) (Figure 3A). Similarly, there was no difference in the 5-year 197 Yamamoto W, et al: Eosinophilia after Allogeneic Hematopoietic Stem Cell Transplantation Turk J Hematol 2016;33:196-201 OS between patients with and without eosinophilia among those not receiving CS therapy (66.3%, 95% CI: 53.6-82.1 vs. 57.4%, 95% CI: 46.5-70.8, respectively; p=0.139) (Figure 3A). Discussion A) Overall survival, B) cumulative incidence of relapse, C) non-relapse mortality. 198 Yamamoto W, et al: Eosinophilia after Allogeneic Hematopoietic Stem Cell Transplantation Turk J Hematol 2016;33:196-201 Table 1. Patient characteristics and predictors of eosinophilia within 100 days after transplantation. Eosinophilia (+) (n=71) Eosinophilia (-) (n=133) Univariate Analysis Multivariate Analysis HR (95% CI) p HR (95% CI) p Age. median (range) 43 (17-65) 43 (18-64) 0.541 Sex Male 31 71 1.0 - Female 40 62 1.5 (0.8-2.8) 0.240 - - Disease Acute myelogenous leukemia 45 81 1.0 - Acute lymphoblastic leukemia 25 38 1.2 (0.6-2.3) 0.633 - - Myelodysplastic syndrome 1 14 0.1 (0.0-0.9) 0.022 - - Disease risk at transplantation Standard risk 51 77 1.0 - High risk 20 56 0.5 (0.3-1.0) 0.068 - - Conditioning regimen Myeloablative 49 104 1.0 - Reduced-intensity 22 29 1.6 (0.8-3.2) 0.175 - - Total body irradiation No 14 9 1.0 1.0 Yes 57 124 0.3 (0.1-0.8) 0.009 0.4 (0.2-1.0) 0.058 GVHD prophylaxis* Cyclosporine+sMTX 25 35 1.0 - Tacrorimus+sMTX 45 98 0.6 (0.3-1.3) 0.196 - - Donor type Related 37 49 1.0 1.0 Unrelated 34 84 0.5 (0.3-1.0) 0.039 0.7 (0.4-1.3) 0.236 Stem cell source Bone marrow 49 83 1.0 - Peripheral blood 13 21 1.0 (0.4-2.4) 1.000 - - Cord blood 9 29 0.5 (0.2-1.3) 0.174 - - HLA disparity* Match 51 86 1.0 - Mismatch 19 47 0.7 (0.3-1.3) 0.272 - - Cytomegarovirus† Other 61 122 1.0 - Recipient negative and donor positive 6 4 2.9 (0.7-14.9) 0.098 - - Sex mismatch Other 61 108 1.0 - Female to male 10 25 0.7 (0.3-1.7) 0.441 - - Acute GVHD Grade 0-I 42 70 1.0 - Grade II-IV 29 63 0.8 (0.4-1.4) 0.381 - - CS administration 100 days after transplantation No 47 67 1.0 1.0 Yes 24 66 0.5 (0.3-1.0) 0.038 0.6 (0.3-1.1) 0.090 *Data is uncertain in one case. †Data is uncertain in eleven cases. HR: Hazard ratio, CI: confidence interval, GVHD: graft-versus-host disease, sMTX: short-term methotrexate, CS: corticosteroid. 199 Yamamoto W, et al: Eosinophilia after Allogeneic Hematopoietic Stem Cell Transplantation Turk J Hematol 2016;33:196-201 to systemic CS administration, but there was no significant impact of eosinophilia on the outcome in either group. Finally, multivariate analysis showed that high-risk disease, unrelated donor, and CS therapy were adverse predictors of survival with statistical significance. Discussion However, there is a limitation in that we did not treat eosinophilia and CS administration as time- dependent covariates in multivariate analyses. Only one study showed that there was no correlation between eosinophilia and the outcome of cord blood transplantation in adults [8]. In the present study, eosinophilia was associated with a better outcome by univariate analysis, but this was not confirmed by multivariate analysis. Since the incidence of eosinophilia differs among patients with or without CS treatment, we also analyzed its effect on prognosis in patients stratified according Only one study showed that there was no correlation between eosinophilia and the outcome of cord blood transplantation in adults [8]. In the present study, eosinophilia was associated with a better outcome by univariate analysis, but this was not confirmed by multivariate analysis. Since the incidence of eosinophilia differs among patients with or without CS treatment, we also analyzed its effect on prognosis in patients stratified according Table 2. Distribution of patients with acute graft-versus-host disease. Acute GVHD Corticosteroid (+) (n=90) Corticosteriod (-) (n = 114) Grade Eosinophilia (+) (n=24) Eosinophilia (-) (n=66) p Eosinophilia (+) (n=47) Eosinophilia (-) (n=67) p 0 2 4 0.709 21 36 0.424 I 2 6 17 24 II 13 26 8 7 III 6 24 1 0 IV 1 6 0 0 GVHD: Graft-versus-host disease. Table 3. Prognostic factors for overall survival. Variables Univariate Analysis Multivariate Analysis HR (95% CI) p HR (95% CI) p Age (years) <50 1.0 - ≥50 1.1 (0.7-1.7) 0.600 - - Sex mismatch Other 1.0 1.0 Female to male 1.6 (1.0-2.5) 0.068 1.4 (0.8-2.2) 0.224 Disease risk Standard risk 1.0 1.0 High risk 2.8 (1.9-4.2) <0.001 2.7 (1.8-4.2) <0.001 Conditioning regimen Myeloablative 1.0 - Reduced-intensity 0.9 (0.5-1.4) 0.550 - - GVHD prophylaxis Cyclosporine+sMTX 1.0 - Tacrorimus+sMTX 1.0 (0.7-1.6) 0.882 - - Donor type Related 1.0 1.0 Unrelated 1.8 (1.2-2.6) 0.006 1.9 (1.2-2.9) 0.003 Cytomegarovirus Other 1.0 - Recipient negative and donor positive 0.7 (0.3-2.0) 0.547 - - Eosinophilia Present 1.0 1.0 Absent 1.7 (1.1-2.6) 0.017 1.2 (0.8-1.9) 0.440 CS administration Absent 1.0 1.0 Present 2 1 (1 4-3 1) <0 001 1 8 (1 2-2 6) 0 006 Table 2. Distribution of patients with acute graft-versus-host disease. Discussion Acute GVHD Corticosteroid (+) (n=90) Corticosteriod (-) (n = 114) Grade Eosinophilia (+) (n=24) Eosinophilia (-) (n=66) p Eosinophilia (+) (n=47) Eosinophilia (-) (n=67) p 0 2 4 0.709 21 36 0.424 I 2 6 17 24 II 13 26 8 7 III 6 24 1 0 IV 1 6 0 0 GVHD: Graft-versus-host disease. Table 3. Prognostic factors for overall survival. Variables Univariate Analysis Multivariate Analysis HR (95% CI) p HR (95% CI) p Age (years) <50 1.0 - ≥50 1.1 (0.7-1.7) 0.600 - - Sex mismatch Other 1.0 1.0 Female to male 1.6 (1.0-2.5) 0.068 1.4 (0.8-2.2) 0.224 Disease risk Standard risk 1.0 1.0 High risk 2.8 (1.9-4.2) <0.001 2.7 (1.8-4.2) <0.001 Conditioning regimen Myeloablative 1.0 - Reduced-intensity 0.9 (0.5-1.4) 0.550 - - GVHD prophylaxis Cyclosporine+sMTX 1.0 - Tacrorimus+sMTX 1.0 (0.7-1.6) 0.882 - - Donor type Related 1.0 1.0 Unrelated 1.8 (1.2-2.6) 0.006 1.9 (1.2-2.9) 0.003 Cytomegarovirus Other 1.0 - Recipient negative and donor positive 0.7 (0.3-2.0) 0.547 - - Eosinophilia Present 1.0 1.0 Absent 1.7 (1.1-2.6) 0.017 1.2 (0.8-1.9) 0.440 GVHD: Graft-versus-host disease. Table 3. Prognostic factors for overall survival. Variables Univariate Analysis Multivariate Analysis HR (95% CI) p HR (95% CI) p Age (years) <50 1.0 - ≥50 1.1 (0.7-1.7) 0.600 - - Sex mismatch Other 1.0 1.0 Female to male 1.6 (1.0-2.5) 0.068 1.4 (0.8-2.2) 0.224 Disease risk Standard risk 1.0 1.0 High risk 2.8 (1.9-4.2) <0.001 2.7 (1.8-4.2) <0.001 Conditioning regimen Myeloablative 1.0 - Reduced-intensity 0.9 (0.5-1.4) 0.550 - - GVHD prophylaxis Cyclosporine+sMTX 1.0 - Tacrorimus+sMTX 1.0 (0.7-1.6) 0.882 - - Donor type Related 1.0 1.0 Unrelated 1.8 (1.2-2.6) 0.006 1.9 (1.2-2.9) 0.003 Cytomegarovirus Other 1.0 - Recipient negative and donor positive 0.7 (0.3-2.0) 0.547 - - Eosinophilia Present 1.0 1.0 Absent 1.7 (1.1-2.6) 0.017 1.2 (0.8-1.9) 0.440 CS administration Absent 1.0 1.0 Present 2.1 (1.4-3.1) <0.001 1.8 (1.2-2.6) 0.006 HR: Hazard ratio, CI: confidence interval, GVHD: graft-versus-host disease, sMTX: short-term methotrexate, CS: corticosteroid. Table 3. Prognostic factors for overall survival. Table 3. Prognostic factors for overall survival. 200 Yamamoto W, et al: Eosinophilia after Allogeneic Hematopoietic Stem Cell Transplantation Turk J Hematol 2016;33:196-201 Atsuo Maruta, Yoshiaki Ishigatsubo, Heiwa Kanamori; Writing: Wataru Yamamoto, Eriko Ogusa, Kenji Matsumoto, Atsuo Maruta, Yoshiaki Ishigatsubo, Heiwa Kanamori. Atsuo Maruta, Yoshiaki Ishigatsubo, Heiwa Kanamori; Writing: Wataru Yamamoto, Eriko Ogusa, Kenji Matsumoto, Atsuo Maruta, Yoshiaki Ishigatsubo, Heiwa Kanamori. Ethics 9. Basara N, Kiehl MG, Fauser AA. Eosinophilia indicates the evolution to acute graft-versus-host disease. Blood 2002;100:3055. Ethics Committee Approval: Retrospective study; Informed Consent: It was taken. 10. McNeel D, Rubio MT, Damaj G, Emile JF, Belanger C, Varet B, Brousse N, Hermine O, Buzyn A. Hypereosinophilia as a presenting sign of acute graft-versus-host disease after allogeneic bone marrow transplantation. Transplantation 2002;74:1797-1800. References 1. Rothenberg ME. Eosinophilia. N Engl J Med 1998;338:1592-1600. 1. Rothenberg ME. Eosinophilia. N Engl J Med 1998;338:1592-1600. 2. Imahashi N, Miyamura K, Seto A, Watanabe K, Yanagisawa M, Nishiwaki S, Shinba M, Yasuda T, Kuwatsuka Y, Terakura S, Kodera Y. Eosinophilia predicts better overall survival after acute graft-versus-host-disease. Bone Marrow Transplant 2010;45:371-377. 3. Aisa Y, Mori T, Nakazato T, Shimizu T, Yamazaki R, Ikeda Y, Okamoto S. Blood eosinophilia as a marker of favorable outcome after allogeneic stem cell transplantation. Transpl Int 2007;20:761-770. In addition to the relationship between eosinophilia and acute GVHD, there have been several reports on the pathogenesis of eosinophilia in the setting of allo-HSCT. Akhtari et al. reported that eosinophilia is observed in patients with eosinophilic pulmonary syndrome after allo-HSCT [14], but there were no specific causes of eosinophilia in our cohort. 4. Kim DH, Popradi G, Xu W, Gupta V, Kuruvilla J, Wright J, Messner HA, Lipton JH. Peripheral blood eosinophilia has a favorable prognostic impact on transplant outcomes afterallogeneic peripheral blood stem cell transplantation. Biol Blood Marrow Transplant 2009;15:471-482. 5. Sato T, Kobayashi R, Nakajima M, Iguchi A, Ariga T. Significance of eosinophilia after stem cell transplantation as a possible prognostic marker for favorable outcome. Bone Marrow Transplant 2005;36:985-991. In conclusion, eosinophilia after allo-HSCT was not related to the outcome of transplantation or the incidence of acute GVHD in patients with or without systemic CS therapy, although this study had some limitations because it was a retrospective investigation conducted at a single institution. 6. Nakane T, Nakamae H, Hirose A, Nakamae M, Koh H, Hayashi Y, Nishimoto M, Umemoto Y, Yoshimura T, Bingo M, Okamura H, Yoshida M, Ichihara H, Aimoto M, Terada Y, Nakao Y, Ohsawa M, Hino M. Eosinophilia, regardless of degree, is related to better outcomes after allogeneic hematopoietic stem cell transplantation. Intern Med 2012;51:851-858. 7. Ahmad I, Labbé AC, Chagnon M, Busque L, Cohen S, Kiss T, Lachance S, Roy DC, Sauvageau G, Roy J. Incidence and prognostic value of eosinophilia in chronic graft-versus-host disease after nonmyeloablative hematopoietic cell transplantation. Biol Blood Marrow Transplant 2011;17:1673-1678. Discussion Especially after allo-HSCT, systemic CS administration is done in patients who develop various complications, such as acute GVHD or pulmonary complications. Imahashi et al. reported that eosinophilia has an independent influence on the prognosis of patients with acute GVHD receiving CS treatment, but not that of patients without CS treatment [2]. Taking our results together with these findings raises the possibility that the severity of acute GVHD and CS therapy for GVHD may strongly influence transplantation outcomes regardless of eosinophilia. Furthermore, systemic CS administration is often done for allo- HSCT patients with severe complications in addition to acute GVHD, and eosinophilia may be suppressed in those patients. Our findings about non-relapse mortality in the presence or absence of CS treatment support this interpretation. Conflict of Interest: The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included. Acknowledgment This study was supported by a grant from the Kanagawa Health Foundation. 8. Tomonari A, Takahashi S, Ooi J, Tsukada N, Konuma T, Kato S, Kasahara S, Iseki T, Tojo A, Asano S. Blood eosinophilia after unrelated cord blood transplantation for adults. Bone Marrow Transplant 2008;42:63-65. Authorship Contributions 11. Walsh GM. Eosinophil apoptosis: mechanisms and clinical relevance in asthmatic and allergic inflammation. Br J Haematol 2000;111:61-67. Surgical and Medical Practices: Wataru Yamamoto, Heiwa Kanamori; Concept: Wataru Yamamoto, Heiwa Kanamori; Design: Wataru Yamamoto, Heiwa Kanamori; Data Collection or Processing: Wataru Yamamoto, Eriko Ogusa, Kenji Matsumoto, Atsuo Maruta, Yoshiaki Ishigatsubo, Heiwa Kanamori; Analysis or Interpretation: Wataru Yamamoto, Heiwa Kanamori; Literature Search: Wataru Yamamoto, Eriko Ogusa, Kenji Matsumoto, Atsuo Maruta, Yoshiaki Ishigatsubo, Heiwa Kanamori; Analysis or Interpretation: Wataru Yamamoto, Heiwa Kanamori; Literature Search: Wataru Yamamoto, Eriko Ogusa, Kenji Matsumoto, 12. Schleimer RP. Effects of glucocorticosteroids on inflammatory cells relevant to their therapeutic applications in asthma. Am Rev Respir Dis 1990;141:59- 69. 13. Przepiorka D, Weisdorf D, Martin P, Klingemann HG, Beatty P, Hows J, Thomas ED. 1994 Consensus Conference on Acute GVHD Grading. Bone Marrow Transplant 1995;15:825-828. 14. Akhtari M, Langston AA, Waller EK, Gal AA. Eosinophilic pulmonary syndrome as a manifestation of GVHD following hematopoietic stem cell transplantation in three patients. Bone Marrow Transplant 2009;43:155- 158. 201
W3017606716.txt	https://link.springer.com/content/pdf/10.1007/s10341-020-00485-y.pdf	de		Natürliche Frostschutzmechanismen bei Obstgehölzen – von Supercooling bis Anti-Freeze Proteinen	Erwerbs-Obstbau/Der Erwerbsobstbau	2,020	cc-by	5,924	Erwerbs-Obstbau (2020) 62:163–174 https://doi.org/10.1007/s10341-020-00485-y ORIGINALBEITRAG Natürliche Frostschutzmechanismen bei Obstgehölzen – von Supercooling bis Anti-Freeze Proteinen Laura Hillmann1,2 · Todd Einhorn2 · Michael Blanke1 Eingegangen: 10. August 2018 / Angenommen: 25. März 2020 / Online publiziert: 22. April 2020 © Der/die Autor(en) 2020 Zusammenfassung Aus Anlass des starken Spätfrostes am 20. April 2017 in Europa wurden im Rahmen einer Masterarbeit der Prozess des Erfrierens und die Mechanismen der Frosttoleranz von Obstgehölzen untersucht. Bakterien, Staub, Schmutz, kleine Sandkörner u. a. auf dem Blatt können als Eiskeimbildner fungieren. Die an diesen Eiskeimen gebildeten Eiskristalle können in die Pflanze bzw. Zelle eindringen und als intra-zelluläre Eiskristalle das Erfrieren verursachen. Kleine, unregelmäßige intra-zelluläre Eiskristalle sind für die Zelle vergleichsweise harmlos, solange sie schmelzen, bevor sie die Zellwand beschädigen. Schnelle Gefriergeschwindigkeit führt dabei zu einer hohen Anzahl kleiner, langsames Gefrieren zu einer geringen Anzahl großer Eiskristalle; schnelles Gefrieren hindert das Wasser daran, zu bevorzugten extra-zellulären Stellen zu diffundieren. Dies führt zur intra-zellulären Eiskristallbildung, wodurch die Pflanzenzellen mechanisch belastet und geschädigt werden können, so dass intra-zelluläre Eisbildung zum Erfrieren führt. Folgende natürliche Frosttoleranz-Mechanismen, die diese intrazellulare Eiskristallbildung z. T. verhindern können, wurden für (Obst-)Gehölze ermittelt 1) Reduzierung des Wassergehaltes in der Zelle einschl. der Reduzierung des „freien Wassers“ und Bindung von freiem Wasser an Dehydrine (Proteine) über Winter 2.) Anreicherung von osmotisch wirksamen Substanzen wie Zucker (Glukose, Fruktose, Saccharose, Sorbitol), Mineralstoffen wie K und Mg und Aminosäuren wie z. B. Prolin; 3.) Anti-Freeze Proteine (AFP) haften an den Oberflächen der Eiskristalle; ihre Struktur und Wachstum beeinflussen und verhindern ihr Eindringen in die Pflanzenzelle; 4) ‘supercooling‘, d. h. einem Prozess, bei dem die Eiskeimbildung und die intrazelluläre Eiskristallbildung (Frostschäden) vermieden wird; 5) Strukturelle Eiskristallbarriere – keine Verbindung zum (wasserführenden) Xylem 6) Nachblüte bzw. zeitliche Verzögerung zwischen Blüten am ein- und zweijährigen Holz Die Entstehung der Eiskristalle an den Eiskeimen hängt ab von der Verfügbarkeit von Anti-Freeze Proteinen (AFP) und Eiskeim fördernden Proteinen, die an der Membran von Eiskeim fördernden Bakterien wie Pseudomonas syringae und Erwinia amylovora (Feuerbrand) Bakterien verankert sind. Ohne heterogene Eiskeime besitzt Wasser die Fähigkeit zum ‘supercooling‘. Im Temperaturbereich von 0 bis ca. –5 °C wird ‘supercooling‘ vorwiegend durch das Vorhandensein von Eiskeim fördernden Bakterien beschränkt; sie gelten daher als verantwortlich für die Eiskristallbildung und damit für die Gewebeschädigung. Voraussetzung für die effektive Wirkung dieser natürlichen pflanzenphysiologischen Schutzmechanismen im Winter ist ein vorhergehendes langsames Abhärten (Kälte-Akklimation) im Herbst. Besonderes Interesse gilt ihrem Erhalt bei steigenden Temperaturen im Frühling während der Enthärtung (De-Akklimation), wobei der Verlust an Frost- bzw. Kältetoleranz mit dem Aufbrechen der Knospenschuppen der sich entwickelnden Blüten einhergeht. Insgesamt verfügen die Pflanzen/-zellen über eine Reihe von Anpassungsmechanismen an Frosttemperaturen, die bisher zu wenig genutzt wurden. Schlüsselwörter Apfel · Obst · Blütenfrost · Süßkirsche (Prunus avium L) · Sauerkirsche (Prunus cerasus) · Anti-freeze Proteine (AFP) · Dehydrine · Eiskeime · Eis-Nukleotide · Frost · Frostschutz · Kälteschutz · Supercooling · Winterruhe 1 INRES-Gartenbauwissenschaft, Universität Bonn, Bonn, Deutschland 2 Dept of Horticulture, Michigan State University, MI, USA Michael Blanke mmblanke@uni-bonn.de K 164 L. Hillmann et al. Endogenous Physiological Mechanisms of Frost Hardiness in Flower Buds of Fruit Trees Abstract The devastating spring frost on 19–20 April 2017 throughout Europe sparked off the present study. The objective of this work, as part of a master thesis, was to investigate and review current knowledge on the process of ice crystallization and endogenous plant protection mechanisms. The process of ice formation or crystallization depends on the availability of antifreeze proteins (AFP) and ice nucleation active substances (INAS), which anchor on the membrane of INAS bacteria. INAS such as small sand and dirt particles, tiny mineral crystals and INAS bacteria such as Pseudomonas and Erwinia can serve as nucleotides for ice nucleation and crystallization. Without ice nucleotides, water exhibits the capacity of supercooling; in the 0 to –5 °C temperature range, supercooling is prevented by the presence of INAS bacteria, which become ultimately responsible for the tissue damage. Flowers of fruit trees with bacteria have been reported to freeze at –2 °C compared with –5 °C without Pseudomonas as INAS bacteria. When the ice crystals penetrate the plant via the cells, intra-cellular ice formation causes the freezing or frost damage. Small, irregularly shaped intra-cellular ice crystals are supposedly harmless to the cell, particularly if they melt before reaching (and damaging) the cell wall. Rapid freezing prevents the water to diffuse out of the cells to extra-cellular sites, results in a large number of small harmless ice crystals, whereas slow freezing ends up in a small number of large harmful ice crystals. Cold tolerance can include the following physiological mechanisms for frost hardiness, which can prevent or reduce intra-cellular ice formation (freezing) 1) Reduction of (free and unbound) water content including free water bound to dedicated proteins (dehydrins) in the cold period 2) Accumulation of osmotically active substances such as sugar (glucose, fructose, sucrose, sorbitol), minerals (potassium etc.) and amino acids (proline etc.). 3) Availability of antifreeze proteins (AFP), which bind to the surface of the ice crystals and positively influence their size, shape and growth, thereby preventing them from penetrating the plant cell 4) Supercooling, a process, which prevents intra-cellular ice formation (frost damage) by preventing the formation of ice nucleotides 5) Structural ice barrier—no connection to the xylem tissues 6) Delayed flowering and time difference between flowering on one or two-year-old wood Effective functioning of these natural endogenous plant protection mechanisms in winter requires slow cold acclimation in autumn and their maintenance in spring. Overall, the challenge for future fruit breeding, selection and growing in a situation with late spring frosts due to climate change is a) to encourage these above endogenous physiological cold hardiness mechanisms in the orchard and b) to maintain them, as they are otherwise lost with increasing temperatures and concomitant bud break and bud scale unfolding during de-acclimation in spring. Keywords Apple · Fruits · Spring fros · Sweet cherry (Prunus avium L) · Sour cherry (Prunus cerasus) · Antifreeze proteins (AFP) · Bud break · Dehydrins · Dormancy · Flower bud · Frost · Frost protection · Ice nucleotides · Ice nucleation active substances (INAS) · Supercooling Einleitung Anlass für die vorliegende Arbeit war der verheerende Spätfrost am 20. April 2017, der die Obsternte europaweit um über 60 % reduziert hat (Schwartau 2018; Tab. 1). Solche Spätfröste können zum Erfrieren von Knospen, Blüten (Abb. 1) sowie junger Früchte (Abb. 2) (Rodrigo 2000; Stepulaitiene et al. 2013) und zu hohen wirtschaftlichen Schäden führen (Mathers 2004; Dalhaus et al. 2020), wobei die Blühstadien vom Knospenschwellen (BBCH 52) bis Fruchtansatz (BBCH 70) am frostempfindlichsten sind (Rodrigo 2000; Wenden et al. 2017). In einer Zeit des rezenten Klimawandels bleibt die Zahl der Spätfröste in den hiesigen Obstbaugebieten gleich oder kann sogar zunehmen (Blanke und Kunz 2009). Daher ist das Verständnis, K wie sich die Gehölze auf natürliche Weise vor Kälte bzw. Frost schützen können, von zunehmendem Interesse (Wenden et al. 2017). Dabei ist die Eisbildung bzw. -kristallisation in den Blütengeweben von besonderer Bedeutung für das Überleben einer Pflanzenart und bestimmt die Grenzen seiner geographischen Ausbreitung (Wisniewski et al. 2014). Die meisten Forschungsarbeiten beschäftigen sich mit der chemischen oder physikalischen Frostabwehr, während es kaum Untersuchungen zu natürlichen Frost- bzw. Kältetoleranzmechanismen der Pflanzen gibt, die neue, bisher kaum genutzte Möglichkeiten z. B. in der Züchtung oder bei Kulturmaßnahmen in der Obstanlage bieten könnten. Vor allem der Verlust der Kältetoleranz von laubabwerfenden Obstgehölzen im Frühling der gemäßigten Klimazonen Natürliche Frostschutzmechanismen bei Obstgehölzen – von Supercooling bis Anti-Freeze Proteinen Tab. 1 Geschätzte Frostschäden in 2017. (Schwartau 2018; Walther 2017) Ergebnisse Gruppierung Frostschäden (%) Region Äpfel Süßkirschen Pflaumen 65 90 60 Deutschland Deutschland, Hessen Frankreich Frostempfindlichkeit der verschiedenen Blütengewebe – anatomische Grundlagen (Rodrigo 2000) kann während der Enthärtung (KälteDe-Akklimation) zu erhöhtem Frostrisiko führen. Die langsame, entwicklungsbedingte Enthärtung ermöglicht der Pflanze eine gewisse Kältetoleranz zu bewahren, obwohl die Temperaturen das Wachstum bereits begünstigen (Proebsting 1970). Ziel dieses Übersichtsreferates ist es, die natürlichen pflanzenphysiologischen Schutzmechanismen der Obstgehölze am Beispiel der früh und einzeln blühenden, und daher besonders Frost gefährdeten Kirsche herauszuarbeiten, um sie ggf. mit geeigneten Maßnahmen zu fördern und oder bei der Züchtung stärker mit zu berücksichtigen. 165 Die sichtbaren unspezifischen Symptome des Erfrierens sind Verbräunen der Blüte und vermindertes Fruchtwachstum bei Apfel und Kirsche (Abb. 1), Verbräunen der Frucht bei Kirsche (Abb. 2) oder Frostzungen oder Missbildungen der Früchte beim Apfel. Bei den Prunus-Arten sind die Fruchtknoten die frostempfindlichsten Teile der Blüten und ihr Verbräunen ist symptomatisch, ohne dass Schäden an den Blütenblättern auftreten (Rodrigo 2000); durch starken Wind oder Sonneneinstrahlung können Schäden wie Frostbrand entstehen (Pearce 2001). Anpassungsmechanismen Die Frosttoleranz einer Obstart beschreibt die Fähigkeit eines Gewebes, sich an eine Umgebung mit kalten (Frost-)Temperaturen bis zu einem gewissen Grad anzupassen, schwankt von Obstart zu Obstart und ist genetisch vorgegeben (Szabó et al. 1996), d. h. ihre Fähigkeit zum sog. ‘supercooling‘ (Tab. 2). Prunus-Arten weisen eine Abb. 1 Frostschaden an Blütenknospen bei Kirsche: Aufgeschnittene ‘Montmorency’ Sauerkirschblüte nach stufenweiser Abkühlung um je ca. 1 °C pro Stunde auf –4 °C; Frostschaden erkennbar am braunen Fruchtknoten (roter Pfeil) (a), ohne dass von außen starke Schäden/ Erfrieren erkennbar sind (b). (Aufnahmen: L Hillmann) Abb. 2 Frostschaden an einer erfrorenen Kirsch-Frucht: a von außen sichtbarer Frostschaden einer ‘Regina‘ Süßkirsche (rechte Frucht); b nach Aufschneiden sichtbares, erfrorenes Endosperm einer ‘Montmorency‘ Sauerkirsche. (Aufnahmen a M. Blanke und b L. Hillmann) K 166 L. Hillmann et al. Tab. 2 Übersicht über Anpassungsstrategien bei Prunus und anderen Obstarten Tab. 3 Osmotisch wirksame Substanzen Zucker Mineralstoffe Aminosäure Strategie Glukose, Fruktose, Saccharose, Sorbitol Kalium, Magnesium Prolin Charakterisierung Vermeidung Die Entstehung von intrazellulären Eiskristallen wird verhindert durch ‘supercooling‘, Frostschutzproteine und Anreicherung von osmotischen Substanzen Toleranz Eiskristalle werden – aber nur extrazellulär – in ihrem Gewebe toleriert, d. h. kein ‘supercooling‘ Reihe von Anpassungsstrategien auf (Abb. 3); in einem Extremfall überlebten geschlossene Knospen in der Winterruhe bis –47 °C an der Pazifikküste in Nordamerika (Quamme 1985; Kadir und Proebsting 1994). Die Obstarten der gemäßigten Breiten haben sich über Jahrzehnte an einen Standort und kalte Temperaturen angepasst und Überlebensstrategien entwickelt, um kalte (Frost-)temperaturen bis zu einem gewissen Grad zu überstehen. Bei diesen kann man zwei Anpassungsstrategien unterscheiden, die erste, die die Entstehung von Eiskernen in ihrem Gewebe durch ‘supercooling‘, Frostschutzproteinen und Anreicherung von osmotisch wirksamen Substanzen vermeiden und eine zweite, die nur extrazelluläre Eiskristalle in ihrem Gewebe tolerieren (Tab. 2). Die Temperatur, bei der sich Eiskristalle in einer Zelle oder Pflanze bilden, hängt vom Wassergehalt und Gehalt osmotisch wirksamer Substanzen ab (Modlibowska 1962). Zu den osmotisch wirksamen Substanzen gehören Zucker (Glukose etc.), Mineralstoffe (Kalium, Magnesium) und die Aminosäure Prolin (Marquat et al. 1999; Bonhomme et al. 2005) (Tab. 3). Bildung von Eiskristallen als Voraussetzung für Erfrieren und Frostschäden Eiskristalle auf der Zelloberfläche oder zwischen Protoplast und Zellwand (‘extrazellulär‘) entziehen der Zelle oder dem Protoplasten (Frost-Plasmolyse) Wasser in flüssiger Form entlang dem Gradienten freier Energie (Sakai und Larcher 1987). Damit ist diese Form der Frost- oder Kältetoleranz mit Wasserentzug eine Art Trockenstress (Burke et al. 1976). Dabei bestimmt die Konzentration an osmotisch wirksamen Substanzen in der Zelle (Tab. 3), wann und wieviel Wasser durch die Zellmembran (Plasmalemma) diffundiert. Dieser Wasserverlust der Zelle führt zur Dehydrierung und Zellkontraktion (Sakai und Larcher 1987; Rodrigo 2000) und kann zu gleichzeitigen Zellveränderungen wie Faltenbildung der Membran, Eiweiß-Denaturierung und Anreicherung an toxischen Stoffwechselprodukten führen (Wisniewski et al. 2003) – der eigentliche Frostschaden entsteht und wird erst beim Auftauen sichtbar. Dabei entscheidet die Form und Größe der Eiskristalle darüber, ob sie die Zellmembran durchdringen können (Ashworth und Abeles 1984). Kälte- bzw. frosttolerante Zellen können das Eindringen der Eiskristalle verhindern (Sakai und Lar- K cher 1987), weil ihre Membran elastischer ist (Levitt 1972; Quamme 1995). Intrazelluläre Eiskristallbildung = Erfrieren Die intrazelluläre Eiskristallbildung entsteht beim plötzlichen Erfrieren der Zelle, setzt sich wie eine Kettenreaktion von Zelle zu Zelle fort, wobei sich Eiskristalle im Protoplasten und in der Vakuole bilden (Burke et al. 1976). Die hohe Geschwindigkeit dieses Vorganges erlaubt keine vorherige Absenkung des Wassergehaltes in der Zelle und führt damit häufig zu größeren Frostschäden an den Blütenknospen (Wenden et al. 2017). Intrazelluläre Eiskristallbildung wird bei der extrazellulären Kristallbildung durch rechtzeitigen Wasserentzug verhindert (Rodrigo 2000; Wisniewski et al. 2003; Wenden et al. 2017). Ob eine Zelle extra- oder intrazellulär erfriert oder nicht, hängt ab von der Flexibilität der Zellwand (Protoplasma) (Chambers und Hale 1932). Daher erfrieren kälte- bzw. frostempfindlichere Gewebe meistens intrazellulär, wobei die Zellmembranen und damit die Zellen durch die Bildung der Eiskristalle erst physikalisch zerstört werden und die freigesetzten Enzyme und Stoffwechselprodukte dann das Gewebe schädigen (Burke et al. 1976). Früher ging man dagegen davon aus, dass Erfrieren bzw. die Eiskristallisation in der Vakuole beginnt; heute weiß man, dass die Vakuole zum Schluss gefriert (Friedrich und Fischer 2000). Wirkungsmechanismus und Verbreitung des ‘supercooling‘ Unter ‘supercooling’ versteht man generell die Fähigkeit eines Gewebes oder von Pflanzenzellen, die Bildung von Eiskristallen und damit Frostschäden zu verhindern (Wisniewski et al. 2014) (Tab. 4). Ein geringer Wassergehalt und Anreicherung osmotisch wirksamer Substanzen ermöglichen ‘supercooling‘ und erhöhen damit die Kältetoleranz (Quamme 1985). Sinkt der Wassergehalt einer Pflanzenzelle um 40–70 %, erhöht sich die Frosttoleranz des Knospenmeristems um 10 °C (Dereuddre 1979 in Sakai und Larcher 1987). Während des ‘supercooling’-Prozesses werden generell weniger Eiskeime gebildet und die Eiskristallbildung unterbunden (Burke et al. 1976). Das Phänomenon des ‘supercooling‘ ist unter laubabwerfenden Obstgehölzen weit verbreitet und tritt im Xylem des Baumstammes und in Blütenknospen auf (Burke et al. 1976; Palonen und Bus- Natürliche Frostschutzmechanismen bei Obstgehölzen – von Supercooling bis Anti-Freeze Proteinen Tab. 4 Arten des ‘supercooling‘, Temperaturen und Verbreitung unter Gehölzen 167 Arten des Supercooling Niedr. Temp (°C) Art Mechanismen der Kältetoleranz Supercooling (generell) n.a. n.a. Persistent supercooling Deep supercooling –80 Birke –47 Prunus Transitory supercooling –5 Prunus Verhindert die Fortsetzung von Eiskristallisation in der Pflanzenzelle Abgrenzung/Ablagerung von Eis an bevorzugten Stellen Geringe Anzahl von Eiskeimen (Quamme 1985, 1995) ‘supercooling‘ während der ganzen Kältephase (Modlibowska und Rogers 1955) ’supercooling‘ spezieller Pflanzengewebe, z. B. geschlossene Knospe mit Knospenschuppen im Winter oder Parenchymzellen der Xylem-Gefäßbündel (Quamme et al. 1982; Quamme 1985) ‘supercooling‘ der verholzten Teile des Baumes nach Blattabwurf (Burke et al. 1976; Quamme 1985) n.a. nicht angegeben zard 1997). ‘Deep supercooling’ kann bei Temperaturen von –15 bis –60 °C vorkommen, wobei die tiefsten Werte im Xylem des Baumstammes im Winter und mittlere Werte in den Knospen gemessen wurden (Wisniewski et al. 2014). ‘Deep supercooling’ ist damit eine der effektivsten Überlebensstrategien für pflanzliches Gewebe auch bei tiefen Frosttemperaturen und setzt eine vorangegangene Abhärtung (Kälte-Akklimation) voraus. Effektives ‘supercooling‘ beruht auf einer Kombination von geringem Wassergehalt der Zelle, kleiner Zellgröße ohne Eiskristallisationskerne (Ishikawa 1984; Quamme 1985; Sakai und Larcher 1987). Bestimmte Pflanzenorgane, wie geschlossene Knospen in Winterruhe, können Temperaturen bis –80 °C ohne größere Schädigung überstehen; diese Art des ‘supercooling’ wird als ‘persistent supercooling’ bezeichnet (Modlibowska und Rogers 1955). auf Grund der schnellen Protonenbewegung im MRT nur langsam (lange T2 Relaxationszeiten) und erzeugt somit kontrastierende Bilder (Faust et al. 1991). Knospen von Apfelbäumen in der Winterruhe enthielten viel gebundenes Wasser, während Knospen im Frühling vor allem freies Wasser aufwiesen. Unter gebundenem Wasser versteht man das Wasser, das beim Erfrieren der Zelle nicht kristallisiert bzw. nicht zu Eis erstarrt. Basierend auf den Ergebnissen der MRT-Analysen konnte man keine Unterschiede im Wassergehalt von Knospen im Herbst und Frühling ausmachen, da beide freies Wasser in den Zellen enthielten. Nur während der Winterruhe ist gebundenes Wasser in den Zellen zu finden. Gebundenes Wasser in den Knospen wird graduell in freies Wasser umgewandelt, wenn kalte Temperaturen herrschen und die Pflanze sich in der Phase der echten Winter- Reduzierung des Wassergehaltes in der Ruhephase und Dehydrine In der Winterruhe schützen hydrophile Proteine, sog. Dehydrine, die Zellen der Bäume vor kalten Temperaturen, indem sie das Wasser in den Zellen der Knospen binden (Erez et al. 1998; Hillmann et al. 2016). Die Bildung dieser hydrophilen Proteine wird von kalten Temperaturen und kurzen Tagen im Herbst aktiviert. Die Dehydrine schützen die Knospen vor Frost und werden während der Winterruhe langsam abgebaut. Gebundenes Wasser kann außerdem das Wachstum der Knospen verhindern, da jeglicher Nährstofffluss unterbunden ist. Erst im Frühling, wenn warme Temperaturen den Abbau der hydrophilen Proteine bewirken, wird ein deutliches Anschwellen der Knospen sichtbar und kennzeichnet somit die Wiederaufnahme des Wachstums (Faust et al. 1997). Magnetresonanztomographie (MRT) ermöglicht eine zerstörungsfreie Untersuchung der Pflanzenorgane hinsichtlich ihres Wasserstatus. Freies Wasser erholt sich Abb. 3 Mechanismen der physiologischen Frosttoleranz in der Pflanzenzelle K 168 L. Hillmann et al. Abb. 4 T2 Relaxationszeit in Blütenknospen von Pfirsich (Prunus persica) (oben ‘Coral‘ mit geringem Kältebedürfnis, unten ‘Kansuke Hakuto‘ mit hohem Kältebedürfnis), in Japan. Die Farbskala kennzeichnet T2 Werte für jeweils 2 ms. (Yooyongwech et al. 2008) ruhe befindet. Diese Phase ist in den MRT-Analysen durch die kurzen T2 Relaxationszeiten (0–ca. 12 ms) gekennzeichnet (Abb. 4). Eine Umwandlung von gebundenem zu freiem Wasser scheint nur möglich, wenn die Knospen eine ausreichende Anzahl Stunden kalten Temperaturen ausgesetzt sind; im Frühling steigt der Anteil freien Wassers stark an, besonders bei Knospen, die zuvor das Kältebedürfnis erfüllt haben (Faust et al. 1991, 1997). Anatomische und saisonale Entwicklung des ‘supercooling‘ bei Prunus Die Kältetoleranz nimmt im Laufe der kühleren Herbsttemperaturen zu und nimmt im Frühling mit steigenden Temperaturen wieder ab. Die Überlebensrate der Zellen nach einem Frost hängt davon ab, ob und wo sich Eiskeime und -kristalle bilden (Rodrigo 2000). So unterliegt auch die Fähigkeit zum ‘Deep supercooling‘ saisonalen Schwankungen, nimmt im Herbst zu, erreicht im Winter sein Maximum und nimmt zum Frühling wieder ab (Wisniewski et al. 2003; Wisniewski et al. 2014). Bei Prunus-Arten nimmt die Fähigkeit zum ‘Deep supercooling’ im Frühling mit steigenden Temperaturen ab, wenn die Blüten-Primordien Wasser aufnehmen, so dass der Wassergehalt der Blüten-Primordien zu einem Bioindikator für schädigende Temperaturen der Süßkirschen-Knospen wird (Quamme 1995; Andrews und Proebsting 1987). Bei den Prunus-Arten hängt ‘Deep supercooling‘ von der Entwicklung von Leitbündeln ab und damit der Fähigkeit, den Wassergehalt in den Blütenknospen zu regulieren (Andrews und Proebsting 1987; Wisniewski et al. 2014). Blütenknospen mit der Fähigkeit K zum ‘Deep supercooling‘ bestehen aus undifferenziertem Gewebe mit procambialen Zellen ohne Xylemgefäße. Das Fehlen einer Verbindung zwischen Primordium und umgebendem Gewebe (Abb. 5) ist die strukturelle Voraussetzung, die ‘Deep supercooling‘ ermöglicht, und wirkt als strukturelle Eisbarriere und Schutz der reproduktiven Knospengewebe vor Frostschäden (Ashworth et al. 1992; Wisniewski et al. 2014). Diese strukturellen Barrieren verhindern die fortschreitende Eisbildung von den Trieben zu den Blüten oder Früchten und kann ihre Ausbreitung aufhalten (Pearce 2001); bei Apfel mit seinen Blütenbüscheln verbreitet sich der Frostschaden von Blüte zu Blüte (Wisniewski et al. 1997). Abb. 5 Längsschnitt durch eine Kirschblüte mit Fruchtknoten Narbe, Griffel, Kelch- und Blütenblätter. (ILA, LehrplanPlus 2016) Natürliche Frostschutzmechanismen bei Obstgehölzen – von Supercooling bis Anti-Freeze Proteinen Sobald die Verbindung mit dem Xylem während des Enthärtens bzw. der Kälte-Deakklimatisierung im Frühling etabliert ist, und die Knospen anfangen zu schwellen, verlieren sie die Fähigkeit zum ‘Deep supercooling‘ (Ashworth und Rowse 1982; Palonen und Buszard 1997). Dies stützt die Hypothese von Andrews und Proebsting (1987), dass die Fähigkeit der Prunus Blütenknospen zum ‘supercooling‘ ein physikalischer Prozess ist, der umso höher ist, je geringer der Wassergehalt der Blüten-Primordien. Frostversuche mit Blüten an abgeschnittenen Ästen zeigen häufig eine größere Frostempfindlichkeit, weil sich die Eiskristallisation über das Xylem fortsetzt, und können so zu Fehlinterpretationen (Überschätzung) der Frosttoleranz führen. 169 Abb. 6 Saisonale Entwicklung der Frosttoleranz im Laufe der Knospenentwicklung Prozess der Eiskristallbildung Faktoren, die Kältetoleranz beeinflussen Wenn Blüten- und Blattknospen im Frühling anschwellen, hängt der Grad der Kältetoleranz zudem auch von der Entwicklungsstufe des Baumes bzw. der Blüte ab (Burke et al. 1976; Proebsting und Mills 1972). Der Verlust an Frostbzw. Kältetoleranz geht dabei mit dem Aufbrechen der Knospenschuppen der sich entwickelnden Blüten einher (Roberts 1917). Andrews et al. (1983) unterscheiden für Kirschen die vier in Abb. 6 dargestellten Stadien während des Enthärtens bzw. der Kälte-De-Akklimation im Frühling. In der Ruhephase besitzen noch geschlossene Blütenknospen die Fähigkeit zum ‘Deep supercooling’. In der Übergangsphase beginnen die Knospen zu schwellen und erreichen schließlich das Stadium des Knospenaufbruchs. Sie verlieren dann langsam die Fähigkeit zum ‘supercooling‘ (Abb. 6). In der dritten Phase, vor der Vollblüte – Kelchblätter geöffnet – liegt die Frosttoleranz bei –4,6 °C. In der letzten, vierten frostempfindlichen Phase – Kelchblätter geöffnet bis Blütenfall –, sind Blüten und junge Früchte am frostempfindlichsten und erfrieren bei –2,7 °C (Andrews et al. 1983). Voraussetzung für die Eiskristallisation in einer Pflanzenzelle ist, dass Wassermoleküle einen ersten Kristallansatz vorfinden. Das Verständnis dieses Prozesses könnte dazu beitragen, den Prozess so zu beeinflussen, dass Frostschäden an einer ganzen Reihe landwirtschaftlicher Kulturen vermindert oder verhindert werden (Wisniewski et al. 2003). Die osmotisch wirksamen Substanzen in den Pflanzenzellen (Tab. 3) reduzieren den Gefrierpunkt und damit die Eisbildung unter 0 °C, wobei die Zahl der freien Wassermoleküle eine Rolle spielt und mit sinkenden Temperaturen abnimmt (Franks und Jones 1987; Pearce 2001). Wenn sich die Eiskristalle homogen bilden, geschieht dies autonom, d. h. von einem Eiskeim aus. Dagegen ist die Eisbildung durch Eiskeim fördernde Substanzen heterogen (Wisniewski et al. 2014) und kann durch Eiskeim fördernde Bakterien, Schmutzteilchen, kleinste Sandkörner und Staub über einen breiten negativen Temperaturbereich von 0 bis –38 °C hervorgerufen werden (Friedrich und Fischer 2000; Pearce 2001). Eiskeim fördernde Substanzen auf (oder in) der Pflanzenzelle müssen mit freien Wassermolekülen in Kontakt kommen, um Eiskeimbildung auf der Pflanze oder Zelle auszulösen. Die Eiskristalle dringen dann entweder durch die Pflanzenoberfläche durch Kutikularrisse oder Stomata und bewirken Eis-(keim-) und Kristallbildung in Abb. 7 Eiskristallbildung: extrazellulär (in den Interzellularen, links) und intrazellulär in der Zelle (rechts) K 170 der Pflanzenzelle (Pearce 2001; Gusta und Wisniewski 2013). Die Entstehung der Eiskristalle an den Eiskeimen hängt ab von der Verfügbarkeit von Anti-Freeze Proteinen (AFP) und Eiskeim fördernden Proteinen, die an der Membran von Eiskeim fördernden Bakterien wie Pseudomonas syringae (Hirano und Upper 2000) und Erwinia amylovora (Feuerbrand) Bakterien verankert sind. Ohne heterogene Eiskeime besitzt Wasser die Fähigkeit zum ‘supercooling‘. Im Temperaturbereich von 0 bis ca. –5 °C wird ‘supercooling‘ vorwiegend durch das Vorhandensein von Eiskeim fördernden Bakterien beschränkt; sie gelten daher als verantwortlich für die Eiskristallbildung und damit Gewebeschädigung (Hirano und Upper 2000). Birnen erfrieren ohne solche Bakterien bei –5 °C, aber mit ihnen bereits bei –2 °C. Der Grad der Frostschädigung ist damit eine Funktion der vorhandenen Bakterien-Population. Sie können den Prozess der Eiskeimbzw. Kristallbildung beeinflussen und möglicherweise die Fähigkeit des Wassers in den Xylemgefäßen des Baumstammes zum ‘supercooling‘ verringern (Lee et al. 1995; Wisniewski et al. 2014). Regulierung der Eiskristallbildung – Anti-Freeze Proteine (AFP) Wenn die Pflanzen beginnen zu erfrieren, bewegt sich das Wasser aus der Pflanzenzelle und gefriert extrazellulär, d. h. auf der Oberfläche von Pflanzenzellen bzw. deren Interzellularräumen (Abb. 7) (Burke et al. 1976). Bei langsamen extrazellulären Gefriervorgängen können große Mengen Eiskristalle entstehen, die die Pflanzenzellen in der Rinde, in Knospenschuppen und am Blütenboden (Blütenknospen als auch vegetative Knospen) von Bäumen in gemäßigten Klimaten aber nicht schädigen (Ashworth et al. 1992; Pearce 2001; Wisniewski et al. 2014). Gefrier-Kinetikstudien von Olien (1965) deuten auf ein Zusammenspiel der Polymere der Zellmembran mit der EisFlüssigkeitsphase. Anti-Freeze Proteine (AFP) adsorbieren an der Eiskristalloberfläche. Auch wenn ihr Einfluss für die Gefrierpunkterniedrigung unter 1 °C liegt und sie nicht das Erfrieren verhindern können, beeinflussen sie aber die Größe und Form der Eiskristalle (Griffith et al. 2005) derart, dass sich kleine und unregelmäßige, für die Zelle vergleichsweise harmlose intrazelluläre Eiskristalle bilden, die schmelzen, bevor sie die Zellwand erreichen (Olien 1965; Burke et al. 1976). Die Größe der Eiskristalle spielt eine große Rolle bei extrazellulärem Erfrieren (Abb. 6) von Blütenorganen mit der Fähigkeit zum ‘supercooling‘ (bis zum Knospenschwellen; Tab. 4). Da Wasser im Zustand des ‘supercooling‘ als flache, runde Kristalle gefriert, sind diese Eiskristalle zu groß, um die Membran oder Zellwand zu durchdringen (Sakai und Larcher 1987). K L. Hillmann et al. Muster der Eiskristallbildung Der Gefrierverlauf in der Pflanzenzelle hängt ab von der Zell-Anatomie, dem Grad der Kältehärtung des Baumes (in Abhängigkeit des Entwicklungsstadiums), des Wassergehaltes und von der Geschwindigkeit des Abkühlens/ Gefrierprozesses (Sakai und Larcher 1987). Die Eiskristallbildung kann dann in drei verschiedenen Mustern erfolgen: 1. Kontinuierliches Gefrieren: In homogenen Pflanzenorganen wie den Parenchymzellen oder den uniform aufgebauten Blättern mit kohärenten Interzellularräumen, verläuft das Gefrieren kontinuierlich sobald Eiskristallbildung stattfindet (Sakai und Larcher 1987). 2. Sequenzielles Gefrieren: Ein mit Wasser gefülltes Pflanzenorgan gefriert als erstes, was charakteristisch für die Wurzeln und Sprossachsen krautartiger Pflanzen ist. In Blättern gefrieren normalerweise nur die Interzellularräume, sodass das Blatt auch nach dem Frost noch (partiell) funktionsfähig ist (Sakai und Larcher 1987). 3. Diskontinuierliches Gefrieren: Knospen und Samen von Bäumen, die ‘supercoolen‘, gefrieren diskontinuierlich. Während Zellteile, die ‘supercoolen‘ (z. B.: BlütenPrimordien), bei tiefen Temperaturen gefrieren, werden Zellteile, die nicht ‘supercoolen‘ können (z. B.: Knospenschuppen), bei höheren Temperaturen gefrieren (Sakai und Larcher 1987). Mit der Entwicklung der Knospen (Tab. 5) werden die Gewebestrukturen zunehmend komplex. Die Gewebemassen erhöhen sich um ein Vielfaches, der Wassergehalt steigt und die Farbe der Knospen verändert sich von braun zu rosa und weiß (Proebsting 1982). Bei einem Frostereignis können nun, anstatt der ganzen Blüte, einzelne Blütenorgane durch unterschiedliche Temperaturen verbräunen und sterben (Proebsting 1982). Dies bedeutet, dass sich die verschiedenen Organe des Blüten-Primordiums in ihrer Frosthärte unterscheiden sobald das Knospenschwellen beginnt. Dabei scheint der Griffel am empfindlichsten zu sein, gefolgt von den Antheren, der Blütenkrone, des Blütenkelches und des Blütenstiels (Quamme 1974). Gefriergeschwindigkeit Die Gefriergeschwindigkeit beeinflusst das Muster der Eiskristallbildung und die Größe der Eiskristalle (Burke et al. 1976). Während eine schnelle Gefriergeschwindigkeit zu einer hohen Anzahl kleiner Eiskristalle führt, resultiert eine langsame Gefriergeschwindigkeit in weniger, dafür aber großen Eiskristallen. Schnelles Gefrieren hindert das Wasser außerdem daran, zu bevorzugten extra-zellulären Stellen zu diffundieren (Abb. 8). Dies führt zur intrazellulären Eiskristallbildung, wodurch die Pflanzenzellen mechanisch belastet und geschädigt werden (Olien 1967). Um den Zell- Natürliche Frostschutzmechanismen bei Obstgehölzen – von Supercooling bis Anti-Freeze Proteinen Tab. 5 Phänologiestadien der ‘Montmorency’ Sauerkirsche und BBCH-Skala nach Meier et al. (1994) als internationale Referenz mit kritischen Erfrier-Temperaturen Entwicklung 171 Entwicklungsstadium BBCH (Meier et al. 1994) Kritische Erfrier-Temperaturen Winterruhe 00 –28 °C Knospenschwellen 51 –9 °C Knospenaufbruch 53 –5 °C Kelchblätter geöffnet 57 –5 °C Ballonstadium 59 –2 °C Vollblüte 65 –2 °C Abgehende Blüte 67 –2 °C Fruchtwachstum 75 –2°C K 172 L. Hillmann et al. Abb. 8 Verlauf der Eiskristallisation bei langsamer (a) und schneller (b) Unterkühlung tod durch mechanische Überlastung der Zellwand durch Eiskristallbildung zu verhindern, sind niedrige Gefriergeschwindigkeiten wichtig. Freies Wasser in den Pflanzenzellen kristallisiert bzw. gefriert zwischen 0 °C und –10 °C (Burke et al. 1976); bei langsamen Gefriergeschwindigkeiten bilden sich extra-zelluläre Eiskristalle an bevorzugten Orten (Wiegand 1906). Nachblüte und Blüte am einjährigen Holz Viele Obstbäume reagieren auf einen Spätfrost mit einer Nachblüte zusätzlich zur zeitlichen Blühfolge, z. B. beim Apfel mit bis zu 10 Tagen späterer Blüte am einjährigen Holz. Maßnahmen in der Obstanlage Einfache Maßnahmen in der Obstanlage könnten bei verfrühtem Temperaturanstieg im Winter/Frühling die Nutzung vorhandener Unterkronenberegnung (Abb. 9a) oder Überdachungen zur Kühlung sein, sofern kein Schneerisiko vorliegt. So werden im Nordwesten Chiles bei unzureichendem Abb. 9 (a) Unterkronenberegnung und (b) Aufspannen von Hagelnetzen über Süßkirschen im Winter in Valparaiso, im Nordwesten Chiles – damit verdoppelt sich hier in warmen Wintern der Kältereiz bzw. die Chilling hours. (Foto: R. Ferreyra) K Kältereiz (Chilling) in warmen Wintern (Yuri und Blanke 2020) die Hagelnetze aufgespannt (Abb. 9b). Der Verlust der Frosttoleranz-Mechanismen während der De-Akklimation kann so möglicherweise verzögert werden. Danksagung Wir danken dem DAAD für das Promos-Stipendium für die Abschlussarbeiten für den ersten Autor zur Durchführung der Versuche an der Michigan State University und Abschluss ihrer Masterarbeit, Rodrigez Ferreyra für Abb. 9b und Daniel Zinsmeister für kritische Durchsicht des Manuskriptes. Funding Open Access funding provided by Projekt DEAL. Interessenkonflikt L. Hillmann, T. Einhorn und M. Blanke geben an, dass kein Interessenkonflikt besteht. Open Access Dieser Artikel wird unter der Creative Commons Namensnennung 4.0 International Lizenz veröffentlicht, welche die Nutzung, Vervielfältigung, Bearbeitung, Verbreitung und Wiedergabe in jeglichem Medium und Format erlaubt, sofern Sie den/die ursprünglichen Autor(en) und die Quelle ordnungsgemäß nennen, einen Link zur Creative Commons Lizenz beifügen und angeben, ob Änderungen vorgenommen wurden. Die in diesem Artikel enthaltenen Bilder und sonstiges Drittmaterial unterliegen ebenfalls der genannten Creative Commons Lizenz, sofern sich aus der Abbildungslegende nichts anderes ergibt. Sofern das be- Natürliche Frostschutzmechanismen bei Obstgehölzen – von Supercooling bis Anti-Freeze Proteinen treffende Material nicht unter der genannten Creative Commons Lizenz steht und die betreffende Handlung nicht nach gesetzlichen Vorschriften erlaubt ist, ist für die oben aufgeführten Weiterverwendungen des Materials die Einwilligung des jeweiligen Rechteinhabers einzuholen. Weitere Details zur Lizenz entnehmen Sie bitte der Lizenzinformation auf http://creativecommons.org/licenses/by/4.0/deed.de. Literatur Andrews PK, Proebsting EL (1987) Effects of temperature on the deep supercooling characteristics of dormant and deacclimating sweet cherry flower buds. 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In: Hampson (Hrsg) II International Cherry Symposium Acta Hort, Bd. 410, S 315–322 K 174 Walther E (2017) Landesbetrieb Hessen: In: Süddeutsche Zeitung Ausgabe 28. Juni 2017 Wenden B, Campoy JA, Jensen M, Lopez-Ortega G (2017) Climatic limiting factors: temperature. In: Quero-Garcia J, Iezzoni A, Pulawska J, Lang G (Hrsg) Cherries: botany, production and uses. COST and CAB international, Wallingford, S 166–188 Wiegand KM (1906) Some studies regarding the biology of buds and twigs in winter. Bot Gazette 41(6):373–424 Wisniewski M, Bassett C, Gusta LV (2003) An overview of cold hardiness in woody plants: seeing the forest through the trees. HortScience 38(5):952–959 Wisniewski M, Gusta LV, Neuner G (2014) Adaptive mechanisms of freeze avoidance in plants: a brief update. Env Expt Bot 99:133–140 Wisniewski M, Lindow SE, Ashworth EN (1997) Observations of ice nucleation and propagation in plants using infrared video thermography. Plant Physiol 113(2):327–334 Yooyongwech S, Horigane AK, Yoshida M, Yamaguchi M, Sekozawa Y, Sugaya S, Gemma H (2008) Changes in aquaporin gene expression and magnetic resonance imaging of water status in peach tree flower buds during dormancy. Physiol Plant 134:522–533 Blanke MM, Yuri A (2020) Chile – Exportrekorde im Obstbau im Schatten der Anden. Erwerbs-Obstbau. https://doi.org/10.1007/ s10341-020-00474-1 K L. Hillmann et al. Laura Hillmann studiert seit dem WS 2012/13 an der Universität Bonn Agrarwissenschaften und macht ihren Master mit dem Schwerpunkt Nutzpflanzenwissenschaften. Im Rahmen ihrer Bachelorarbeit bearbeitete sie als Literaturarbeit mögliche Bioindikatoren zur Bestimmung des Dormanzstatus der Obstgehölze, die 2016 im ErwerbsObstbau veröffentlicht wurde. Mit Unterstützung eines Promos-Stipendiums des DAAD untersuchte sie im Rahmen ihrer Masterarbeit die Frostanfälligkeit von Sauerkirschen an der Michigan State University in den USA. Nach dem Masterabschluss in Nutzpflanzenwissenschaften strebt sie eine Promotion/Post-Graduate an; ihr besonderes Interesse liegt in der Gartenbauwissenschaft.
W2981887135.txt	https://www.frontiersin.org/articles/10.3389/fvets.2019.00357/pdf	en		A Clinical, Radiographic and Histological Study of Unerupted Teeth in Dogs and Cats: 73 Cases (2001–2018)	Frontiers in veterinary science	2,019	cc-by	12,311	ORIGINAL RESEARCH published: 08 November 2019 doi: 10.3389/fvets.2019.00357 A Clinical, Radiographic and Histological Study of Unerupted Teeth in Dogs and Cats: 73 Cases (2001–2018) Emma Bellei 1,2 , Silvia Ferro 3 , Eric Zini 4,5,6 and Margherita Gracis 7,8* 1 Department of Veterinary Medical Science, Alma Mater Studiorum-Bologna University, Bologna, Italy, 2 “I Portoni Rossi” Veterinary Hospital, Bologna, Italy, 3 Department of Comparative Biomedicine and Food Science, University of Padova, Padova, Italy, 4 Clinic for Small Animal Internal Medicine, Vetsuisse Faculty, University of Zurich, Zurich, Switzerland, 5 Department of Animal Medicine, Production and Health, University of Padova, Padova, Italy, 6 Department of Internal Medicine, Istituto Veterinario di Novara, Novara, Italy, 7 Department of Dentistry and Oral Surgery, Istituto Veterinario di Novara, Novara, Italy, 8 Department of Dentistry and Oral Surgery, San Siro Veterinary Clinic, Milan, Italy Edited by: Boaz Arzi, University of California, Davis, United States Reviewed by: Maria M. Soltero-Rivera, University of Pennsylvania, United States Christopher John Snyder, University of Wisconsin-Madison, United States Correspondence: Margherita Gracis marghegracis@mac.com Specialty section: This article was submitted to Veterinary Dentistry and Oromaxillofacial Surgery, a section of the journal Frontiers in Veterinary Science Received: 18 June 2019 Accepted: 27 September 2019 Published: 08 November 2019 Citation: Bellei E, Ferro S, Zini E and Gracis M (2019) A Clinical, Radiographic and Histological Study of Unerupted Teeth in Dogs and Cats: 73 Cases (2001–2018). Front. Vet. Sci. 6:357. doi: 10.3389/fvets.2019.00357 Lack of dental eruption may be accompanied by development of dentigerous cysts and has also been rarely associated with neoplasia. However, little information is available on prevalence of unerupted teeth and associated lesions in dogs and cats. The main objective of this study was to describe the epidemiologic data of canine and feline dental patients with unerupted teeth, and assess the prevalence of associated dentigerous cysts and tumors. Secondary aims included the evaluation of possible factors implicated in cystic development, and description of the histological features of dentigerous cysts. Medical and dental records, intraoral photographs, intraoral radiographs of client-owned dogs and cats with clinically missing teeth examined between 2001 and March 2018 were reviewed. Collected data included signalment, reason for presentation, number, type, depth of inclusion and angulation of unerupted teeth, presence of cystic lesions or tumors, abnormalities affecting involved teeth, histopathological findings, performed treatment and outcome. Seventy-three animals (69 dogs and 4 cats) with 113 unerupted teeth were included. The most frequent unerupted tooth in dogs was the first premolar teeth (78%), followed by the canine and third molar teeth. Dentigerous cysts were diagnosed associated with 48 (44.4%) teeth in dogs and one out of five unerupted teeth in cats. The affected teeth in dogs were predominantly in horizontal inclination (40%) and in soft tissue inclusion (77%). Brachycephalic canine breeds were overrepresented. The only unerupted tooth in boxer dogs was the first premolar tooth (32 teeth). Ninety percentage of boxers with unerupted teeth developed associated lesions (25 dentigerous cysts and one tumor). Two ameloblastomas (one in a dog and one in a cat) and one osteosarcoma (in a dog) were diagnosed in association with three unerupted teeth. Histology was essential in diagnosing two odontogenic cysts not evident on radiographs. In all cases that were followed-up, treatment (i.e., extraction, extraction and surgical curettage, or operculectomy) appeared successful. Untreated dentigerous cysts showed progression at re-examination. None of the unerupted teeth without evidence of cyst at the time of diagnosis showed incipient cystic development. None of the evaluated factors were associated with lack of eruption and/or development of associated lesions. Keywords: unerupted teeth, dentigerous cyst, neoplasia, eruption, dog, cat Frontiers in Veterinary Science \| www.frontiersin.org 1 November 2019 \| Volume 6 \| Article 357 Bellei et al. Unerupted Teeth in Dogs and Cats INTRODUCTION obtain in case of small lesions. A method comparing the size of the periodontal ligament space of a reference tooth and the pericoronal lucency of unerupted teeth has been suggested to differentiate between small dentigerous cysts and normal dental follicles (14). Nevertheless, the applicability of this criterion remains to be confirmed. Extraction of embedded and impacted teeth is recommended in veterinary patients when related pathology (i.e., dentigerous cyst, bone, and root resorption) is diagnosed (14). However, indications for the correct approach to unerupted teeth in the absence of clinical and radiographic signs of complications are lacking in the veterinary literature. The main purpose of this retrospective study was to describe canine and feline dental patients with unerupted teeth and assess the prevalence of associated dentigerous cysts and tumors. As secondary aim, possible factors implicated in cystic development were evaluated. Furthermore, the radiographic and histological features were defined and compared in 10 cases to evaluate if the size of the pericoronal radiolucency in radiographically doubtful cases could help reaching a definitive diagnosis of cystic development. An unerupted tooth is a tooth that does not emerge into the mouth within the expected time frame because of lack of space, malposition, and other physical impediments such as the presence of a tumor or fibrous tissue along the path of eruption (impacted tooth), or because of a lack of eruptive forces (embedded tooth) (1). Clinical, radiographic and pathological features associated with unerupted teeth are well-described in humans (2), but only a few reports are present in the veterinary literature. In human patients, the prevalence varies considerably in different countries (from 8 to 38%) (3, 4). The most frequently affected teeth are the mandibular and maxillary third molar (up to 68.6% of the cases), followed by the maxillary canine, mandibular and maxillary premolar teeth, and maxillary central incisor teeth (5). In humans, 1.4% of unerupted teeth develop dentigerous cysts (3) and <1% may be associated with tumor development (6). Unerupted teeth are typically asymptomatic, but pain may be reported because of inflammation of the soft tissues surrounding the crown (pericoronitis), or pressure resorption of adjacent roots (1). Diagnostic imaging (i.e., radiography or computed tomography) is required to confirm the presence of an unerupted tooth in edentulous areas (3). Extraction is the treatment of choice in case of loss of bone around the impacted teeth, presence of cysts or tumors, displacement of the adjacent teeth, or chronic pain (1). Sometimes, treatment is recommended even in the absence of clinical or radiographic pathologic findings, as a preventive method against the development of cystic lesions that can lead to severe loss of surrounding bone and resorption of adjacent teeth (7). However, because of the proximity of many unerupted third molar teeth to the inferior alveolar nerve and the risk of nerve injury during extraction, in humans coronectomy and intentional root retention of healthy, unerupted teeth has also been recommended (8). Furthermore, surgical exposure and orthodontic treatment may be attempted in select cases (5). Little information is available on prevalence of unerupted teeth in companion animals. Only a few cases have been described in cats (9–12). In dogs, most of the studies and case reports have focused on the description of associated dentigerous cysts, with a reported prevalence of 29.1% (13, 14). Brachycephalic breeds (e.g., boxer, Boston terrier, pugs, and Shi-tzu) have been described as more frequently affected, but a variety of breeds of different morphology (e.g., Belgian malinois, German shepherd, Labrador retriever, Dalmatian dog, and greyhound) have also been reported. Based on the available information, the permanent first premolar tooth seems to be the most affected tooth in dogs, followed by the canine teeth (13, 14). Several criteria, including the presence of a coronal radiolucency extending apically beyond the cementoenamel junction, cortication, bone expansion, and root or bone resorption of adjacent teeth, have been suggested for the radiographic identification of cystic lesions (14). However, in absence of more sensitive diagnostic imaging systems such as cone beam computed tomography (CBCT) or standard CT, the radiographic diagnosis of cystic development may be difficult to Frontiers in Veterinary Science \| www.frontiersin.org MATERIALS AND METHODS Medical and dental records, intraoral photographs and radiographs of client-owned dogs and cats examined between February 2001 and March 2018 at different veterinary clinics in northern Italy were reviewed. All patients were presented because of an oral or maxillofacial problem, but not necessarily because of missing teeth. Complete examination of the head and oral cavity was performed under general anesthesia. Patients were included in the study if full mouth intraoral radiographic examination or at least radiographic examination of all edentulous areas had been performed, and if at least one unerupted tooth was radiographically present. Data collected for each patient included: signalment (species, breed, sex, age, and body weight at the time of diagnosis), main reason for presentation (i.e., related to missing tooth/teeth or not), number and type of unerupted teeth, presence or absence of clinically and/or radiographically visible signs indicative of cystic lesions (i.e., a well-defined radiolucency around or beside the crown of an unerupted tooth), presence of neoplastic lesions associated with unerupted teeth, histopathological findings (for biopsied lesions), depth of inclusion (i.e., gingival vs. bone inclusion) and inclination of the unerupted teeth in the jaw, radiographic abnormalities affecting unerupted tooth/teeth and adjacent teeth, treatment performed, treatment outcome, and follow-up findings. To describe the inclination of the unerupted tooth within the jaw, the classification system by Winter was utilized in this study, typically used to score the difficulty of impacted molar extraction in humans (15). The inclination, measured as the angle between the long axis of the tooth and the occlusal plane, may be horizontal (0◦ -30◦ in mesial direction), mesioangular (31◦ 60◦ in mesial direction), vertical (61◦ -90◦ in mesial direction), distoangular (91◦ -180◦ in distal direction), inverted (negative 2 November 2019 \| Volume 6 \| Article 357 Bellei et al. Unerupted Teeth in Dogs and Cats angle < 0◦ , with the crown pointing in the opposite direction to the occlusal plane) (15) (Figure 1). Teeth were considered in soft tissue inclusion when one side (normally the side toward the alveolar margin) was covered only by gingival tissue, and in bone inclusion when they were completely encased in bone (tooth not visible after cutting the overlying gingiva and/or radiographically surrounded by bone) (16). Treatments performed included: operculectomy (for teeth in gingival inclusion), extraction of unerupted teeth only (if cystic lesions were not evident clinically and/or radiographically), extraction of unerupted teeth and curettage of the cystic lining, and maxillectomy/mandibulectomy including the unerupted teeth and cystic lesion (in case of concomitant presence of neoplasia). In some cases, treatment was not performed, but radiographic monitoring was recommended. For histological evaluation, samples were fixed in 10% buffered formalin and paraffin embedded. Then, 4 µ sections were stained with hematoxylin and eosin, and observed with an optical microscope (Olympus BX40, Segrate, Milan, Italy). Mineralized tissues were decalcified after fixation with a rapid decalcifying solution (Biodec R, Bio-opotica, Milan, Italy). A single pathologist (SF) examined all histological samples. Outcome of treatment was evaluated clinically and whenever possible radiographically at the last follow-up available. The possible outcomes were: complete healing, absence of healing, or recurrence. The lesion was classified as completely healed if, at the time of the last follow-up examination available, there was absence of swelling or any other type of soft tissue abnormality [except for the presence of mild inflammation and suture material along the surgical wound, which was considered normal in case of short-term (<4 weeks) follow-ups] and, when available, the radiographic examination showed evidence of new bone formation and absence of any radiolucent lesions indicative of a cystic process at the previous surgical site. Absence of healing at the time of the last follow-up examination available was determined by clinical evidence of flap’s dehiscence, presence of swelling, persistent (>4 weeks) soft tissue inflammation or any other type of soft tissue abnormality at the surgical site, and/or radiographic persistence of a radiolucent lesion or a radiopaque, tooth-like material following treatment. If, following complete soft tissue healing, a radiolucent lesion was evident at a later time, a recurrence was recorded. Untreated cases had follow-up evaluations to assess for development of new, previously absent, cystic lesions (defined as incipient cysts), progression of pre-existing lesions (progressive cyst), or if the clinical and radiographic appearance of the unerupted tooth with or without a previously diagnosed cyst was unchanged (defined as static or no cyst, respectively). Finally, it was evaluated if the size of coronal radiolucencies present around unerupted teeth could predict the nature of the lesions (i.e., dentigerous cyst vs. dental follicle), as recently suggested (14). In particular, it was assessed if pathological cystic lesions always appeared larger than three times the width of the periodontal space of a normal canine tooth in the same patient (14). Statistical Analysis Descriptive analyses were performed to characterize the population of dogs and cats if at least five cases per group were present. Contingency tables were used to verify if the type of teeth was associated with the presence of a cyst. Chi2 test was used to identify an association between the type of inclusion of unerupted teeth and the presence of cysts. The above tests were also used to identify an association between the type of inclination of unerupted teeth and the presence of cysts, and to FIGURE 1 \| Angle of inclination of unerupted teeth relatively to the alveolar margin, using a modified Winter classification (15). This example shows an unerupted right mandibular first premolar tooth (asterisk) in mesioangular inclination. DA, distoangular; H, horizontal; I, inverted; MA, mesioangular; V, vertical; Blue dotted line, unerupted tooth long axis; Red line, alveolar margin. Frontiers in Veterinary Science \| www.frontiersin.org 3 November 2019 \| Volume 6 \| Article 357 Bellei et al. Unerupted Teeth in Dogs and Cats Number and Type of Unerupted Teeth compare the frequency of unerupted teeth between types of teeth. The overall number of first, second and third incisor, canine, first and fourth premolar teeth was considered to be 276, assuming that all of the listed teeth were present in the 69 dogs (i.e., 69 × 4 = 276); the overall number of third molar teeth was considered to be 138, assuming that each of the 69 dogs had 2 third molar teeth (i.e., 69 × 2 = 138). To evaluate differences between type of most frequent unerupted teeth (i.e., canine, first premolar, and third molar) and body weight, the Kruskal–Wallis test was applied. To compare the age of dogs with and without dentigerous cysts, the Mann–Whitney test was applied. Significance was considered for P < 0.05. In cats, five unerupted teeth were diagnosed in four patients (Figure 2). In one case, both maxillary second premolar teeth were unerupted. In dogs, 108 unerupted teeth were diagnosed: 85 (78.7%) first premolar teeth, 8 (7.4%) canine teeth, 8 (7.4%) mandibular third molar teeth, 5 (4.6%) mandibular incisor teeth, 1 (0.9%) mandibular second molar tooth, and 1 (0.9%) maxillary fourth premolar tooth (Figure 3). Multiple unerupted teeth were diagnosed in 29 (42%) dogs (Table 1). Lack of eruption was more frequent for first premolar teeth compared to all other teeth (p < 0.0001, for each contrast), and for third molar teeth compared to second molar teeth (p = 0.0009), fourth premolar teeth (p = 0.0009), second incisor teeth (p = 0.0031), and first incisor teeth (p = 0.008) (Table 2). The median body weight of dogs demonstrating unerupted canine, first premolar and third molar teeth was 3 (min 2.6–max 18), 28 (min 1.8–max 35), and 5.4 (min 2.7– max 9.6) kg, respectively. Body weight of dogs with RESULTS Seventy-three cases [69 (94.5%) dogs and 4 (5.5%) cats] with a total of 113 unerupted teeth were included in the study (Supplemental Table 1). Signalment Three cats were European short hair and one was a Maine coon. There were one female and three male cats, all neutered. The ages at the time of diagnosis were 1, 2.5, 5, and 10 years, respectively. The body weight was 4, 5 (2 cats), and 8 kg. Canine breeds included boxer [20 dogs (28.9%)], Labrador retriever [6 dogs (8.7%)], Maltese, Chihuahua and mix breed [5 dogs each (7.2%)], pugs [4 dogs (5.8%)], Bernese mountain dog, Yorkshire terrier, Shih-tzu, spitz, and miniature schnauzer [2 dogs each (2.9%)], bull terrier, bull mastiff, Czechoslovakian wolfdog, English setter, Epagneul papillon, French bulldog, German shepherd, golden retriever, Irish setter, Italian shepherd, miniature poodle, Pekingese dog, pinscher, pitbull [1 dog each (1.4%)]. Forty-one dogs (59.4%) were males (38 intact and 3 neutered) and 28 (40.6%) females (13 intact and 15 neutered). The mean and median ages at the time of diagnosis in dogs was 4.4 and 3.6 years, respectively (range from 5 months to 10.5 years). The mean body weight for dogs was 21.0 kg (median 25 kg, range from 1.8 to 59.5 kg). Chief Complaint The main reasons for dental consultation included the presence of an oral swelling/mass at or near the area of a missing (unerupted) tooth [8 cases, including 1 cat (10.9%)], which could be due to a cystic or neoplastic development, a missing tooth that was in fact unerupted [7 cases (9.6%], and discoloration of a tooth near the area of a missing (unerupted) tooth [1 case (1.4%)]. The remaining 57 cases (78.1%), including three cats, presented for unrelated reasons (e.g., periodontal disease, palatal defect, tooth fracture) and the presence of the unerupted tooth/teeth was an incidental finding diagnosed during the routine intraoral radiographic examination. Frontiers in Veterinary Science \| www.frontiersin.org FIGURE 2 \| Number and type of unerupted teeth in cats included in the study. AA, associated ameloblastoma; DTC, teeth with radiographically or histologically confirmed dentigerous cyst. 4 November 2019 \| Volume 6 \| Article 357 Bellei et al. Unerupted Teeth in Dogs and Cats FIGURE 3 \| Number and type of unerupted teeth in dogs included in the study. CAA, associated canine acanthomatous ameloblastoma; DTC, teeth with radiographically or histologically confirmed dentigerous cyst; OSA, associated osteosarcoma; SN, supernumerary tooth. TABLE 2 \| Comparison of the frequency of unerupted teeth in dogs. TABLE 1 \| Multiple unerupted teeth (T/I) in dogs (numbering based on the modified Triadan system). UT Total number of dogs with multiple T/I 2 Total number of T/I 4 Number of dogs and location of T/I PM1 3 I1 p < 0.0001 p < 0.0001* p < 0.0001* p < 0.0001* 0.0009X p = 0.008X p = 0.0031X NS NS M2 p < 0.0001* p = 0.0009X 1: 304-305-404 I1 p < 0.0001* p = 0.008X NS 1: 305-311-405 I2 p < 0.0001* p = 0.0031X NS NS 1: 104-204-405 PM4 p < 0.0001* p = 0.0009X NS NS 1: 105SN-205SN-305-405 I2 NS NS UT, unerupted tooth; * statistically significant for PM1; X statistically significant for M3; NS, not significant. Statistical test: r × c contingency table. 1: 205SN-305-405 2 M2 p < 0.0001* 1: 301-301SN-401SN 22 M3 M3 1: 305-311-405-411 5 PM1 19: 305-405 1: 204-305 1: 208-411 Neoplastic Lesions and Dentigerous Cysts 1: 311-411 A 10-year-old, neutered male, 7.8 kg, domestic short hair cat presented with an unerupted right maxillary second premolar tooth associated with a cystic lesion and an ameloblastoma. This was the only cystic lesion diagnosed in cats. The other unerupted teeth in cats were not evaluated histologically, but appeared radiographically negative for cystic development. SN, supernumerary tooth. unerupted first premolar teeth was greater than in dogs with unerupted canine and third molars (p = 0.005 and p = 0.004, respectively). Frontiers in Veterinary Science \| www.frontiersin.org 5 November 2019 \| Volume 6 \| Article 357 Bellei et al. Unerupted Teeth in Dogs and Cats Chihuahuas (3 cysts) and Maltese dogs (5 cysts); 2 of 4 pugs (4 cysts), and mix breed dogs (2 cysts); and 1 of 2 Shih-tzu dogs (2 cysts). Each of the single Italian shepherd, German shepherd, bull terrier, Czechoslovakian wolfdog, and pinscher dogs developed one cyst each. Twenty-nine of 48 dentigerous cysts (60.4%) were diagnosed in intact male dogs, 6 (12.5%) in neutered male dogs, 5 (10.4%) in intact female dogs, and 8 (16.7%) in female spayed dogs. The mean age at the time of diagnosis of the dogs showing dentigerous cysts was 4 years (median 3.3 years; range from 1.2 to 9.2 years). The mean body weight of dogs with dentigerous cyst was 26.1 kg (median 30.8 kg; range from 3 to 46 kg). Concomitant neoplasias and cystic lesions were also histologically diagnosed in two dogs with unerupted teeth: a 5 months old, intact male, English setter dog with a canine acanthomatous ameloblastoma (CAA) (associated with an unerupted right maxillary canine tooth) and a 9 years old, female spayed, Boxer dog with an osteosarcoma (associated with an unerupted left mandibular first premolar tooth). Because of the impossibility to determine if the neoplastic and cystic lesions developed independently or were related changes, these teeth were not considered in the following analysis of cases with cysts. Therefore, apart from these oncological cases, the presence of a dentigerous cyst was diagnosed in association with 48 teeth (44.4% of the unerupted teeth in dogs) in 34 canine patients, by radiography, and/or histology (Supplemental Table 1). Thirty-four cysts (77.3%) developed in 25 brachycephalic dogs (boxer, pug, Maltese, Shih-tzu, and Chihuahua dogs), and 14 cysts (22.7%) in nine non-brachycephalic dogs (Labrador retriever, miniature pinscher, Italian shepherd, Bull terrier, Czechoslovakian wolf dog, and German shepherd dogs). Histopathologic Findings Histological examination was performed on excised tissues associated with 47 (40.4%) unerupted teeth (one from a cat and 46 from dogs), including the three neoplastic cases. Submitted histological samples included: the lining of the cystic lesion only (nine teeth), the lining of the cystic lesion and the extracted teeth (29 teeth), soft tissue (i.e., gingiva) covering the crown of the unerupted teeth (when operculectomy was performed) (three teeth), gingiva and lining of the cyst (two teeth), the unerupted tooth only (one tooth), an incisional biopsy of a suspected neoplastic lesion associated with an unerupted tooth (one tooth), or the surgical excised maxillary portion including the neoplastic lesion and the unerupted tooth (two cases, of which one was a cat). Three cases presented concomitant cystic and neoplastic diseases associated with unerupted teeth. One cat had an ameloblastoma, one dog had a CAA and one dog an osteosarcoma. Histologic diagnosis of dentigerous cyst was made for 38 other teeth. All samples showed histological similarities. The cystic lesions were often multilocular, lined by a variably thick, multilayered, squamous epithelium with multifocal or diffuse keratinization and multifocal parakeratosis (Figure 4). Keratin was scarce or moderate, never filling the lumen. Often the stratum basale was described as palisading. Occasionally, apoptosis and superficial cytoplasmic vacuolar degeneration were present. The epithelium was often multifocally detached, with hemorrhage and fibrin deposition. The wall was composed of dense fibrous tissue frequently showing a multifocal, mild to severe, mononucleated infiltrate (lymphocytes, plasma cells, macrophages, hemosiderophages), and sometimes cholesterine crystals (a sign of cellular damage). Neutrophils were sometimes present. Multifocal, small irregular areas of newly formed cementum/osseous tissue were also a common finding. Six of 47 histological samples were negative for cystic or neoplastic development. Radiographic vs. Histological Diagnosis of Dentigerous Cyst in Dogs In dogs, full-mouth radiographs were available for 66 cases, and partial radiographic examination (including radiographs of all edentulous areas) for remaining three cases. Excluding the cases with neoplastic disease, a welldefined radiolucency compatible with a dentigerous cyst was radiographically evident for 44 teeth. The diagnosis was histologically confirmed in 34 (77.3%) of these cases, while in the remaining 10 cases histology was not performed. Four teeth were radiographically equivocal (two teeth) or not evident (two teeth), however cystic lesions were confirmed histologically. The presence of a dentigerous cyst was suspected for 19 other teeth, but the radiolucency was too small to reach a definitive radiographic diagnosis and histology was not performed. Finally, the radiographic examination was considered negative for cystic changes in the remaining 39 unerupted teeth. Only six of these cases were evaluated histologically, confirming the absence of cystic tissues. Six of eight (75%) unerupted canine teeth and 41 of 85 (48.8%) unerupted first premolar teeth developed a radiographic and/or histological change confirming a dentigerous cyst (Figure 3). The remaining cystic lesion was associated with a mandibular third molar tooth. There was a statistical association between the type of teeth and the absence of cyst (p = 0.010); in particular, the mandibular third molar tooth associated with the absence of cyst (p = 0.038). No statistical association was documented between the type of teeth and the presence of cyst, although a trend was observed for canine and first premolar teeth (p = 0.089 and p = 0.094, respectively). Differentiation Between Small Dentigerous Cysts and Normal Dental Follicle Ten of the 62 teeth that were either radiographically equivocal or negative for the presence of cystic lesions were evaluated histologically (Table 3). Four of these samples histologically demonstrated evidence of a dentigerous cyst, even though the size of their pericoronal Characteristics of Dogs Showing Dentigerous Cyst Dentigerous cysts were diagnosed in 18 of 20 (90%) boxer dogs (25 cysts), 2 of 6 (33.3%) Labrador retrievers (2 cysts); 2 of 5 Frontiers in Veterinary Science \| www.frontiersin.org 6 November 2019 \| Volume 6 \| Article 357 Bellei et al. Unerupted Teeth in Dogs and Cats FIGURE 4 \| Histologic representation of a typical dentigerous cyst from one of the canine cases included in the study. (A) The wall of the cyst is lined by multistratified epithelium (black arrows) surrounded by moderate fibrous stroma with multifocal chronic infiltrates. In some areas the epithelium is detached (white arrows). Black asterisk, lumen of the cyst. Bar = 100 µm. (B) An irregular area of cementum (white asterisk) is visible in the stroma near the cyst. Bar = 100 µm. (C) In this field, the epithelium shows a basilar palisade similar to odontogenic epithelium (black bars). Bar = 25 µm. (D) The epithelium is characterized by parakeratotic keratosis (arrowheads). Black asterisk, lumen of the cyst. Bar = 25 µm. TABLE 3 \| Differential diagnosis between small dentigerous cysts and normal dental follicle. Breed Age (months) Weight (Kg) T/I Pericoronal space (mm) PDL space of canine tooth (mm) Pericoronal space: PDL space of canine tooth ratio Rx Histology Mix breed 84 25 405 0.9 0.5 1.8 Dubious Cyst Bull terrier 19 18 204 0.9 0.6* 1.5 No cyst Cyst Maltese dog 19 5.4 305 0.8 0.4 2 Dubious Cyst Pinscher 36 4 405 0.3 0.3 0 No cyst Cyst Mix breed 92 11 305 0.8 0.2 4 No cyst No cyst Miniature poodle 8 5.8 305 0.7 0.2 3.5 No cyst No cyst Miniature poodle 84 5.8 405 0.6 0.2 3 No cyst No cyst Boxer 54 46 305 0.5 0.2 2.5 No cyst No cyst Chihuahua 18 2 405 0.3 0.3 0 No cyst No cyst Chihuahua 18 2 305 0.3 0.3 0 No cyst No cyst Width of pericoronal radioluncency and periodontal ligament space of a reference tooth [the adjacent or contralateral (* ) canine tooth] for radiographically dubious or negative teeth evaluated histologically. Numbering based on the modified Triadan system. Rx, radiographic diagnosis for cystic lesion; T/I, unerupted tooth. The histological examination of the remaining three teeth (two with pericoronal radiolucency and periodontal ligament space of the reference tooth of identical dimensions, one with a pericoronal radiolucency that was 2.5 times the size of the periodontal ligament space of the referenced tooth) were negative for histologic signs consistent with a cyst. radiolucency was equal or less than three times the width of the periodontal ligament space of the reference tooth. Three teeth histologically consistent with dentigerous cysts demonstrated a pericoronal radiolucency that was in fact three times or more the width of the periodontal ligament space of the adjacent canine tooth. Frontiers in Veterinary Science \| www.frontiersin.org 7 November 2019 \| Volume 6 \| Article 357 Bellei et al. Unerupted Teeth in Dogs and Cats Depth of Inclusion of Unerupted Teeth in vertical inclination and the teeth with a different inclination, and cystic development (p = 0.879). The radiographic inclination of unerupted teeth that developed dentigerous cysts was horizontal in 21 cases (43.8% of teeth with dentigerous cysts; 51.2% of teeth in horizontal position), vertical in 12 cases (25% of teeth with dentigerous cysts; 44.4% of teeth in vertical position), mesioangular in 10 cases (20.8% of teeth with dentigerous cysts; 33.3% of teeth in mesioangular position), and inverted in four cases (8.3% of teeth with dentigerous cysts; 57.1% of teeth in inverted inclination); distoangular in one case (2.1% of the teeth with dentigerous cysts). There was no statistical association found between the type of inclination and the presence of cysts (p = 0.484). In feline patients, one of five unerupted teeth was in complete bone inclusion, and four teeth were in soft tissue inclusion, including one tooth associated with an ameloblastoma (Table 4). In dogs, 25 (23.6%) of 106 unerupted teeth were in complete bone inclusion (10 teeth with evidence of cystic development), and 81 teeth (76.4%) were in soft tissue inclusion at the time of diagnosis (38 teeth with cystic development) (Table 5). The remaining two teeth, associated with neoplastic lesions, were also considered in soft tissue inclusion. No statistical association was found between the type of inclusion and the presence of cysts (p = 0.686). Inclination of Unerupted Teeth All five unerupted feline teeth were in vertical inclination, including the tooth associated with the ameloblastoma (Table 4). In dogs, 43 (39.8%) teeth were positioned horizontally, 31 were angled [30 (27.8%) in mesioangular and 1 (0.9%) in distoangular direction], 27 (25%) were in vertical position, and 7 (6.5%) were inverted (Table 5). The remaining two teeth, associated with neoplastic lesions, were in vertical and horizontal inclination. There was no statistical difference between the teeth Abnormalities Affecting Unerupted and Adjacent Teeth External resorption of the canine tooth adjacent to the neoplastic lesion was evident in the cat with ameloblastoma associated with an unerupted right maxillary second premolar tooth. Thirty-six dogs demonstrated abnormal unerupted and/or adjacent teeth. External root resorption was commonly TABLE 4 \| Inclination and type of inclusion of unerupted teeth with and without confirmed dentigerous cyst in cats. Tooth inclination MA With DTC DA Without DTC With DTC Type of inclusion V Without DTC H I Bone inclusion With DTC Without DTC With DTC Without DTC With DTC Without DTC With DTC Without DTC Soft tissue inclusion With DTC Without DTC 0 203 0 0 0 0 0 1 0 0 0 0 0 1 0 106–206 0 0 0 0 1* 3 0 0 0 0 0 0 1* 3 Total 0 0 0 0 1 4 0 0 0 0 0 1 1 3 Numbering based on the modified Triadan system. DA, distoangular; H, horizontal; MA, mesioangular; V, vertical; I, inverted; DTC, dentigerous cyst; * tooth associated with cyst and neoplasia. TABLE 5 \| Inclination and type of inclusion of unerupted teeth with and without confirmed dentigerous cyst in dogs. Tooth inclination MA DA Type of inclusion V H I Bone inclusion Soft tissue inclusion With DTC Without DTC With DTC Without DTC With DTC Without DTC With DTC Without DTC With DTC Without DTC With DTC Without DTC With DTC Without DTC 301–401 0 0 0 0 0 1 0 0 0 2 0 0 0 3 302–402 0 0 0 0 0 0 0 2 0 0 0 1 0 1 104–204 0 0 0 0 0 0 4* 1 0 0 1 0 3* 1 208 0 0 0 0 0 1 0 0 0 0 0 0 0 1 304–404 0 0 0 0 0 0 3 0 0 0 0 0 3 0 105–205 0 0 0 0 0 0 2 1 0 0 0 0 2 1 305–405 10 20 1 0 13* 8 12 13 4 0 9* 11 31 31 310–410 0 0 0 0 0 0 0 2 0 0 0 2 0 0 311–411 0 0 0 0 0 4 1 2 0 1 0 1 0 6 Total 10 20 1 0 13 14 22 21 4 3 10 15 39 44 Numbering based on the modified Triadan system. DA, distoangular; H, horizontal; MA, mesioangular; V, vertical; I, inverted; DTC, dentigerous cyst; * tooth associated with cyst and neoplasia. Frontiers in Veterinary Science \| www.frontiersin.org 8 November 2019 \| Volume 6 \| Article 357 Bellei et al. Unerupted Teeth in Dogs and Cats In the dog with an osteosarcoma associated with an unerupted left mandibular first premolar tooth, the untreated dentigerous cyst associated with its right mandibular first premolar tooth showed progression at the 3-month radiographic follow-up, but treatment was again refused by the owner. Another untreated cyst associated with a mandibular first premolar tooth showed progressive changes at the 6-month follow-up, and surgical excision was then performed. All other nine untreated unerupted teeth did not show clinical evidence of an incipient cyst, but none was radiographically re-examined. The cases with the remaining four untreated unerupted teeth (two with dentigerous cyst) were lost at follow-up. The 12-months follow-up of the dog with OSA showed significant radiographic enlargement of the neoplasia and the associated cystic lesion, despite palliative treatment. The maxillectomy site of the dog with CAA at the 1-month follow-up appeared clinically healed. associated with large cystic lesions or tumors (42 teeth). Four unerupted teeth were malformed. One dog was affected by generalized dentin dysplasia, also involving the unerupted tooth. Three teeth that were secondarily involved by the expanding cystic lesion were non vital. Three teeth adjacent to unerupted teeth were missing and 15 teeth showed morphological abnormalities (i.e., fused roots). Thirty-three dogs did not have evidence of morphological or structural abnormalities of unerupted or adjacent teeth. Treatment In cats, two unerupted teeth were extracted from the same patient, and two unerupted teeth (with no signs of associated complications) were left untreated, with the recommendation to radiographically monitor it over time. Partial maxillectomy including the unerupted tooth was performed for treatment of the ameloblastoma. In dogs, operculectomy was performed to treat seven (6.5% of unerupted teeth) first premolar teeth, one of which showed radiographic and histological signs of cystic development. Fortyseven (43.5%) teeth without signs of dentigerous cyst were simply extracted, 38 (35.2%) teeth were extracted and curettage of the cystic lining was performed, and one (0.9%) tooth was excised during a rostral maxillectomy performed to treat a CAA. The remaining 15 (13.9%) unerupted teeth (four with radiographic signs of cystic development and one associated with an osteosarcoma) were not treated. The large OSA was treated by palliative intralesional excision and radiation therapy. DISCUSSION AND CONCLUSIONS Tooth eruption is the axial movement of the tooth from its non-functional position in the bone to functional occlusion (17). It is a multifactorial, not yet fully understood process, starting from the reduced enamel epithelium (REE) covering the crown of the developing tooth, which fuses with the gingiva during the process. The REE produces proteases that, by breaking down connective tissue, decrease the tissue resistance to eruption. The dental follicle is also essential in the creation of an eruptive pathway for the tooth by modulating bone remodeling. It produces growth factors promoting the differentiation of monocytes into osteoclasts, that create a path to eruption. At the same time the production of osteoblasts from mesenchymal stem cells is accentuated at the base of the alveolar crypt (17). Teeth may fail to erupt because of mechanical obstruction by a physical barrier (e.g., gingiva, bone, neoplastic tissue, and tooth crowding) (defined as tooth impaction), or because of an impaired mechanism of eruption [e.g., a dysfunction of REE cells (18)] (defined as dental retention, or embedded teeth) (19). In humans a discrepancy between jaw and tooth size is described as a possible cause of the frequent impaction of the third molar teeth (5). It is interesting to note that 6 of 8 unerupted canine teeth (in Maltese and Chihuahua dogs) and 7 of 8 unerupted mandibular third molar teeth (in Yorkshire terrier, Maltese, Shihtzu, Epagneul papillon, and pug dogs) in the present study were diagnosed in small breed dogs. The reason could be related to the limited jaw size typical of these breeds, frequently also showing congenitally missing third molar and other teeth (20). In agreement with the literature, in the present study it was also found that brachycephalic dogs were overrepresented (40 dogs; 57.9%). Boxer dogs in particular accounted for 28.9% of the canine study population. Interestingly, they only showed unerupted first premolar teeth, and 18 out of 20 boxer dogs developed at least one dentigerous cyst. Furthermore, Outcome of Treatment In cats, clinically complete soft tissue healing was evident at the last follow-up visit for three teeth (two extracted teeth, 1 month follow-up; one tooth included in a maxillectomy procedure, 2 months follow-up). The clinical and radiographic re-examinations on two untreated teeth, performed in other two cats 3 months and a 3 years following diagnosis, showed no changes on the affected tooth or nearby bone or teeth. In dogs, only clinical (43 teeth), clinical and radiographic (35 teeth), or clinical and tomographic (one tooth) follow-ups were available. Follow-ups were not available for the remaining 29 teeth. Follow-up visits were performed from 1 to 68 months (mean 8 months; median 1.5 months) following diagnosis and/or treatment. In 62 of 85 cases treated by extraction and/or curettage, treatment appeared successful with complete healing of soft tissues and (in cases where diagnostic imaging was performed) new bone formation at the surgical site (100%) (Figure 5). No cases of recurrence were reported. The remaining 23 extracted teeth were lost to follow up. Short-term follow-up re-examination was available for four teeth that were treated by operculectomy: a 1-month clinical reexamination in two cases and 3-month clinical and radiographic re-examination in other two cases. The procedure appeared successful (the crown of the treated tooth being still exposed above the gingival margin at follow-up), however further eruption was not apparent. Frontiers in Veterinary Science \| www.frontiersin.org 9 November 2019 \| Volume 6 \| Article 357 Bellei et al. Unerupted Teeth in Dogs and Cats FIGURE 5 \| (A) Intraoral radiograph showing a dentigerous cyst (arrowheads) developed around the right mandibular first premolar tooth (asterisk) in a 3-years-old boxer dog. (B) Intraoral radiograph at the 1-year follow-up visit, following extraction of the right third incisor, canine, first and second premolar teeth, and cystic lining curettage. the only three unerupted maxillary first premolar teeth (all being supernumerary teeth) were found in this breed. Another interesting finding was the high prevalence of dentigerous cyst formation in boxer dog (78% of unerupted teeth in this breed developed a cyst, and overall 52% of cysts associated with unerupted teeth were diagnosed in boxers). Moreover, in the entire study period a total of 39 boxer dogs were anesthetized and evaluated radiographically by the dental and oral surgery departments of the involved veterinary clinics (data not shown). Therefore, it is notable that more than 50% of boxers presented for various dental and oral problems had at least one unerupted tooth (20 dogs), and that most of these developed associated lesions (18 dentigerous cysts and 1 OSA). Brachycephalic dogs often show tooth rotation, tooth crowding, and reduced interproximal spaces. However, in the cases described here most unerupted teeth were located at normally wide interproximal spaces. Also, the gingival tissues appeared clinically normal in consistency and thickness, the alveolar bone did not seem abnormally dense, either radiographically or during extraction, and only in a few cases dental crowding (six unerupted teeth being supernumerary) or the presence of neoplastic tissues over the affected teeth could justify a mechanical obstruction to eruption. Therefore, other pathogenetic mechanisms may have been involved. Histologically normal gingival tissues overlying numerous impacted teeth were described in a 7-month-old Bedlington terrier dog (21). A genetic basis is hypothesized for the primary failure of eruption (PFE) in humans. PFE is a non-syndromic condition that occurs without any obvious evidence of obstruction, most commonly affecting non-succedaneous teeth (i.e., the molar teeth) (22). This condition seems to be linked to a mutation of the PTH1R (parathyroid hormone receptor 1) gene that, Frontiers in Veterinary Science \| www.frontiersin.org in association with the parathyroid like hormone, seems to be able to affect number, quality and function of osteoclasts and osteoblasts (23, 24). Reduced bone resorption by osteoclast cells and bone formation by odontoblasts could contribute to dental eruption failure (23). Genetic studies would be necessary to evaluate if a similar mechanism could be involved in particular for failure of eruption of the first premolar and molar teeth (nonsuccedaneous teeth) in dogs and if a genetic breed predisposition exists. In the present study all cases with multiple unerupted teeth showed lack of eruption in the same jaw (mandibles or maxillae), or unilaterally and isolated to one side. Only one of 29 cases demonstrated unerupted teeth in diagonally opposite jaws (left maxillary fourth premolar tooth and right mandibular third molar tooth). These findings may support the potential for an underlying genetic influence contributing to the condition. One of the important limitations of the present study is the lack of data of the general population visited during the same period of time at the same veterinary clinics. It is therefore difficult to demonstrate definite associations between lack of eruption and development of associated lesions (i.e., cysts and tumors), and breed, age, body weight, sex, and neutering status of the animals. However, based on data provided by the Italian kennel club (ENCI) on dogs registered in Italy in the last 5 years, the distribution ratio between boxers and Labrador retrievers in the country is greatly in favor of the latter breed (1 boxer: 2.6 Labrador retrievers) (ENCI)1 . Therefore, the present findings (few unerupted teeth in a few Labrador retrievers, despite their wide distribution in the country, and numerous unerupted teeth 1 http://www.enci.it/ 10 November 2019 \| Volume 6 \| Article 357 Bellei et al. Unerupted Teeth in Dogs and Cats mandibular first premolar teeth in Shih-tzu dogs (20). In the present study, a higher prevalence (44.4% dentigerous cysts formation) was reported. This difference could be related to the study methodology (i.e., diagnosis based on radiographic as well as histologic findings), and possibly to epidemiologic factors (e.g., possible predisposition in certain breeds, that may be more common in Italy as compared to the USA or Korea). Most teeth (76.4%) were found in soft tissue inclusion, and almost half of these developed dentigerous cysts. However, cystic lesions enlarge over time, causing progressive bone resorption. Therefore, these teeth may have originally been in bone inclusion, and it can not be determined if depth of inclusion is actually a predisposing factor to cyst development. Also, there was no statistical association between inclination angle and the presence of cystic lesions. In the present study the mandibular first premolar tooth was not only the most commonly diagnosed unerupted tooth (85 of 108 unerupted teeth), but was also most commonly associated with cyst development (41 of 48 dentigerous cysts). This is also in agreement with the data extrapolated from the literature, with cysts described in association with 49 out of 71 (69%) reported unerupted first premolar teeth (13, 14, 20, 29, 50, 54, 57, 58, 60, 70). Interestingly, six of eight unerupted canine teeth in the present study were associated with a cystic lesion, one with a neoplastic lesion, and one showed a radiographically equivocal pericoronal appearance. However, only one of eight unerupted third molar teeth had a radiographically evident and histologically confirmed cyst, and another one had a radiographically equivocal appearance. Even though it could be speculated that the high prevalence of cyst formation at the canine teeth as compared to mandibular third molar teeth could be related to the greater REE surface of larger teeth, the high prevalence of cysts associated with first premolar teeth would contradict this theory. Typical histological characteristics of dentigerous cysts associated with unerupted teeth were repetitively observed. However, the diagnosis is sometimes challenging, particularly in case of small biopsy samples, or because of the cystic nature of the tissues, that can be difficult to sample or may be incorrectly oriented. Also, the lining epithelium may be partially lost from the samples. In these cases, it is important to look for and evaluate other typical characteristics, like the presence of a cystic space combined with the presence of fibrin or cholesterin crystals, keratin, and/or cementum/osseous tissue in a fibrotic wall, and/or a chronic infiltrate. Finally, as always, histopathological findings should be correlated to the radiological and clinical presentation. In humans, tumors may be associated with unerupted teeth with or without dentigerous cysts (75, 76). The epithelial component seems to be essential for neoplastic transformation, but the exact pathogenetic mechanism is still unclear (74, 75). Odontomas are among the most common associated tumors (77, 78), and are mainly diagnosed in the second decade of life (77, 79). In about one fourth of these patients, dentigerous cysts are also present (76). Surgical excision of the tumor and extraction of the associated unerupted teeth is the recommended treatment, however tumor excision and denudation of the unerupted teeth, followed by orthodontic treatment, is sometimes possible (78). in a relatively large number of boxer dogs, in spite of their limited distribution) may support a genetic predisposition in boxers. Similarly to the cases published in the veterinary literature (13, 14, 20, 21, 25–61), the first premolar tooth was by far the most frequently unerupted tooth, followed by the maxillary/mandibular canine teeth. In our study population, the mandibular third molar tooth was also frequently affected. Also, the great majority of unerupted teeth in dogs in this study were mandibular teeth (91%), similarly to the epidemiologic data reported in humans (4, 62, 63). Tooth inclination and depth of inclusion into hard or soft tissues in human patients are important features used by surgeons to predict the degree of difficulty of surgical extraction of unerupted teeth (15). Seventy-five percentage of impacted mandibular third molar teeth are reported in mesioangular and horizontal inclination, but the inclination of those developing into dentigerous cysts is mainly vertical (64%) or horizontal (41%) (64, 65). In the present study, unerupted teeth in dogs were also mainly in horizontal (41%) and mesioangular (30%) inclination, with a total of 75.9% of teeth in a position different from the physiologic (vertical) inclination. Considering how the tooth germs normally develop and erupt following an axial path, it could therefore be speculated that tooth inclination may represent a possible predisposing factor to eruption failure (66, 67). However, tooth inclination at the time of diagnosis may reflect the original (abnormal) position of the tooth germ (i.e., representing a cause for eruption failure) or be a result of an attempt of eruption against a physical barrier (e.g., gingiva or bone) forcing the tooth in a different position. Therefore, this hypothesis remains to be elucidated. The development of dentigerous cysts is commonly associated with unerupted teeth in humans and animals (68, 69). Several veterinary case reports (21, 25, 28–30, 32–34, 37, 42, 50, 52– 54, 57, 60, 70) and a few retrospective studies (13, 14, 20) describe dentigerous cysts associated with unerupted teeth in dogs. Cysts may develop by accumulation of fluid between the REE and enamel, or between the layers of enamel epithelium itself, but the exact pathogenetic mechanism remains unclear (71). Inflammation affecting a deciduous tooth has also been involved in the development of dentigerous cysts around the corresponding unerupted permanent teeth and in causing a volume increase of the cyst by exudate formation in humans (71– 73). None of the cases reported here had history of trauma or other lesions affecting the deciduous dentition, but they can not be completely excluded. In humans, the prevalence of DTC associated to unerupted third molar teeth (the most commonly unerupted teeth) is low, ranging from 1.4 to 2.31% (74, 75). Babbit et al. reported a 29.1% prevalence (62 teeth) for dentigerous cysts development in a selected canine population of 136 patients with unerupted teeth (14). A radiographic study performed in 233 small breed dogs to determine the prevalence of dental abnormalities described 25 unerupted teeth (0.3% of the total number of existing teeth) with a 32% prevalence of cystic development (8 teeth, the 0.1% of the total number of existing teeth) (20). In this publication all dentigerous cysts were diagnosed associated with Frontiers in Veterinary Science \| www.frontiersin.org 11 November 2019 \| Volume 6 \| Article 357 Bellei et al. Unerupted Teeth in Dogs and Cats (and permanent) teeth are also a relatively common feature in cats showing atraumatic patellar fracture, possibly caused by a still undefined primary bone disorder [the so called “Knees and Teeth Syndrome” (KaTS) or “Patellar Fracture and Dental Anomaly Syndrome” (PADS)], but these teeth do not seem to develop dentigerous cysts (89–91). Finally, three cases of feline inductive odontogenic tumors associated with unerupted canine teeth have been published (92–94). In the present study only an unerupted maxillary second premolar tooth developed a cystic lesion, which was histologically associated with an ameloblastoma and which exact origin could not be determined. The other four unerupted teeth in the remaining three cats did not show evidence of cystic development. However, given the extremely small feline study population, no conclusion can be drawn on incidence and possible predisposing factors to cystic development in this species. In humans, a higher incidence of tooth impaction is reported in women (3, 4, 63). This could be explained by the smaller size of the jaws as compared to men (63). On the other hand, a male predisposition is described in developing dentigerous cysts associated with unerupted third molar teeth (68, 69). Sex and/or neuter status have not been reported to be associated with either lack of eruption or cystic development in veterinary patients, and this observation was confirmed by the present study. The relatively high number of intact male dogs reported is likely due to the uncommon habit of neutering male dogs in Italy. The relatively older age of the animals at the time of diagnosis in this study may be explained by the fact that dogs and cats are rarely brought to veterinary attention because of a missing tooth (13). In fact, the main reason for presentation in this study was related to the unerupted teeth (i.e., clinical absence of a tooth, presence of an oral tumor or a visible cyst in an edentulous area) in only 22% of the cases. In most instances the diagnosis was incidental, following routine intraoral radiographic examination. Furthermore, it must be considered that even if a full dentition is clinically present, unerupted supernumerary teeth may be present. Therefore, this study highlights the usefulness of a complete radiographic examination in all dental patients, to obtain an accurate and early diagnosis. The use of more advanced diagnostic imaging modalities (CT or CBCT) may also be indicated to assess the presence of cystic lesions that may be too small to diagnose radiographically. One of the main aims of this study was to try to elucidate if unerupted teeth should always be extracted or if conservative treatment is an acceptable approach in those cases that at the time of diagnosis do not show clinical or radiographic signs of pathology. In humans, indications for extraction are the presence of pathology of the dental follicle (cysts and tumors), recurrent pericoronitis, cellulitis, abscessation, resorption of adjacent roots or pain (95). As expected, in the present study the few cases initially diagnosed with dentigerous cysts but left untreated showed enlargement of the lesion at the follow-up visit performed up to 6 months after diagnosis. On the other hand, in two adult cats teeth that at the time of diagnosis were radiographically negative for cystic formation did not show any changes at the 3 months (1 tooth) and 3 years (1 tooth) follow-ups. In dogs, 9 teeth without radiographic evidence of The risk of developing tumors within the wall of a dentigerous cyst in humans is rare (6, 75). Ameloblastomas, ameloblastic fibromas, fibro-odontomas and fibrosarcomas, adenomatoid odontogenic tumors, keratocystic odontogenic tumors, calcifying epithelial odontogenic tumors, mucoepidermoid carcinomas, odontogenic carcinomas, and squamous cell carcinomas have been described associated with dentigerous cyst, with a 0.14–2% incidence (3, 6, 74, 80–82). In these cases, chronic inflammation, keratin metaplasia or the continuous intracystic pressure are suggested as possible causes of tumor development (83–85). Neoplastic lesions associated with unerupted teeth have previously been described in 16 dogs: 13 odontomas (7 compound odontomas, 1 ameloblastic odontoma, 1 ameloblastic fibro-odontoma, and 4 non-specified odontomas), 1 squamous papilloma, 1 peripheral odontogenic fibroma, and 1 squamous cell carcinoma (14, 26, 31, 32, 38, 39, 44, 47, 52, 56, 59, 86). Only in six out of 16 cases (37.5%) a cystic component was also described (32, 39, 59, 86). The dogs with reported age were all very young (<12 months), except for a 4 years old dog with a cystic peripheral odontogenic fibroma. When reported, the treatment involved surgical excision of the tumor, including the unerupted tooth. In our study, two ameloblastomas and one osteosarcoma were diagnosed closely associated with unerupted teeth. In all three cases cystic lesions were also present. In the 5 month-old dog, lack of eruption could have been secondary to the presence of the neoplastic tissue, with secondary cyst formation. On the other hand, canine ameloblastoma is an epithelial tumor of the adult age (87). Therefore, the development of this lesion in such a young patient may support the hypothesis of a direct origin from the DTC. In the other two cases the tumors developed at an older age, several years after tooth eruption should have taken place, and the presence of the neoplastic tissue was unlikely the cause of tooth impaction. Moreover, the possibility for the osteosarcoma, a mesenchymal tumor, to originate from the epithelial elements of a DTC is very low and the two lesions were likely independent entities. Histologically, it is difficult or often impossible to determine if a neoplastic lesion is concomitant (but independent from) or if it is directly associated with the adjacent cyst. It is reported that if there is a direct connection between the wall of the cyst and an epithelial neoplasia, the latter arises from the cyst (85). However, to reveal a potential connection between the cyst and the tumor it would be necessary to histologically examine the entire cystic wall, which is rarely possible. Unerupted teeth have rarely been reported in cats. A prevalence of 2.3% teeth was described in a study on value of full mouth radiography in 115 cats (88). Unerupted teeth (4 maxillary canine teeth and 1 maxillary third incisor tooth) were also reported in 5 of 50 brachycephalic cats in a prospective study on oro-dental anomalies in Persian and Exotic cats (12). Dentigerous cysts were not described in these studies. Two clinical cases described the development of dentigerous cysts associated with unerupted canine teeth (9, 10). One further case report described multiple unerupted teeth in a cat with abnormal eruption of the entire dentition, supposedly due to regional odontodysplasia (11). Retained and persistent deciduous Frontiers in Veterinary Science \| www.frontiersin.org 12 November 2019 \| Volume 6 \| Article 357 Bellei et al. Unerupted Teeth in Dogs and Cats dentigerous cyst were left untreated. However, they were only followed-up clinically rather than radiographically. Therefore, even if there was not clinical evidence of complications, these can not be completely excluded. A large debate exists regarding the prophylactic extraction of asymptomatic unerupted third molars in humans, because of the high risk of iatrogenic injury to the inferior alveolar and lingual nerves, as well as the morbidity associated with the surgical procedure (1, 95). Coronectomy with intentional root retention is suggested in some of these cases (8). The guidelines by the American Association of Oral and Maxillofacial Surgeons (AAOMS) recommend the extraction of unerupted teeth showing evidence of or at risk of developing pathology, and strict clinical and radiographic control of untreated teeth (95). However, 50% of radiographically normal impacted teeth show histological evidence of cystic lining (64). In the present study, only 40.4% of unerupted teeth were submitted for histological evaluation, because of owners’ preference or surgeon’s recommendation (based on lack of radiographic changes). The prevalence of cystic development could therefore be higher than reported. In fact, histology revealed the presence of dentigerous cysts in two radiographically negative and two radiographically equivocal cases, based on the definition proposed by the available literature (14). Therefore, relying only on the radiographic examination to diagnose the presence of dentigerous cysts for the diagnosis may be misleading. Thus, extraction and histological evaluation of all unerupted teeth is recommended by the authors. The limited diagnostic yield of radiology and the risk of not treating asymptomatic teeth should also be discussed with the owners, and radiographic (or tomographic) follow-ups should be recommended in these cases. The mean age of the dogs without clinical and radiographic evidence of cystic development was 4.8 years, which was statistically not significantly different from the animals with diagnosed dentigerous cysts (data not shown). Also, the age of the oldest dogs with and without cysts was very similar (9.2 and 10 years, respectively). Therefore, no conclusion can be drawn about the likelihood of cystic development based on age. However, it may be reasonable to consider radiographic surveillance of unerupted teeth diagnosed in older dogs if complications are not visible and extraction is considered complicated. Clear indications on appropriate follow-ups intervals are not available in the literature, but the authors in these cases recommend yearly re-checks. More frequent radiographic re-evaluations may be indicated in situations considered at higher risk (e.g., unerupted first premolar teeth in boxer dogs). Few reports describe operculectomy as a treatment modality for unerupted teeth (21, 33). In the present study, the four cases treated this way and for which a follow-up re-examination was available showed a successful outcome. However, because of the small number of patients treated with this procedure and their short follow-up, definitive conclusions of the validity of this treatment can not be determined. Therefore, the risk of applying such non-standard approach should be discussed with the owners. DATA AVAILABILITY STATEMENT All datasets generated for this study are included in the article/Supplementary Material. ETHICS STATEMENT This study reviewed data and treatments offered to clientowned dogs and cats accordingly to the current guidelines for treatment of cases affected by unerupted teeth. Each procedure was performed after obtaining an informed consent from the owners as indicated in the Legislative decree of 4 March 2014 No. 16, art. 2 and subsequent law No. 17710 from 26 July 2017 on non-experimental Veterinary Clinical Practices. AUTHOR CONTRIBUTIONS EB: collection and interpretation of data, and draft of the manuscript. MG: study design and critical revision of the manuscript. SF: evaluation and interpretation of histological samples and critical revision of histopathological part of the study. EZ: statistical analysis of the data. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fvets. 2019.00357/full#supplementary-material REFERENCES 4. Chu FCS, Li TKL, Lui VKB, Newsome PRH, Chow RLK, Cheung LK. 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Diagnostic imaging in veterinary dental practice. J Am Vet Med Assoc. (2017) 251:1139–42. doi: 10.2460/javma.251.10.1139 Rafetto LK. Managing impacted third molars. Oral Maxillofac Surg Clin North Am. (2015) 27:363–71. doi: 10.1016/j.coms.2015.04.004 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. Copyright © 2019 Bellei, Ferro, Zini and Gracis. 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. 15 November 2019 \| Volume 6 \| Article 357
https://openalex.org/W3022221457	https://europepmc.org/articles/pmc7249050?pdf=render	Latin	null	Realizing Efficient Security and Privacy in IoT Networks	Sensors	2,020	cc-by	12,435	ived: 22 March 2020; Accepted: 30 April 2020; Published: 3 May 2020 Received: 22 March 2020; Accepted: 30 April 2020; Published: 3 May 2020 Received: 22 March 2020; Accepted: 30 April 2020; Published: 3 May 2020 Abstract: In recent times, security and privacy at the physical (PHY) layer has been a major issue of several communication technologies which comprise the internet of things (IoT) and mostly, the emerging ﬁfth-generation (5G) cellular network. The most real-world PHY security challenge stems from the fact that the passive eavesdropper’s information is unavailable to the genuine source and destination (transmitter/receiver) nodes in the network. Without this information, it is difﬁcult to optimize the broadcasting parameters. Therefore, in this research, we propose an efﬁcient sequential convex estimation optimization (SCEO) algorithm to mitigate this challenge and improve the security of physical layer (PHY) in a three-node wireless communication network. The results of our experiments indicate that by using the SCEO algorithm, an optimal performance and enhanced convergence is achieved in the transmission. However, considering possible security challenges envisaged when a multiple eavesdropper is active in a network, we expanded our research to develop a swift privacy rate optimization algorithm for a multiple-input, multiple-output, multiple-eavesdropper (MIMOME) scenario as it is applicable to security in IoT and 5G technologies. The result of the investigation show that the algorithm executes signiﬁcantly with minimal complexity when compared with nonoptimal parameters. We further employed the use of rate constraint together with self-interference of the full-duplex transmission at the receiving node, which makes the performance of our technique outstanding when compared with previous studies. Keywords: privacy capacity; IoT; 5G; physical layer security; MIMOME; jamming Article Joseph Henry Anajemba 1 , Yue Tang 1 , Celestine Iwendi 2 , Akpesiri Ohwoekevwo 3, Gautam Srivastava 4,5,,† and Ohyun Jo 6, Joseph Henry Anajemba 1 , Yue Tang 1 , Celestine Iwendi 2 , Akpesiri Ohwoekevwo Gautam Srivastava 4,5,,† and Ohyun Jo 6, 1 Department of Communication Engineering, College of Internet of Things, Hohai University, Nanjing 210098, China; herinopallazo@ieee.org (J.H.A.); 20141933@hhu.edu.cn (Y.T.) 1 Department of Communication Engineering, College of Internet of Things, Hohai University, 1 Department of Communication Engineering, College of Internet of Things, Hohai University, Nanjing 210098, China; herinopallazo@ieee.org (J.H.A.); 20141933@hhu.edu.cn (Y.T.) 2 Department of Electronics BCC of Central South University of Forestry and Technology, Changsha 410004 China; celestine iwendi@ieee org 2 Department of Electronics BCC of Central South University of Forestry and Technology, Changsha 410004, China; celestine.iwendi@ieee.org 3 Computer Science and Technology, Xidian University, Shaanxi 710126, China; gamaliel@stu.xid 4 Research Centre of Interneural Computing, China Medical University, Taichung 404472, Taiwan 3 Computer Science and Technology, Xidian University, Shaanxi 710126, China; gamaliel@stu.xidian.edu.cn 4 Research Centre of Interneural Computing, China Medical University, Taichung 404472, Taiwan 5 C ll f I f ti d El t i l E i i A i U i it T i h 41354 T i p g y g 5 College of Information and Electrical Engineering, Asia University, Taichung 41354, Taiwan 6 Department of Computer Science, College of Electrical and Computer Engineering, Chungbuk National University, Cheongju-si 28644, Korea * Correspondence: srivastavag@brandonu.ca (G.S.); ohyunjo@chungbuk.ac.kr (O.J.) † Department of Math and Computer Science, Brandon University, Brandon, R7A 6A9, Canada. 1. Introduction With the recent swift advancement of wireless communication networks and the advent of the ﬁfth generation (5G) cellular network, interconnected devices are embedded into the environment through the IoT paradigm to enhance constant quality of service (QoS) and connectivity [1]. However, security of wireless transmissions has become a vital concern [2]. Unfortunately, in wireless technology, security risks are unavoidably inherent. Recently, network intrusion and eavesdropping, known as Eves, has become Sensors 2020, 20, 2609; doi:10.3390/s20092609 www.mdpi.com/journal/sensors www.mdpi.com/journal/sensors Sensors 2020, 20, 2609 2 of 24 the major cradle of security risks in 5G wireless communications. The resources (e.g., battery) of most IoT devices (such as handheld/mobile communicating devices) are constrained, thus resulting in the devices having limited power for transmission, which may warrant the use of frailer cryptographic techniques as power savers. Therefore, devices may be prone to several attacks by some prevailing adversaries. Security in wireless networks is typically employed through cryptographic methods using the upper layers of the open system interconnection (OSI) model [2,3]. However, these adversaries, which are considered as unauthorized users or network Eves, may inﬁltrate the network system, exhaust the networks bandwidth, taint the transmission data, reduce transmission performance, and inject data harms that thwart easy access of network data by authentic users. As a result of wireless link’s unprotected nature, most wireless connectivity is susceptible which makes it easy for them to be attacked by jamming technology. These jamming attacks can result in a problem of Denial-of-Service (DoS), which can cause numerous other higher-layer security glitches in IoT and 5G technologies [3]. The 5G wireless network is predicted to activate the smart hyperlinked environment, enhancing the evolution and the growth of several sectors, such as energy and railway, where huge amounts of accessibility and reliability is essential. In contrast, this evolution may warrant that future mobile transmitting devices be exposed to cyber-attacks, which can destabilize system accessibility [4]. Because of the nature of wireless communications, vulnerability to Eve’s attacks is inevitable. Therefore, transmission conﬁdentiality cannot be guaranteed. In this paper, our work is focused on utilizing the networks physical layer to address countermeasures towards tackling conﬁdentiality attacks in wireless network. 5G communications will headline what is being called the 4th Industrial Revolution, where the mobile wireless broadband, pervasive sensing, and artiﬁcial intelligence (AI) promises to lead to major changes in academia, industry, and society itself. 1. Introduction This coming generation of wireless communications globally focuses on many aspects as standards, policy, and infrastructure are still being shaped. 5G deﬁnitely promises to make the IoT network a reality. In wireless networks, conﬁdentiality attacks mostly stem from jamming. This is considered as the interruption of the ﬂow in wireless transmissions by diminishing the signal-to-noise ratio (SNR) at the receiver end over a wireless signal’s transmission interference. This clearly varies from normal network interferences because it explains a deliberate injection of wireless signals into an existing transmission with the intention of interrupting communications. However, network interference is said to be an accidental kind of disruption during transmission [5]. Recently, through conventional means, the implementation of different encryption approaches has been used to handle this security challenge at the advanced communications layers. That being said, a lot of attention has been drawn towards the security of the physical (PHY) layer lately. From a PHY security perspective, the authors of [6] investigated the impact of saturation nonlinear energy harvesting (EH) and activation threshold on the multiuser wireless powered sensor networks (WPSNs) from the physical layer security (PLS) perspective, and with respect to the generalized multiuser scheduling (GMS), they examined the improvement in the secrecy performance in WPNs. Their work explored and presented an exact closed-form expressions for secrecy outage probability (SOP) under linear EH (LEH), saturation nonlinear EH (SNEH), and saturation nonlinear EH with activation threshold (SNAT), respectively, through ﬁnding a solution for the maximization problem of secure energy efﬁciency (SEE). The basis for tackling jamming attacks is hinged on Wyner’s architecture. This architecture presented and demonstrated a wiretap channel when the channel of the eavesdropper (Eve) is a tainted version of the authentic receiver’s channel, a secret message can be sent from the transmitter to the destination, while Eve is kept unaware of the content of the transmitted message [7]. The concept of privacy capacity is described as the optimal attainable rate of transmission of private data from the transmitting source (node) to its receiving destination. In [8], the Wyner’s wiretap method was generalized by assuming the private message transmission over channels broadcast. Lately, substantial studies have analyzed privacy 3 of 24 Sensors 2020, 20, 2609 in wiretap channels under multiple antennas networks [9–11]. Particularly, the authors of [11,12] described the performance capacity of privacy in a multiple-input, multiple-output (MIMO) wiretap transmission channel. 1. Introduction However, the authors of [13] investigated a joint effect of multiple jamming signals and noise at the eavesdropper in a MIMO network by assuming the implementation of antenna selection technique by the transmitter, while both the eavesdropper and legitimate receiver make use of a maximal-ratio combining scheme to achieve spatial diversity in reception. Their results show the effects of imperfect feedback and other key system parameters on the secrecy performance. Basically, security at the PHY layer guarantees an optimal level of transmission privacy against Eve’s as well as establishing an anticipated reception standard and quality as expected by the receiver(s). This research area comprises both signal processing analysis and theoretical information study. Although the latter involves more bounds and asymptotical limits, the former inclines towards innovating primal designs of algorithms and architectures to tackle security issues in a wireless network. Therefore, the focus and motivations of this paper is wrapped around the former with respect to Eve’s growing quantity of transmitting antennas on privacy. All the above-mentioned studies, except for [10,11], failed to explore a MIMOME scenario wish illustrates the effects of Eve’s growing quantity of transmitting antennas on privacy. Although the authors of [6,11,13] proposed proﬁcient privacy, their technique is reliant only on the assumption of a scenario where several factors such as the quantity of Eve’s antennas that are accessible for transmitter usage for artiﬁcial noise subspace, are constrained in a MIMO and MIMOME network. By implication, it is essential that Eve’s multiple antenna usage needs be considered and addressed. In this research, this is believed to be an issue of importance, therefore these problems are ﬁrst extensively described, then techniques to tackle such problems are investigated and proposed.The main contributions of this research are as follows. • A primal outlook into the investigation and optimization of wireless communications security with respect to IoT networks is presented. • A primal outlook into the investigation and optimization of wireless communications security with respect to IoT networks is presented. 1. Introduction • Using a mathematical model, an analysis of a novel transmission system where the eavesdroppers attack is tackled by injecting artiﬁcial noise which is transmitted by the receiver who has full-duplex aptitude with the same frequency in the channel which reduces Eve’s reception quality aptitude with the same frequency in the channel, which reduces Eve’s reception qual • Proposal of a new jamming mitigation technique and developed a sequential convex estimation and optimization (SCEO) algorithm for an optimized and enhanced privacy to solve the optimization problem in a three-node network where network users do not have knowledge of Eve’s channel state information (CSI). • The vulnerability of the proposed privacy enhancing scheme to Eve’s increasing number of antennas is characterized and explored, while the performance of the proposed algorithm in a three-node network is established. • Finally, because privacy capacity in a three-node network have been over-studied by several authors, this research is expanded to cover a MIMOME scenario, justifying its applicability in secured IoT network transmission. The remainder of this paper is organized as follows. The privacy capacity model in wireless networks is described in Section 2. Review of recent works related to the research is presented in Section 3. System model is described in Section 4. Numerical analysis and results are provided in Section 5, and ﬁnally conclusions are outlined in Section 6. 2. Privacy Capacity Model in Wireless Networks In this section, the privacy capacity model of the transmitting network is expressed. In the privacy capacity model of the network, it is assumed that the malicious node may eavesdrop the source as well as well as the receiver. However, in order to get a full use of the signals transmitted from the transmitter, 4 of 24 Sensors 2020, 20, 2609 the eavesdropper must be fully synchronized in the network. According to traditional privacy deﬁnitions in [8], the channel of communication can be modeled as a channel of broadcast in line with the wiretap channel as illustrated in Figure 1. Tx Rx Main Channel Encoder Decoder Wiretap Channel m C n A m B m E Eve Figure 1. A Wiretap Channel for Privacy Capacity model Figure 1. A Wiretap Channel for Privacy Capacity model Considering the wiretap channel, the transmitting channel’s message is represented as an ∈An and is encoded and broadcasted as a codeword cm ∈Cm. The receiver (Rx) and eavesdropper (Eve) receive bm ∈Bm and em ∈Em, respectively. Eve’s received information via her receiving signal is then modeled and described as in Equation (1), I (em; an) = g (an) −g (an \|em ) (1) (1) where I(em; an) represents the mutual information shared by the transmitter and the legitimate receivers, while g is considered as the entropy. As long as Eve cannot decode any bit of the transmitted information, then perfect privacy is achieved. Thus, Equation (2), where I(em; an) represents the mutual information shared by the transmitter and the legitimate receivers, while g is considered as the entropy. As long as Eve cannot decode any bit of the transmitted information, then perfect privacy is achieved. Thus, Equation (2), I (em; an) = 0 ↔g (an) = g (an \|em ) (2) (2) This implies that the quantity of uncertainty about Eve’s private information is not altered after em is received. This implies that the quantity of uncertainty about Eve’s private information is not altered after em is received. By deﬁnition, the probability of experiencing an error Pi in the message estimation of message an, and ˆan is deﬁned as the estimate of an; therefore, Pi = P {an ̸= ˆan} . Notations We denoted the column vectors and matrices by boldface letters (both at upper and lower cases), while the determinant, inverse of a matrix and column-wise vectorization of the matrix X are all represented as XH, X−1, \|X\|, respectively. In a diagonal vectorization, Y is denoted as the column vector of y , whereas the random variable of y is represented as Cy[.] and the probability of event occurrence is denoted as P{X}. Using the same probability space, we deﬁned the random variables Y and Ym, if Ym converges to Y, we transposed Ym a.s →Y as m →∞. We used I to represent the matrix identity of supplied size, as I(y; z) represents the information which is mutually and randomly transmitted between y and x variables. 2. Privacy Capacity Model in Wireless Networks (3) (3) Eve’s rate of uncertainty about message an is term as the rate of equivocation and can be described as Eve’s rate of uncertainty about message an is term as the rate of equivocation and can be described as ate of uncertainty about message an is term as the rate of equivocation and can be described Qi = 1 m g (an \|em ) (4) (4) where 0 ≤Qi ≤g(an)/m (5) (5) 5 of 24 Sensors 2020, 20, 2609 Evidently, if Qi = g(an)/m, then perfect privacy, which is related to perfect privacy rate Qs, is realized. For each ε > 0, a particular Qs is assumed to be realizable, and there is a sequence of (2mQs, m) codes such that for any m > m(ε) the following states are obtained.      Pi < ε Qs −ε < Qi (6) (6) The ﬁrst state is the constraint for the realizable rate, whereas the second is the equivocation rate constraint which guarantees prefect privacy. In summary, privacy capacity Sc is the optimal realizable rate of privacy in a network transmission. Thus, in [8], it is established that the difference between the main channel capacity Cmc and the wiretap channel capacity Cwc is the privacy capacity Sc, in other words, Sc = (Cmc −Cwc)+, (7) (7) as (.)+ ∆= max(0, .), where negative rate is meaningless. Although Wyner demonstrated this for the distinct no-memory channel, the principle that the capacity of privacy is the difference of the capacity of the legitimate channel and that of the eavesdropper’s is established to be accurate for several systems like in multiple-input, multiple-output (MIMO) set-ups [12,13]. as (.)+ ∆= max(0, .), where negative rate is meaningless. Although Wyner demonstrated this for the distinct no-memory channel, the principle that the capacity of privacy is the difference of the capacity of the legitimate channel and that of the eavesdropper’s is established to be accurate for several systems like in multiple-input, multiple-output (MIMO) set-ups [12,13]. 3. Related Works In a wireless network, jammers intentionally introduce radio frequency (RF) interference to distort wireless communications. This is achieved by occupying the transmitting channel and keeping it busy, thereby triggering the transmitter to withdraw each time it senses a busy wireless channel, or a tainted signal acknowledged at the receiver’s end. In this section, in an attempt to understand the jamming attack on wireless networks, different kinds of jamming in wireless communications as proposed by several researchers are explored. Primarily, network jamming can occur in different ways based on the type of jammer. Therefore, different kinds of jammers and their jamming mechanisms are reviewed in this section. The established secrecy capacity rate principle by Wyner in [8] preceded several other theoretical proposals by different researchers. So far, the wireless channels rate of secrecy has been explored from several perspectives like in [9,10]. The different perspectives explored are as follows, the fading channels secrecy [9–11], the analysis of Gaussian wiretap channels secrecy of [11], the multiple antenna systems secrecy [13–15], the broadcast channels secrecy [16], the analysis of secure degrees of freedom [17–21], and the secrecy of cooperative jamming techniques coupled with helpers or relays [22–29]. Additionally, the theoretical information [30] forms guarantees deﬁned by secrecy capacity of the communication channel; the nodes that legitimate can also assume active approaches to improve their communication secrecy. One important approach for improving the secrecy of wireless communication was developed in [31]. The approach proposes that the transmitter (Alice) injects artiﬁcial noise into legitimate 6 of 24 6 of 24 Sensors 2020, 20, 2609 channels null-space together with the data signal. This scheme is intended to actively reduce the reception quality of any Eve’s presence without altering the quality of legitimate receivers (Bobs) channel. Several other works [32,33] have also investigated this approach. Equipping Bob with the capacities of full-duplex radio aptitude, which enables him broadcast jamming noise counter to Eve, while both Bob and Eve attempt to acquire information from Alice [34,35] is another approach. The combination of the aforementioned two jamming approaches is investigated in most part of this study to attain secrecy at higher levels. Moreover, this combination was examined in [36], as it proposed a cooperative optimization algorithm to generate decent parameters for the transmission. Separately, two different sets of antennas are considered for broadcasting and for full-duplex reception. Meanwhile, the research did not fully consider the residual self-interference at Bob. 3. Related Works Further, several other works were interested with comparable systems in [37,38]. The system proposed in [39] presents a scenario with Alice and Bob communicating simultaneously and are furnished with full-duplex radio with full consideration of residual self-interference. The study target was to discover the maximum beamforming transmit trajectories for artiﬁcial noise and signal considering some constraints of quality of service (QoS) with Eve’s CSI recognizable to users. With Eve’s exact CSI unrecognizable to the user in [40], a closed-form lower bound on the ergodic rate of secrecy is realized. The authors of [41,42] considered a case of a single-antenna Bob and multiple antenna base station coupled with a colluding and non-colluding single-antenna Eves. As an alternative to beamforming, an antenna selection scheme is used by the study and it is of the research assumption that Eves are dispersed in accordance to the process of Poisson point. For cooperative relaying networks, several authors have also investigated different techniques for the security of systems physical layers [43]. In cooperative relaying networks, even destinations or relays are used as assistants to offer jamming signals to complicate the eavesdropper’s transmission. This method is known as cooperative jamming. The noise-forwarding approach which was introduced in [24], and applied in a channel comprising of four-terminal relay-eavesdropper also considered a full-duplex relay, which independently transmits secret messages codewords which are targeted at complicating the eavesdropper’s transmission. The authors of [44] investigated a two-stage cooperative jamming scheme (TSCS) which involves multiple relay nodes acting as the single-antenna’s source node extension. The relays in this study do not transmit the information signals as they only function as a helper. However, for a single-antenna relay network, the authors of [45] proposed three different cooperative communication methods. On the other hand, for the second hop, they attempted to optimize secrecy by deriving a power allocation approach and its corresponding relay weights. The study in [46] investigated a decode-and-forward (DF) relays performance based on an optimal beamforming strategy; however, the investigation is limited because it only considered a transmission where the Eve monitors just a single connection linking the transmission destination and relay. The investigation of [47] was based on different privacy enhancing technologies (PETs) for IoT devices which have resulted in a much efﬁciency and convenience to our daily life. 3. Related Works Their survey claim to have identiﬁed current state of improvement of the PETs in several turfs and also analyzed how the current technologies adhere with the modern legal ideologies and privacy criteria in curtailing the threats to privacy. A secured IoT-based healthcare system, which operates through the body sensor networks (BSN) architecture, was examined by the authors of [48]. The main focus of their system is to concurrently realize system robustness of transmission and efﬁciency within publicly transmitting IoT-based communication networks. Utilizing a vigorous utilize crypto-primitives, they constructed two communication schemes to ensure conﬁdentiality in transmission and support entity authentication among smart objects. As most of the IoT data is relevant to personal privacy, it is necessary to pay attention to data transmission security. The authors of [49] investigated an IoT-oriented ofﬂoading method (IOM) which is enclave with privacy preservation to solve the problem of privacy in Cloudlet-enabled Wireless Metropolitan Area Networks (CWMAN). Their research and that of [50] adopted the non-dominated sorting differential evolution algorithm (NSDE) in order to optimize the multi-objective problem. Sensors 2020, 20, 2609 7 of 24 By contrast to the works analyzed above, the work in [51] proposed a cooperative jamming approach for a half-duplex two-hop wireless MIMO relay scheme where the eavesdropper can bug the channels throughout the phases of transmission. The study investigation considered both single and multiple streams of data transmissions. However, for jamming support, due to the absence of an “outer” helpers, the relay, destination, and source must depend on themselves. Whether the eavesdropper is in proximity to the source or the destination, the strategy guarantees that it is jammed. In this strategy, both the source and the destination nodes perform as provisional assistants for transmitting jamming signals throughout the phases of transmission where they are generally inactive. p y g y In summary, the event of optimizing privacy without the users recognizing Eve’s CSI is sparsely contained in literature; however, a few works like [52–57] gave more attention to the use of only enough energy to authenticate a particular QoS for Bob and is this energy is estimated in terms of signal-to-interference-plus-noise-ratio (SINR). Using the remaining energy, artiﬁcial interference is generated to jam Eve not minding the effect of Eve’s location. This process is implemented in place of making attempts to optimize the rate of secrecy, which is impossible without the users having a knowledge of Eve’s CSI [58,59]. 3. Related Works Furthermore, as new telecommunication technologies emerge, the use of several security and privacy techniques proposed earlier becomes over exploited and obsolete; therefore, it is vital to develop advanced state-of-the-art techniques and algorithms that can mitigate against network jamming and eavesdropping attacks and ensure constant quality of service (QoS) in the network. Considering that this is the optimal focus of this research, therefore, the relevance of this research to cutting-edge telecommunication technologies like the IoT and 5G cellular network cannot be underestimated. 4. System Model In this section, the system model is described. There are similarities in the model to what was given in [8]. For the set-up, a three-node-based wireless transmission is illustrated in Figure 2. Tx Eve Rx gse gde gdd gsd Figure 2. A single-antenna three-node wireless transmission with a full-duplex destination. Figure 2. A single-antenna three-node wireless transmission with a full-duplex destination. In the set-up, all three nodes are attached to a single transmitting antenna. The intention of the transmitter (Source) is to broadcast some private information to the receiver (Destination) while Eve (a passive eavesdropper) attempts to gain access to the sensitive private information. We assume that each link channel entails several M orthogonal subcarriers and each subcarrier fading is ﬂat. Tackling eavesdropping attacks in this set-up entails that artiﬁcial noise is broadcasted by Rx with the same frequency and in the same channel, which reduces Eve’s reception quality. Considering that this jamming attack operates at the same frequency and time in which information is transmitted from Tx to Rx; thus, it is Sensors 2020, 20, 2609 8 of 24 assumed that Rx has full-duplex aptitude. It is well known that there is no perfect full-duplex system, therefore Rx always manifests some level of residual self-interference. y Considering ys(x) ∈Mx1 is the signal vector independent and identically distributed (i.i.d) zero mean cyphers and unit variance that the Tx will be transmitting while yd(x) ∈Mx1 is assumed to be the jamming noise vector of i.i.d. zero mean cyphers and unit variance the transmitter (Tx) will be transmitting. Thus, the signal vectors Rx and Eve received can be individually described, Considering ys(x) ∈Mx1 is the signal vector independent and identically distributed (i.i.d) zero mean cyphers and unit variance that the Tx will be transmitting while yd(x) ∈Mx1 is assumed to be the jamming noise vector of i.i.d. zero mean cyphers and unit variance the transmitter (Tx) will be transmitting. Thus, the signal vectors Rx and Eve received can be individually described, zd(x) = gsd ∗√ps ∗ys(x) + √ δgdd ∗√pd ∗yd(x) + md(x), (8) ze(x) = gse ∗√ps ∗ys(x) + gde ∗√pd ∗yd(x) + me(x), (9) (8) (9) where Mx1 comprises the vector, gsd, gse, gde, and gdd are explicitly described in Figure 2, and ps and pd vectors, respectively, represent the power of transmission between the Tx and Rx. 4. System Model md(x) ∈Mx1 and me(x) ∈Mx1 are considered as the mean of independent white Gaussian noise of zero and unit variance, respectively. The multiplication and square root are used as element-wise operators, and δ represents the attenuation factor of self-interference. Denoting the superscript (m) as a vector mth element, we formulate the respective signal-to-noise-ratios (SNRs) of the mth subcarrier at Rx and Eve as γ(m) d = Amym 1+Bmzm andγ(m) e = Cmym 1+Dmzm , (10) γ(m) d = Amym 1+Bmzm andγ(m) e = Cmym 1+Dmzm , (10) (10) where Am = g(n) sd 2 , Bm = δ g(n) dd 2 , Cm = g(n) se 2 , Dm = g(n) de 2 , ym = p(m) s and zm = p(m) d . Recall that as stated earlier, Eve’s CSI (Cm and Dm∀m) is only supposed to be recognizable by legitimate users in this Section. Thus, the privacy capacity of this model is deﬁned as Sc(y, z) = 1 M M ∑ m=1 max {0, ∆Sm(ym, zm)}, (11) (11) where where ∆Sm(ym, zm) = log 1 + γ(m) d −log 1 + γ(m) e . (12) (12) . Formulated Optimization Problem 4.1. Formulated Optimization Problem 4.1. Formulated Optimization Problem In this subsection, our objective is to boost the privacy capacity of the scheme using power and rate constraints with cooperative power distribution between Tx (source) and Rx (destination). As compared with the conventional physical layer security techniques, the proposed SCEO and swift privacy rate optimization algorithms are suitable for the Internet of Things, because the optimization algorithms are energy efﬁcient; therefore, the low-energy consumption necessities of IoT is enormously satisﬁed. Speciﬁcally, in a wireless channel transmission scheme, intrinsic noise is deployed which degrades the quality of the eavesdropper’s received signal; thus, privacy in transmission is guaranteed with no cost of supplementary power. In summary, the application of the proposed technique in IoT technologies is low power capable as it does not necessitated the use of additional energy to guarantee privacy in transmission. The authors in [38] attempted to solve this low power problem through the bisection approach by swapping the source and destination powers optimizations, iteratively. However, in this 9 of 24 Sensors 2020, 20, 2609 work, we provided an improved solution by jointly assigning source and destination powers, thereby formulating the ﬁrst optimization problem as (13): max x,y 1 M ∑ m∈Ψz log(1 + Amyn 1+Bmzm ) −log(1 + Cmym 1+Dmzm ) s.t. 1 M ∑ m∈Ψz log(1 + Amyn 1+Bmzm ) ≥Csd ∑ m∈Ψz zm ≤pd, zm ≥0∀m ∈Ψz ∑ m ym ≤ps, ym ≥0∀m ∈ℵ zm = 0, ∀m ∈Ψ ⊥ z (13) (13) ∑ m ym ≤ps, ym ≥0∀m ∈ℵ zm = 0, ∀m ∈Ψ ⊥ z where Ψz = Θz ∩Φ Θz = n m Am 1+Bmzm > Cm 1+Dmzm , ∀m ∈ℵ o Φ = n m Am Cm > Bm Dm , Bm Dm < 1, ∀m ∈ℵ o Ψ ⊥ z = {m \|m ∈ℵ, m /∈Ψz} (14) (14) The privacy capacity of our system is considered as the objective function, and the approximation is performed over the privacy capacity of the group of subcarriers which warrants a positive capacity of ψz. First, the rate constraint which guarantees the quality of service (QoS) of the network is considered. Although the techniques warrant data exchange or channel feedback between the authentic users, which can result in a slight rate performance degradation, typically IoT devices and applications have very low data rates. Considering the KKT conditions as                                  ∀m ∈ψz    ∂l ∂ym = −ϕm(ym, zm) −1 M Am 1+BmzmAmym −µy(m) + υy = 0 ∂l ∂zm = −ϕm(ym, zm) −1 M Bm 1+BmzmAmym − Bm 1+Bmzm −µz(m) + υz = 0 ym ≥0, µy(m)0, ymµy(m) = 0, ∀m ∈ℵ zm ≥0, µz(m)0, zmµz(m) = 0, ∀m ∈ψz υy ≥0, ∑mym ≤ps, υy (∑mym −ps) = 0 υz ≥0, ∑∈ψyym ≤pd, υz ∑∈ψyym −pd = 0 λ ≥0, ˜Csd ≤1 M ∑m∈ψz log(1 + Amym 1+Bmzm ) λ ˜Csd −1 M ∑m∈ψz log(1 + Amym 1+Bmzm ) = 0 (16) (16) where ϕ(ym, zm) = 1 M Am 1+Bmzm+Amym −1 M Cm 1+Dmzm+Cmym (17) ϑm(ym, zm) = 1 M Bm 1+Bmzm+Amym ) − Bm 1+Bmzm − Dm 1+Dmzm+Cmym + Dm 1+Dmzm (18) (17) (18) Equations (16)–(18) above are the derivations of the KKT conditions which the Lagrangian function of the problem must satisfy for optimal solution to be achieved. Equations (16)–(18) above are the derivations of the KKT conditions which the Lagrangian function of the problem must satisfy for optimal solution to be achieved. Apparently, attempting to ﬁnd solution to the source-to-destination KKT conditions, a two-dimensional (2-D) bisection search estimation is performed on parameters λ, ν, and µ as analyzed in Algorithm 1. Algorithm 1 Algorithm to solve problem (15) by solving the KKT conditions of (16), using 2-D search approach for µ, ν, and λ. pp µ Initialize: Am, Bm, Cm, Dm, ym, ∀m ∈ℵ; ps; sd; CSOD; ε, ζ. Generate: Initialize: Am, Bm, Cm, Dm, ym, ∀m ∈ℵ; ps; sd; CSOD; ε, ζ. Generate: Am, Bm, Cm, Dm, ym, ∀m ∈ℵ; ps; sd; CSOD; ε, ζ. Generate: Am, Bm, Cm, Dm, ym, ∀m ∈ℵ; ps; sd; CSOD; ε, ζ. Generate: 1: Initiate λ = 0 therefore, eliminating the rate constraint), perform search for ν and y. 1: Initiate λ = 0 therefore, eliminating the rate constraint), perform search for ν and y. 2: Compute C(y) as sd capacity. 3: if C(y) > CSOD then 4: revert y (meaning the rate constraint is achieved). 4.1. Formulated Optimization Problem Therefore, this setback of low rate performance does not alter the adoption of the scheme in IoT operations. Second, the jamming mitigation power constraint which has its summation over the set of subcarriers ψz is considered. This is due to the need to properly manage power so no power waste is experienced by subcarriers which might not guarantee a positive gain of privacy. Last, the third constraint which is the power constraint at Source is taken into consideration. Furthermore, the minimum expected rate is represented as Csd, whereas ps and pd are set to represent the optimal sum of powers at Source and Destination. With the aim of exploring the Karush–Kuhn–Tucker (KKT) conditions, we formulated the Lagrangian function of the problem as L(y, z, λ, µy, µz, υy, υz) = −1 M ∑ m∈ψz log(1 + Amym 1+Bmzm ) −log(1 + Cmym 1+Dmzm ) −µR y y −µR z z + µy(∑ m ym −ps) + υz( ∑ m∈ψz ym −pd) +λ ˜Csd −1 M ∑ m∈ψz log(1 + Amym 1+Bmzm ) ! (15) (15) 10 of 24 Sensors 2020, 20, 2609 Considering the KKT conditions as Considering the KKT conditions as Considering the KKT conditions as Considering the KKT conditions as else 2-D search: perform search for ν= 0 to satisfy power constraint till precision ε 5: else 2-D search: perform search for ν= 0 to satisfy power constraint till precision ε is achieved. P f h λ > 0 f i t hi th t t i t til i i ζ i hi d 5: else 2-D search: perform search for ν= 0 to satisfy power constraint till precision ε is achieved. Perform search λ > 0 for every given ν to achieve the rate constraint until precision ζ is achieved. Perform search λ > 0 for every given ν to achieve the rate constraint until precision ζ is achieved. Perform search λ > 0 for every given ν to achieve the rate constraint until precision ζ Given each pair of λ and ν, ym ≥0 is achieved as the solution of (24) for every m ∈ℵ. y (meaning the rate constraint is achieved). 7: end if 7: end if Considering Algorithm 1 (bisection algorithm), the following set of equations which require solution will be encountered. These represent two nonlinear systems of equations:    ∂l ∂ym = −ϕm(ym, zm) −λ M Am 1+BmzmAmym −µy(m) + υy = 0 ∂l ∂zm = −ϕm(ym, zm) −λ M Bm 1+BmzmAmym − Bm 1+Bmzm −µz(m) + υz = 0 . (19) (19) Sensors 2020, 20, 2609 11 of 24 Finding solution for the KKT conditions which contains these two nonlinear equations appears to be unnerving; therefore, we consider applying the approach of sequential convex approximation. Finding solution for the KKT conditions which contains these two nonlinear equations appears to be unnerving; therefore, we consider applying the approach of sequential convex approximation. 4.2. Sequential Convex Approximation 4.2. Sequential Convex Approximation In attempt to realize a sequential convex approximation, the rate constraint and the objective function which constitute the optimization problem formulated in (13) is rewritten as (20) below. f (y, z) = 1 M ∑ m∈Θz log(1 + Amym 1+Bmzm ) −log(1 + Cmym 1+Dmzm ) = 1 M ∑ m∈Θz (log(1 + Bmzm + Amym) + log(1 + Dmzm)) −1 M ∑ m∈Θz (log(1 + Dmzm + Cmym) + log(1 + Bmzm)) = f1(y, z) + f2(y, z) 1 M ∑ m (log(1 + Bmzm + Amym)) −1 M ∑ m (log(1 + Bmzm)) ≥Csd f3(y) + f4(y, z) ≥Csd (20) (20) Evidently, f1 and f4 functions are concave while f2 and f3 functions are convex. Considering ﬁrst-order Taylor series expansion of convex functions as the function’s underestimator. By denoting the ﬁrst-order Taylor series expansion of f2 and f3 around (y(h), z(h)) points and expressing them, respectively, as ˜f (h) 2 , ˜f (h) 3 , the following was realized, f1(y, z) + f2(y, z) ≥f1(y, z)+ ˜f (h) 2 (y, z)∀y, z ∈M f4(y, z) + f3(z) ≥f4(y, z)+ ˜f (h) 3 (z)∀y, z ∈M (21) (21) where where ˜f (h) 2 (y, z) = f2(y(h),z(h)) + ∇f2(y(h),z(h))R( " y z # − " y(h) z(h) # ) ˜f (h) 3 (z) = f3(z(h)) + ∇f3(z(h))R(z −z(h)) ∇f2(y, z) = −1 M   C1 1+D1z1+C1y1 ... CM 1+DMzM+CMyM D1 1+D1z1+C1y1 + B1 1+B1z1 ... DM 1+DMzM+CMyM + BM 1+BMzM   ∇f3(z) = −1 M   B1 1+B1z1 ... BM 1+BMzM   (22) ˜f (h) 2 (y, z) = f2(y(h),z(h)) + ∇f2(y(h),z(h))R( " y z # − " y(h) z(h) # ) ˜f (h) 3 (z) = f3(z(h)) + ∇f3(z(h))R(z −z(h)) ∇f2(y, z) = −1 M   C1 1+D1z1+C1y1 ... CM 1+DMzM+CMyM D1 1+D1z1+C1y1 + B1 1+B1z1 ... DM 1+DMzM+CMyM + BM 1+BMzM   ∇f3(z) = −1 M   B1 1+B1z1 ... 4.2. Sequential Convex Approximation BM 1+BMzM   (22) (22) 12 of 24 Sensors 2020, 20, 2609 Based on work done in [34] and assuming Bm Dm < 1 we further simplify Θz and obtain Θz = n m zm ≥0, Am 1+Bmzm > Cm 1+Dmzm , ∀m ∈ℵ o = {m \|(BmCm −AmDm)zm > Am −Cm, ∀m ∈ℵ} = n m zm ≥ Am−Cm BmCm−AmDm , 1 > Am Cm > Bm Dm , ∀m ∈ℵ o ∪ n m zm ≥0, 1 < Am Cm > Bm Dm , ∀m ∈ℵ o (23) (23) Thus, following iteration h, we formulate the optimization problem as follows, max y, z f1(y, z) + ˜f (h) 2 (y, z) s.t.f4(y, z) + ˜f (h) 3 (z) ≥Csd ∑ m zm ≤pd, zm ≥0∀m ∈ℵ zm = 0∀m ∈ n m Am Cm < Bm Dm < 1, ∀m ∈ℵ o ∪ n m Bm Dm > 1, ∀m ∈ℵ o zm ≥ Am−Cm BmCm−AmDm ∀m ∈ n m 1 > Am Cm > Bm Dm , ∀m ∈ℵ o ∑ m ym ≤ps, ym ≥0∀m ∈ℵ (24) (24) It is observed that a convex optimization problem occurs at each point of iteration. This attempts to optimize a lower bound on the primal objective function and guarantees the rate constraint. As the iterative Algorithm reaches convergence, a decent approximation on the optimal values is expected. The optimization process is detailed in Algorithm 2. Algorithm 2 Sequential Convex Estimation Optimization Algorithm for solving optimization problem. Algorithm 2 Sequential Convex Estimation Optimization Algorithm for solving optimizat Algorithm 2 Sequential Convex Estimation Optimization Algorithm for solving optimization problem. Initialize: (Am, Bm, Cm, Dm) : ∀m ∈ℵ, pd, ps, Csd, ζ. Generate: y(0) = 1 Mps1M z(0) = 1 \|Θx\|pda(i) = 1 1: if (i ∈Θx) 2: else a(i) = 0, t = 1 3: While True do 4: end if Perform the convex optimization problem in (18) to realize y(t) , z(t), if ( [y(t); z(t)] −[y(t−1); z(t−1)] < ζ) then break. else t = t + 1 end if 5: end 6: return y(t) , z(t); Algorithm 2 Sequential Convex Estimation Optimization Algorithm for solving optimization problem. (Am, Bm, Cm, Dm) : ∀m ∈ℵ, pd, ps, Csd, ζ. Generate: (Am, Bm, Cm, Dm) : ∀m ∈ℵ, pd, ps, Csd, ζ. 4.2. Sequential Convex Approximation Generate: y(0) = 1 Mps1M z(0) = 1 \|Θx\|pda(i) = 1 y Mps1M z(0) = 1 \|Θx\|pda(i) = 1 Perform the convex optimization problem in (18) to realize y(t) , z(t), if ( [y(t); z(t)] −[y(t−1); z(t−1)] < ζ) then break. else t = t + 1 end if 5: end 6: return y(t) , z(t); Perform the convex optimization problem in (18) to realize y(t) , z(t), Perform the convex optimization problem in (18) to realize y(t) , z(t), 13 of 24 Sensors 2020, 20, 2609 4.3. Optimization of Swift Privacy Rate in a MIMOME 4.3. Optimization of Swift Privacy Rate in a MIMOME 4.3. Optimization of Swift Privacy Rate in a MIMOME Considering a network scenario where multiple eavesdroppers operate, the ﬁrst assumption in realizing a swift privacy rate will be that Eve has full knowledge of her covariance matrix of noise and interference (ME) just as she has full knowledge of all her entire CSIs (as earlier stated); then, she can make use of the optimal receiving antenna and her achievable rate would be derived as RTE = log ME + aA1ZrAG 1 −log \|ME\| (25) (25) where ME = I+ aPm ATx −r A2AG 2 + ePB ARx BBG (26) (26) Then, the swift realizable privacy rate would be expressed as Then, the swift realizable privacy rate would be expressed as PR = (RTR −RTE)+ (27) (27) However, for a multiple-input multiple-output multiple-antenna eavesdropping (MIMOME) system, assuming there is a constraint on the number of Eve’s antenna (AE), one major issue of the system will be on how can to optimize the parameters of the transmission in such a way that Eve’s interference in the transmission is made difﬁcult. This becomes our focus in this subsection. In this MIMOME system, if there is great increase in of (AE), the artiﬁcial noise does not have a signiﬁcant impact on the rate of transmission between Tx and Eve (RTE). Therefore, the assumption in this subsection is that there is a constraint on Eve’s maximum number of antennas; therefore, for the reason of optimization, a worst case is assumed, and thus this maximum number of antennas is considered as (AE). To achieve this optimization, Tx and Rx cannot utilize the swift CSI at Eve, so as earlier mentioned, PR is inappropriate for this optimization. From some previous research [6,13,18], it is observed that Eve’s symptotic rate is a decent approximation to the tangible rate; thus, we adopted this fact in order to achieve optimization. Suppose that the we make no assumption on the realizable random parameter (r) and assuming that zr is a vector encompassing the transverse elements of Zr, and (AE) is discreetly huge. 4.3. Optimization of Swift Privacy Rate in a MIMOME Then, by rewriting (26), we obtained RTE = log I+ aPm ATx−rA2AG 2 + ePB ARx BBG + aA1zrAG 1 −log I+ aPm ATx−rA2AG 2 + ePB ARx BBG = log I+K3 ¯Θ3KG 3 −log I+K4 ¯Θ4KG 4 , (28) as K3 and K4 are expanded as K3 = 1 √AE [A1, A2A3] , and K4 = 1 √AE [A2, B] . where ¯Θ3 = AEtrans h azP r , aPm ATx−r1ATx −r, bPB ARx 1ARx i , (29) ¯Θ4 = AEtrans h aPm ATx−r1ATx −r, bPB ARx 1ARx i , (30) f h f b f h h l h f RTE = log I+ aPm ATx−rA2AG 2 + ePB ARx BBG + aA1zrAG 1 −log I+ aPm ATx−rA2AG 2 + ePB ARx BBG = log I+K3 ¯Θ3KG 3 −log I+K4 ¯Θ4KG 4 , (28) (28) Tx Rx = log I+K3 ¯Θ3KG 3 −log I+K4 ¯Θ4KG 4 , as K3 and K4 are expanded as K3 = 1 √AE [A1, A2A3] , and K4 = 1 √AE [A2, B] . where ¯Θ3 = AEtrans h azP r , aPm ATx−r1ATx −r, bPB ARx 1ARx i , (29) ¯Θ4 = AEtrans h aPm ATx−r1ATx −r, bPB ARx 1ARx i , (30) K3 and K4 are expanded as K3 = 1 √AE [A1, A2A3] , and K4 = 1 √AE [A2, B] . where d as K3 = 1 √AE [A1, A2A3] , and K4 = 1 √AE [A2, B] . where ¯Θ3 = AEtrans h azP r , aPm ATx−r1ATx −r, bPB ARx 1ARx i , (29) (29) ¯Θ4 = AEtrans h aPm ATx−r1ATx −r, bPB ARx 1ARx i , (30) (30) Deriving from the approximation of RTE, an objective function which entails the approximation of PR is proposed. Note that the proposed objective function does not depend on the CSI of Eve; however, it considers the approximate swift rate of Eve. Likewise, as it is assumed that the transmitter and receiver 14 of 24 Sensors 2020, 20, 2609 has full knowledge of the null space (G), the receivers exact rate is applied but not in its asymptotic form. 4.3. Optimization of Swift Privacy Rate in a MIMOME The objective function is expressed as follows, h(zr, Pm, PB) = −log MB + GV1ZrVG 1 GG + log \|MB\| + AEΩ ¯β3, ¯Θ3, ¯σ3 −AEΩ ¯β4, ¯Θ4, ¯σ4 , (31) (31) where MB implies that the receiver has full knowledge of her covariance matrix of noise and interference, ¯β3 = ATx+ARx AE , ¯β4 = ATx−r+ARx AE , while ¯σ3 and ¯σ4 are solutions to the formulated problem. In order to optimize the swift privacy rate, we proposed an optimization problem as stated below. min r min zr, Pm, PB h (zr, Pm, PB) s.t. r ∑ i=1 zr(i) + Pm ≤Pmax A zr(i) ≥0, ∀i = 1, ..., r Pm ≥0 0 ≤PB ≤Pmax B (32) min r min zr, Pm, PB h (zr, Pm, PB) s.t. r ∑ i=1 zr(i) + Pm ≤Pmax A zr(i) ≥0, ∀i = 1, ..., r Pm ≥0 0 ≤PB ≤Pmax B (32) (32) In the optimization problem above, the constraints are in their convex form; nevertheless, h(.) is yet to assume a convex form. We can achieve this by rewriting the function In the optimization problem above, the constraints are in their convex form; nevertheless, h(.) is yet to assume a convex form. We can achieve this by rewriting the function h(zr, Pm, P) = −log MB + GV1ZrVG 1 GG + log \|MB\| + AE ¯β3υΘ3(¯σ3) −AE ¯β4υΘ4(¯σ4) + AE log ¯σ4 ¯σ3 + AE (¯σ3 −¯σ4) log(c) = −log MB + GV1ZrVG 1 GG + log \|MB\| + N ¯Θ3 ∑ k=1 log (1 + ¯σ3( ¯Θ3)k, k) − N ¯Θ4 ∑ k=1 log (1 + ¯σ4( ¯Θ4)k, k) + AE log ¯σ4 ¯σ3 + AE (¯σ3 −¯σ4) log(c) (33) (33) From Equation (33), we can linearize + ∑ N ¯Θ3 k=1 log (1 + ¯σ3( ¯Θ3)k, k), which is the convex form of h(.) at every iteration point of the optimization algorithm using the expansion method of ﬁrst-order Taylor series. Similarly, we can resolve the reliance state of h(.) on the ¯σ3, ¯σ4 parameters by making them constant with respect to their values and the upgrading them at the end to achieve the following, 1 −¯σj+1 i = ¯βi ¯σj+1 i N ¯Θi N ¯Θi ∑ k=1 ( ¯Θj+1 i )k, k 1 + ¯σj+1 i ( ¯Θj+1 i )k, k , i = 3, 4. 4.3. Optimization of Swift Privacy Rate in a MIMOME (34) (34) note that j + 1 represents the parameter value at j + 1 iteration. Thus, we optimized the preceding convex function at j iteration, gj(yr) = −log MB + GV1ZrVG 1 GG − N ¯Θ4 ∑ k=1 log 1 + ¯σj 3( ¯Θ4)k, k + yr −yj r J∇yr f j yr=yj r , (35) (35) 15 of 24 Sensors 2020, 20, 2609 denoting yr = h zJ r, Pm, PB iJ and f j(yr) = log \|MB\| + N ¯Θ3 ∑ k=1 log 1 + ¯σj 3( ¯Θ3)k, k . Recall that terms that are r = h zJ r, Pm, PB iJ and f j(yr) = log \|MB\| + NΘ3 ∑ k=1 log 1 + ¯σj 3( ¯Θ3)k, k . Recall that terms that a d ( ) b h d h ff h h constant were not used in (25) because they do not have any effect in the optimization. However, at this point, h(.) has assumed a convex function and we can then optimize it using the preceding optimization yj+1 r = arg min yr gj(yr) s.t. r ∑ i=1 zr(i) + Pm ≤Pmax A zr(i) ≥0, ∀i = 1, ..., r Pm ≥0 0 ≤PB ≤Pmax B . (36) yj+1 r = arg min yr gj(yr) s.t. r ∑ i=1 zr(i) + Pm ≤Pmax A zr(i) ≥0, ∀i = 1, ..., r Pm ≥0 0 ≤PB ≤Pmax B . (36) yj+1 r = arg min yr gj(yr) s.t. r ∑ i=1 zr(i) + Pm ≤Pmax A zr(i) ≥0, ∀i = 1, ..., r Pm ≥0 0 ≤PB ≤Pmax B . (36) (36) Similarly, at this point, if any possible values of r is deployed, an optimal output will be recorded. The summary of our proposed optimization procedure can be seen in Algorithm 3. Although different optimization methods to tackle this kind of problem were proposed in [13,18] comparatively, these methods give almost similar outcome, nonetheless, our proposed Algorithm 3 signiﬁcantly executes with minimal complexity. Our ﬁgures illustrate the efﬁciency and out-performance of our swift optimization algorithm as compared with nonoptimal parameters. Algorithm 3 Swift privacy rate optimization algorithm. 4.3. Optimization of Swift Privacy Rate in a MIMOME Input actual: ε, ¯σ0 3, ¯σ0 4, and initiate hmin= 0 1: for r = 1 : ATx do 2: Set yr to satisfy the constraints 3: while yj r−yj−1 r yj−1 r >ε do 4: execute (28) till yj+1 r is realized 5: execute (25) using yj−1 r till ¯σ0 3 , ¯σ0 4 is updated 6: j = j + 1. 7: end while 8: if h yj r < hmin then 9: hmin = h yj r 10: ymin = yj r 11: end if 12: end for 13: Revert ymin. Algorithm 3 Swift privacy rate optimization algorithm. p ε, ¯σ0 3, ¯σ0 4, and initiate hmin= 0 end while 5. Numerical Analysis and Results In this section, MATLAB simulation results based on our proposed SCEO algorithm and the Swift privacy rate optimization algorithm is presented. Our investigations show that the magnitudes of all transmission channels are distributed in a Rayleigh form with an even unit of mean square, while the 16 of 24 Sensors 2020, 20, 2609 attenuation factor of the transmission self-interference ρ is set to be 0.8, except where slight changes are required. The transmission power constraints were set at 20db except where otherwise stated. 5.1. Realized Privacy Capacity 5.1. Realized Privacy Capacity We performed a transmission performance evaluation for a three-node transmission under power and rate constraints as shown in Figures 3 and 4. A comparison of three separate transmission scenarios with respect to the privacy capacity of their transmissions is shown in the experiment. 0.0 0.1 0.2 0.3 0.4 0.5 0.6 Sc(bit/s/Hz) P(dB) Transmission 1 (Sc,1) Transmission 2 (Sc,2) Transmission 3 (Sc,3) 20 30 40 50 60 Figure 3. Realized privacy capacity against eavesdroppers under power and rate constraints. 0 2 4 6 8 10 P (dB) Transmission 1 (CSOD,1) Transmission 2 (CSOD,2) Transmission 3 (CSOD,3) CSOD 20 30 40 50 60 Figure 4. Realized privacy capacity for Source-to-Destination (C∗ SOD = 0.8CSOD,1) transmission under power and rate constraints. 0.0 0.1 0.2 0.3 0.4 0.5 0.6 Sc(bit/s/Hz) P(dB) Transmission 1 (Sc,1) Transmission 2 (Sc,2) Transmission 3 (Sc,3) 20 30 40 50 60 Figure 3. Realized privacy capacity against eavesdroppers under power and rate constraints. Figure 3. Realized privacy capacity against eavesdroppers under power and rate constraints. 0 2 4 6 8 10 P (dB) Transmission 1 (CSOD,1) Transmission 2 (CSOD,2) Transmission 3 (CSOD,3) CSOD 20 30 40 50 60 Figure 4. Realized privacy capacity for Source-to-Destination (C∗ SOD = 0.8CSOD,1) transmission under power and rate constraints. Figure 4. Realized privacy capacity for Source-to-Destination (C∗ SOD = 0.8CSOD,1) transmission under power and rate constraints. Figure 3 indicates privacy capacity against Eve, whereas Figure 4 represents the data rate of the transmission between the source and destination (Source-to-Destination) speciﬁcally realizing for the entire Am < Cm, ∀m ∈ℵ. This clearly implies that, for the entire set of subcarriers, the Eve’s channel is stronger from the Source (Tx) than the Destination (Rx) has from the Source. For Transmission 1 (the ﬁrst transmission scenario), at the Source and Destination terminals, data rate is optimized subject to power constraints. However, this occurs without the constraint for privacy Sensors 2020, 20, 2609 17 of 24 17 of 24 capacity. The subsequent obtainable data rate is signiﬁed by CSOD,1. On the other hand, as expected for this channel, the subsequent privacy capacity Sc,1 is realized as zero. For Transmission 2, the transmissions privacy capacity is optimized subject to power constraints at Source and Destination likewise a rate constraint of Source-to-Destination. 5.1. Realized Privacy Capacity Setting the lesser bound on the rate (i.e., the constrained rate) at C∗ SOD = 0.8CSOD,1, the equivalent rate realized at the channel transmission is signiﬁed by CSOD,2, as anticipated, the curve is vague from that of C∗ SOD. Sc,2 represents the subsequent attained transmission privacy capacity and it is huge and quite close to Transmission 3. In Transmission 3 (the third transmission scenario), the privacy capacity is optimized with only power constraint at Source and Destination without considering any rate constraint. Subsequently, we represented the privacy capacity as Sc,3 while the data transmission rate is signiﬁed as CSOD,3. Considering the three transmission scenarios, it is observed that the result obtained at the second scenario (Transmission 2) outperform the other two transmission scenarios in terms of source-to-destination data rate trade-off and the transmission’s privacy capacity. 5.2. Joint Power Assignment for Multiple Destinations For a multiple transmission destinations scenario, we considered Cn = χC∗ n, representing C∗ n as the optimal data rate attainable from Source to Destination when power ps N is assigned to Source for data broadcast to the nth destination. Considering χ = 0.8 and N = 4, Figures 5 and 6, respectively, represent the maximum attained privacy capacities and data rates from the source-to-destination. Likewise, the equivalent outputs with no rate constraint are represented in the two ﬁgures. It is observed that if rate constraints are applied, a small measure of privacy capacities is lost, however, signiﬁcant data rates are gained and maintained. For both Figures 5 and 6, under rate constraint, both the achieved and constrained rates are separable. 0.0 0.1 0.2 0.3 0.4 0.5 0.6 Sc(bit/s/Hz) P (dB) Transmission 1 without rate constraint Transmission 2 without rate constraint Transmission 3 without rate constraint Transmission 4 with rate constraint Transmission 5 with rate constraint Transmission 6 with rate constraint 20 30 40 50 60 Figure 5. Maximum realized privacy capacity for multiple destinations transmission. Figure 5. Maximum realized privacy capacity for multiple destinations transmission. 18 of 24 Sensors 2020, 20, 2609 0 1 2 3 CSOD P (dB) Transmission 1 (with rate constraint) Transmission 2 (with rate constraint) Transmission 3 (with rate constraint) Transmission 4 (w/o rate constraint) Transmission 5 (w/o rate constraint) Transmission 6 (w/o rate constraint) 20 30 40 50 60 Figure 6. Source-to-Destination data rate for multiple destinations transmission. Figure 6. Source-to-Destination data rate for multiple destinations transmission. 5.3. Joint Power Assignment for Multiple Sources For a multiple transmission source scenario, considering the nth transmission source, we set the rate constraint as Cn = χC∗ n, representing C∗ n as the optimal data rate attainable from Source to Destination when power ps,n = ps M is assigned to Destination for data broadcast from the nth Source. Figures 7 and 8 present the data rates and privacy capacities for all three different sources. The subsequent result shows that although a small measure of privacy capacity is lost in the absence of rate constraint, signiﬁcant data rates are gained and maintained. 5.2. Joint Power Assignment for Multiple Destinations 0.0 0.1 0.2 0.3 0.4 0.5 Sc (bit/s/Hz) P (dB) Transmission 1 (w/o rate constraint) Transmission 2 (w/o rate constraint) Transmission 3 (w/o rate constraint) Transmission 4 (with rate constraint) Transmission 5 (with rate constraint) Transmission 6 (with rate constraint) 20 30 40 50 60 Figure 7. Maximum realized privacy capacity for multiple sources transmission. Figure 7. Maximum realized privacy capacity for multiple sources transmission. 19 of 24 Sensors 2020, 20, 2609 0.0 0.5 1.0 1.5 2.0 2.5 3.0 CSOD P (dB) Transmisson 1 (with rate constraint) Transmisson 2 (with rate constraint) Transmisson 3 (with rate constraint) Transmisson 4 (w/o rate constraint) Transmisson 5 (w/o rate constraint) Transmisson 6 (w/o rate constraint) 20 30 40 50 60 Figure 8. Source-to-Destination data rate for multiple sources transmission. Figure 8. Source-to-Destination data rate for multiple sources transmission. 5.4. Performance Comparison of Difference Algorithms 5.4. Performance Comparison of Difference Algorithms In Figures 9 and 10, we compared our proposed sequential convex estimation optimization (SCEO), which is intended to mitigate the optimization problem in (15) against the Bisection method in [38] and the two-stage cooperative jamming scheme (TSCS) in [44]. A total of 60 transmitting antennas were selected for both experiments. For the rate constraints, about 0.8 of optimal attainable capacity (OAA) was selected for Figure 9, whereas for Figure 10, the power constraint (ps = pd = p) is set at 20 dB. Figure 9 indicates that mostly if the power constraints are low, our proposed SCEO technique is latent enough to obtain optimal values. Comparing the three different techniques in Figure 10 at different variations of rate constraint. Our technique is observed to outperform the other two compared techniques notwithstanding severe rate constraints. Moreover, as the rate constraints becomes more severe, convergence might be difﬁcult for the TSCS and Bisection techniques but our algorithm converges almost seamlessly. 0.0 0.1 0.2 0.3 0.4 0.5 Sc (bit/s/Hz) P (dB) TSCS in [44] Bisection Estimation in [38] Proposed SCEO Algorithm 20 30 40 50 60 Figure 9. Performance of the algorithms under different power constraints. Figure 9. Performance of the algorithms under different power constraints. 20 of 24 Sensors 2020, 20, 2609 0.0 0.2 0.4 0.6 0.8 1.0 0.10 0.12 0.14 0.16 0.18 0.20 0.22 0.24 0.26 Sc (bit/s/Hz) Rate Constraint Proposed SCEO Algorithm Bisection Estimation in [28] TSCS in [44] Figure 10. Performance of the algorithms with different rate constraints for p = 20 dB. Figure 10. Performance of the algorithms with different rate constraints for p = 20 dB. Finally, the complexity analysis of our swift privacy rate optimization algorithm in a multiple transmission and multiple eavesdropper scenario is shown in Figure 11. We set the optimization parameters as AE = ATx, β1 = 6, Pmax n = Pmax B = 20 dB. From the result of our investigation, there were no evident patterns in the optimal values, in addition, the optimal parameters are dependent on the outputs of G. Nevertheless, Pn can be assumed to be frequently and approximately distributed evenly among the channels. Finally, it is observed that as AE becomes larger, the minimal PB and r are respectively favored by the optimization. 5.4. Performance Comparison of Difference Algorithms 0 100 200 300 400 500 600 0 100 200 300 400 500 600 700 800 Average number of Eve's (AE) Number of transmiting Antenna's (ATx) Optimal Pn=PB=20dB Pn=PB=30dB Pn=30dB,PB=10dB Figure 11. Comparison of ¯AE versus ¯ATx with different parameters. Figure 11. Comparison of ¯AE versus ¯ATx with different parameters. References 1. Anajemba, J.H.; Tang, Y.; Ansere, J.A.; Iwendi, C. Performance Analysis of D2D Energy Efﬁcient IoT Networks with Relay-Assisted Underlaying Technique. 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Subject to both the transmission rate and power constraints, we considered and implemented an efﬁcient power allocation optimization algorithm for enhancing privacy capacity in a three-node transmitting network and also in a real life MIMOME scenario. The applications of the resulting research and results can be applied to current wireless communication networks seen in rampant use in both IoT and 5G networks. Our experimental results showed that by using the sequential convex estimation optimization (SCEO) algorithm, a more optimal result and enhanced convergence is achieved. 21 of 24 21 of 24 Sensors 2020, 20, 2609 However, due to possible challenges envisaged when a multiple eavesdropper is active in a network, we expanded our research to develop a swift privacy rate optimization algorithm, which executes signiﬁcantly with minimal complexity when compared with nonoptimal parameters. The use of the rate constraint together with self-interference of the full-duplex at the receiving node makes the performance of our technique outstanding from the recent studies reviewed. Furthermore, we extended our study to consider a scenario where multiple sources and multiple destinations are in use. Finally, our technique indicates that as the iterative algorithm reaches convergence, a decent approximation on the optimal values is achieved. In a future work, we intend to consider a stochastic optimization approach for privacy capacity with Eve’s CSI Unknown to Users. Author Contributions: Conceptualization, J.H.A. and Y.T.; methodology, J.H.A. and C.I.; software, A.O.; validation, J.H.A, Y.T., C.I., and G.S.; formal analysis, Y.T. and J.H.A.; investigation, A.O. and J.H.A; resources, O.J. and G.S.; data curation, J.H.A.; writing—original draft preparation, J.H.A.; writing—review and editing, C.I., and G.S.; visualization, C.I.; supervision, Y.T.; project administration, J.H.A. and O.J.; funding acquisition, O.J. All authors have read and agreed to the published version of the manuscript. Funding: This research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. NRF-2018R1C1B5045013). Acknowledgments: The authors wish to acknowledge the technical, supervision and material supports received from Tang Yue, Department of Communication Engineering, College of Internet of Things, Hohai University, Changzhou Campus, China. Conﬂicts of Interest: The authors declare no conﬂict of interest. 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https://openalex.org/W2742413383	https://www.frontiersin.org/articles/10.3389/fpsyg.2017.01416/pdf	English	null	Human–Animal Interaction and Older Adults: An Overview	Frontiers in psychology	2,017	cc-by	6,260	Edited by: Natalie Ebner, University of Florida, United States Keywords: human–animal interaction, pet ownership, animal-assisted therapy, aging, older adults Human–Animal Interaction and Older Adults: An Overview Nancy R. Gee1,2, Megan K. Mueller3,4 and Angela L. Curl5 1 Department of Psychology, State University of New York, Fredonia, NY, United States, 2 WALTHAM Centre for Pet Nutrition, Leicestershire, United Kingdom, 3 Tufts Institute for Human-Animal Interaction, Department of Clinical Sciences, Cummings School of Veterinary Medicine at Tufts University, North Grafton, MA, United States, 4 Jonathan M. Tisch College of Civic Life at Tufts University, Medford, MA, United States, 5 Family Science and Social Work and Scripps Gerontology Center, Miami University, Oxford, OH, United States Both pet ownership and animal-assisted therapy are becoming increasingly popular in the United States, and the science of human–animal interaction (HAI) seeks to explore how these relationships with animals can impact health and well-being. In particular, one burgeoning area of research is the role of HAI in healthy aging, given the potential for HAI as an important feature of health and well-being in older adults. The purpose of this review is to summarize and evaluate existing research in this innovative area of scholarship, identifying the potential beneﬁts and risks of both pet ownership and animals in therapeutic settings for older adults. We will also identify recommendations for future research and applications in this developing area of scholarship. INTRODUCTION Reviewed by: Marie Jose Enders-Slegers, Open University in the Netherlands, Netherlands Phoebe E. Bailey, Western Sydney University, Australia Reviewed by: Marie Jose Enders-Slegers, Open University in the Netherlands, Netherlands Pet ownership is prevalent in the United States; 65% of households have at least one pet (American Pet Products Association, 2016). Animals are increasingly incorporated into therapeutic settings targeting improved mental and physical health (e.g., Fine, 2015). The science of Human–Animal Interaction (HAI) seeks to understand how our relationships with animals can inﬂuence both human and animal health, and has grown considerably in recent years (McCune et al., 2014). Phoebe E. Bailey, Western Sydney University, Australia Correspondence: Nancy R. Gee nancy.gee@fredonia.edu One particular area for continued growth is the role of HAI in healthy aging, given the potential for HAI as a key component of understanding health and well-being in older adults. Initial work in this area has demonstrated promising outcomes. The purpose of this paper is to describe existing research on HAI and older adults, and identify avenues for future research in this developing area of scholarship. Other reviews of HAI and aging exist, but are more limited in scope (e.g., limited to pet ownership; McNicholas, 2014), pathology (e.g., depression; Virués-Ortega et al., 2012), or are restricted to one type of HAI (e.g., Virués-Ortega et al., 2012; Bernabei et al., 2013) or one population (e.g., persons with dementia; Filan and Llewellyn-Jones, 2006). In this overview, we will draw on these reviews, along with edited volumes and original research, in order to frame the current state of the ﬁeld with regards to HAI in healthy aging. As with much HAI research, the preponderance of the ﬁndings discussed herein is from work undertaken in Western countries. Specialty section: This article was submitted to Emotion Science, a section of the journal Frontiers in Psychology Received: 30 May 2017 Accepted: 04 August 2017 Published: 21 August 2017 Keywords: human–animal interaction, pet ownership, animal-assisted therapy, aging, older adults MINI REVIEW published: 21 August 2017 doi: 10.3389/fpsyg.2017.01416 Citation: Gee NR, Mueller MK and Curl AL (2017) Human–Animal Interaction and Older Adults: An Overview. Front. Psychol. 8:1416. doi: 10.3389/fpsyg.2017.01416 As will become apparent the theme of the discussion below is that the impact of animals on healthy human aging is complicated and fraught with challenges. In this paper, we provide an overview of the state of this research, brieﬂy describe the challenges, and make recommendations for future researchers. August 2017 \| Volume 8 \| Article 1416 1 Frontiers in Psychology \| www.frontiersin.org Human–Animal Interaction and Older Adults Gee et al. OVERVIEW OF HAI RESEARCH dog, and with retaining functionality after stroke (Rondeau et al., 2010). However, more research is needed focusing speciﬁcally on older adult populations. In exploring how HAI can impact older adults, it is important to understand the contexts in which they interact with animals, both as pets and in therapeutic situations. Pet ownership is one of the most common and sustained forms of interaction; pets may live in the home (e.g., cats and dogs) or outside (e.g., horses). An examination of the Health and Retirement Study data revealed that just over half of adults over the age of 50 years have at least one pet (author calculations using 2012 data). In addition, animal-assisted activities (AAAs) and therapy (AAT) are becoming increasingly popular for older adults. Animal- assisted activities involve informal activities such as animal visitation programs, whereas AAT is deﬁned as “a goal oriented, planned, and structured therapeutic intervention directed and/or delivered by health, education, or human service professionals” (Jegatheesan et al., 2015, p. 416). As AAA/T programs designed for older adults become more commonplace, it is increasingly important to build an evidence base about the eﬀectiveness of these practices. Existing research in both pet ownership and AAA/T has demonstrated promising ﬁndings for a variety of outcomes of health and well-being for older adults. However, this evidence base is not strong in all areas of health. We begin by presenting existing evidence for speciﬁc areas of health and well-being, with a focus on older adults, and then discuss the associated challenges. Depression and Anxiety Much HAI research has focused on the role of animals in mitigating mental health disorders such as depression/anxiety. Animal-assisted activities and therapy has been associated with reductions in depression symptoms for a variety of populations (Souter and Miller, 2007), with moderate eﬀect sizes (Virués- Ortega et al., 2012). These eﬀects are particularly apparent with populations in elder-care institutions and assisted-living facilities, such as older adults with no cognitive impairment (Colombo et al., 2006), with dementia (Travers et al., 2013), requiring the use of wheelchairs or walkers (Le Roux and Kemp, 2009), and with mental illness (Moretti et al., 2011). Similarly, AAA/T has been shown to reduce anxiety in patients with Alzheimer’s disease (Mossello et al., 2011), those hospitalized with heart failure (Cole et al., 2007), and residents of long-term care facilities (Le Roux and Kemp, 2009). In addition to evidence of the therapeutic eﬀects of AAA/T, some ﬁndings have indicated a link between pet ownership and reduced depression for older adults (e.g., Garrity et al., 1989); the emotional bond between older adults and their pets seems to be a particularly important component of that relationship (Peretti, 1990). However, several studies have not found diﬀerences in levels of depression between pet owners and non-pet owners (Miller and Lago, 1990; Branson et al., 2016), or have found an increase in depressive symptoms after the loss of a pet (Chan et al., 2012). More research is needed in this area to clarify the impact of pets on depressive symptoms. Cardiovascular Health Some of the strongest research exploring HAI and health outcomes, though not speciﬁcally focused on older adults, has focused on the link between HAI and cardiovascular health and adaptive stress responses. Pet ownership has been associated with lower blood pressure, lower heart rate, and faster recovery during mental stress (Allen et al., 2001, age not speciﬁed; Allen et al., 2002, adults of all ages). Friedmann et al. (2013) demonstrated a link between pet ownership and cardiovascular health, both as a predictor of long-term survival in patients (ages 33–84 years) who had experienced a myocardial infarction (Friedmann et al., 2011), and in improving ambulatory blood pressure in hypertensive older adults. Aiba et al. (2012, participant ages: M = 69, SD = 8) showed pet ownership to be an independent predictor of cardiac autonomic imbalance for patients with conditions such as diabetes, hypertension, or hyperlipidemia. In fact, the American Heart Association (AHA) issued a scientiﬁc statement suggesting that pet ownership (particularly dogs) may reduce the risk of cardiovascular disease (Levine et al., 2013). Speciﬁcally, the AHA statement identiﬁed and summarized the 31 most relevant studies and concluded that there were varying levels of evidence (depending on outcome) for a link between pet ownership and improved outcomes regarding systemic hypertension, hyperlipidemia, physical activity, obesity, autonomic function, cardiovascular reactivity, and survival for individuals with cardiovascular disease. In addition to pet ownership, animal-assisted physical health interventions (primarily dog walking) have been associated with increasing cardiac functioning for older adults (Motooka et al., 2006) compared to when the same individuals were not walking with a Frontiers in Psychology \| www.frontiersin.org Physical Activity and Falls In addition to mental health and well-being outcomes, pet owners (and dog owners in particular) are often more likely to be physically active. Older adult dog owners are signiﬁcantly more likely to walk more than non-pet owners, and have higher levels of physical activity (Dembicki and Anderson, 1996; Thorpe et al., 2006; Toohey et al., 2013; Curl et al., 2016). In addition, one study found less deterioration in ability to perform activities of daily living for older adult pet owners compared to non-pet owners (age 65+; Raina et al., 1999). Recently, a study using the ActivPal accelerometer showed that independent living older adult dog owners spent more time walking, walked for more minutes at a moderate cadence, and spent less time sitting compared to their non-dog-owning matched counterparts (Dall et al., 2017). Within the context of AAA, assisted-living residents who walked with a dog walked farther compared to walking without a dog (Herbert and Greene, 2001). In addition to the physical health beneﬁts of HAI, it is also important to note that animals may be associated with increased risk. Falls are a major factor inﬂuencing mortality and morbidity for aging adults (Ambrose et al., 2013). Dogs and cats may be associated with fall risk, and fall injuries related to pets are highest for people 75 years of age and older (Stevens et al., 2010). In addition, dogs can be a hazard for injuries related to leash-walking (Willmott et al., 2012) and both cats and dogs can be linked to scratches and bites. Although the risk of disease spreading between animals and people (zoonotic disease transmission) is low (e.g., Lowden et al., 2015), it is important to recognize that older adults likely represent a vulnerable population. Additional research is needed to explore the risk/beneﬁt ratio of HAI for older adults, and to establish best practices for ensuring safety and supporting pet ownership, particularly in physical activity interventions involving dog walking. In addition, there are certain methodological challenges speciﬁc to the nature of HAI. For example, it is nearly impossible to conduct double-blind studies involving animals – participants are aware that the animal is present. Finally, in examining the impact of pet ownership, there is a fundamental confound involving causality that is diﬃcult to avoid. Loneliness and Social Support Human–animal interaction may provide social support for older adults, thus reducing the risk of loneliness. Animal-assisted activity has been leveraged as an intervention to improve social functioning in older psychiatric (Haughie et al., 1992) and dementia patients (Sellers, 2005; Filan and Llewellyn-Jones, 2006; Marx et al., 2010). In addition, AAA can contribute to reducing loneliness, and improving quality of life, mood, and social interaction (Banks and Banks, 2002; Vrbanac et al., 2013) in long-term and elder-care facilities. Pet ownership has been hypothesized to have similar beneﬁcial eﬀects. Older pet owners have been found to be less likely to report loneliness (Stanley et al., 2013). Pets may act as catalysts for human social interactions (McNicholas and Collis, 2000), thereby fostering a sense of community (Wood et al., 2007). In a study of adults 50+ years of age, Toohey et al. (2013) found that frequent dog walkers were more likely to report a high sense of community, in addition to the beneﬁts of increased physical activity. In addition, for older adults high attachment to a pet has been associated with lower levels of loneliness (age 55–84 years; Krause-Parello and Gulick, 2013) and appears to mediate the relationship between loneliness and general health for older women (age 55–84 years; Krause-Parello, 2008), perhaps providing a “buﬀering” eﬀect. However, there are also mixed ﬁndings in this area; Gilbey et al. (2007) found no diﬀerence in August 2017 \| Volume 8 \| Article 1416 Frontiers in Psychology \| www.frontiersin.org 2 Gee et al. Human–Animal Interaction and Older Adults Quality of Life and Life Satisfaction Quality of Life and Life Satisfaction Quality of life is a key issue for aging adults as they move through life transitions, and interacting with animals may be a way of mitigating some of the associated stressors (Steed and Smith, 2002). Pet ownership has been linked to smaller decreases in life satisfaction after retirement (Norris et al., 1999). Interestingly, one study found that caregivers of a spouse with dementia reported higher attachment to their pets after onset of their spouse’s dementia (Connell et al., 2007). Animal-assisted activities also have been associated with increased life satisfaction and decreased depression in older adults, including those both with and without dementia or cognitive deﬁcits (Steed and Smith, 2002; Colombo et al., 2006; Olsen et al., 2016). CONSIDERATIONS/CHALLENGES FOR HAI RESEARCH loneliness between pet owners and non-pet owners in a sample of adults 18 years and over. Since these studies have been correlative, it cannot be ruled out that lonely individuals are more likely to get pets; therefore, future work assessing causality is necessary (Pikhartova et al., 2014). Research on HAI has been plagued by methodological shortcomings which call into question the strength of the evidence base. The preponderance of the published research lacks the use of standardized measures for concepts such as pet bonding and for outcomes (e.g., loneliness), relies on small sample sizes, and involves short-term examination of eﬀects (lack of longitudinal studies). The animals involved and the human interaction with animals are frequently not well described and rarely is pet ownership history or degree of involvement with pets included when describing the sample and methodology (for a complete discussion see Kazdin, 2015). In research involving AAA/T, many studies do not employ randomized controlled trials or other rigorous control or comparison procedures such as use of appropriate covariate procedures in data analysis. As HAI researchers begin to more seriously investigate the impact of animals on older adults, it is critical that they carefully examine their planned research approaches and simply put, raise the bar on quality. Further, as with any ﬁeld of investigation, there is a positive publication bias – typically journals are more likely to accept for publication manuscripts that report signiﬁcant ﬁndings (Herzog, 2015). This bias is exacerbated in HAI research because often researchers form hypotheses that test for associated beneﬁts, rather than posing more objective questions, and fail to consider that HAI could have negative eﬀects (Stevens et al., 2010). Physical Activity and Falls In most studies of pet ownership, due to selection eﬀects, it is diﬃcult to ascertain if pet ownership is the cause of certain health outcomes – do pets make people healthier or do healthier people choose to own pets? One could address this by randomly assigning pet ownership to participants (e.g., Allen et al., 2001), but because pet type and ownership are personal choices with long-term implications, few participants are willing to be assigned to a particular pet ownership status. Another approach is to utilize large-scale, longitudinal datasets that allow for the use of comprehensive control variables at the analysis phase to identify some of the factors predicting pet ownership; however, as elegant as this approach may be, it is still not possible to completely determine causality. Frontiers in Psychology \| www.frontiersin.org RECOMMENDATIONS FOR FUTURE RESEARCH Despite the heterogeneity in health status, there are general patterns of age-related declines in health, which can impact research participation: normal age-related declines in hearing, vision, cognition, and dexterity can create diﬃculties for older research participants (Weil, 2015). Age-related health changes can also pose mobility and transportation problems in traveling to research sites if a participant is in a wheelchair and/or cannot drive. These changes might also impact an individual’s ability to participate in HAI, particularly in activities such as dog walking (Toohey et al., 2013). Of critical importance to future research in the intersection of HAI and gerontology is high quality methodology, as has been called for in other areas of HAI (McCune et al., 2014). This nascent research focus will beneﬁt from the broad spectrum of investigative approaches from qualitative and descriptive, to large-scale longitudinal studies, to the gold standard of establishing causation: the randomized controlled trial. In all cases, the key is to implement the best design to appropriately address the question under investigation. The use of high quality, rigorous methodologies is critical to establishing a solid foundational evidence base. Experimental and longitudinal researchers face additional challenges: older participants often drop out of research studies after moving, when experiencing illness and/or hospitalization, because of cognitive decline, or because they simply decide to stop participating (García-Peña et al., 2015a). Attrition can be reduced by allowing someone else (a “proxy”) to provide responses, but this can be problematic when trying to examine change (Weil, 2015). Other options include multiple follow- up attempts, reducing the length of time between observations, and use of other modes of contact (e.g., telephone and internet). Older participants are also at an increased risk of mortality. It is important for researchers to be comprehensive in presenting the relevant background research. In addition to presenting ﬁndings that are published in both HAI and gerontology journals, researchers in both ﬁelds also frequently publish in a wide variety of other disciplines (e.g., psychology, sociology, and anthropology). This breadth of publication outlets may make it challenging to consolidate and organize all the relevant previous research. We recommend that researchers develop interdisciplinary teams that bring the relevant HAI and gerontology expertise to projects. Normal age-related changes and increased risk for health problems must also be considered (García-Peña et al., 2015b). CONSIDERATIONS/CHALLENGES FOR AGING RESEARCH It is important to understand the challenges of integrating HAI research with aging research. When conducting research with older adults, there are a number of design and implementation considerations. For example, researchers must decide how to August 2017 \| Volume 8 \| Article 1416 Frontiers in Psychology \| www.frontiersin.org 3 Human–Animal Interaction and Older Adults Gee et al. deﬁne the lower age limit to be considered an “older adult.” In the United States, age 65 years is used for federal statistical purposes (e.g., Administration on Aging [AoA], 2016), while the United Nations (2015) generally uses 60 years as the cut-oﬀage for “older adulthood.” Alternatively, researchers may deﬁne older adulthood functionally, in terms of being retired or living in a retirement community. In addition, researchers often create age groupings of young–old, old–old, and oldest–old (Garfein and Herzog, 1995), and may vary in how they deﬁne such groups (e.g., Garrity et al., 1989; Toohey et al., 2013). questions may be asked in a way that reduces the measure’s psychometric properties or comparability to other research studies. In aging-related studies, researchers are faced with the disentangling age, period, and cohort eﬀects (Nesselroade and Labouvie, 1985). Age eﬀects refer to chronological age-related changes, while period eﬀects are the historical context and eﬀects of a given timeframe (e.g., natural disaster). Cohort eﬀects describe the way that age generations tend to act and behave. Disentangling these eﬀects can be diﬃcult if not impossible, and researchers should carefully consider which eﬀects are of greatest interest. Researchers should be wary of attributing diﬀerences to age when they could be explained by period or cohort eﬀects. A related consideration is that of continuity versus change – that is, diﬀerences between individuals could have existed for years and extend into older adulthood, or they could have emerged as part of the aging process. Aside from the characteristic of age, there is a great deal of heterogeneity among the older adult population, including gender, marital status, living arrangements and housing, racial and ethnic composition, geographic distribution, income and wealth, employment, education, health, and health care (Administration on Aging [AoA], 2016), all of which may be important variables in understanding HAI in older adults (e.g., living alone; Stanley et al., 2013). Therefore, researchers should consider how to recruit study participants to reﬂect these characteristics, and take care when making statements about the generalizability of ﬁndings (Nesselroade and Labouvie, 1985). Frontiers in Psychology \| www.frontiersin.org REFERENCES Cole, K. 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F., Paul, G., and Hausdorﬀ, J. M. (2013). Risk factors for falls among older adults: a review of the literature. Maturitas 75, 51–61. doi: 10.1016/j. maturitas.2013.02.009 Dembicki, D., and Anderson, J. (1996). Pet ownership may be a factor in improved health of the elderly. J. Nutr. Elder. 15, 15–31. doi: 10.1300/J052v15n03_02 American Pet Products Association (2016). Pet Industry Market Size & Ownership Statistics. Available at: http://www.americanpetproducts.org/press_ industrytrends.asp Filan, S. L., and Llewellyn-Jones, R. H. (2006). Animal-assisted therapy for dementia: a review of the literature. Int. Psychogeriatr. 18, 597–611. doi: 10.1017/S1041610206003322 Banks, M. R., and Banks, W. A. (2002). RECOMMENDATIONS FOR FUTURE RESEARCH For some conditions, it might be more realistic to test whether an intervention is eﬀective in slowing the rate of health decline, rather than resulting in improvements. Similarly, in longitudinal studies, the measurement approaches may change over time. While changes may reﬂect methodological improvements and/or simpliﬁcation to reduce burden, these changes also can create problems when studying change over time. Researchers should carefully consider the amount and nature of changes in measurement when designing studies and publishing ﬁndings. Secondary data sources may also not include all relevant measures for a research topic, and/or Another important aspect of building an evidence base is the use of theory to guide research. There is a need to identify the important issues in both ﬁelds and which theories best provide a framework to guide their investigation. For example, attachment theory may help us to better understand the bond that older adults form with their pets, and social support theory may help us to understand how and for whom pets may serve as alternate support ﬁgures when older adults lose key support ﬁgures such as spouses (e.g., Beetz, 2017). There are many areas within HAI and aging ripe for future research. For example, within the context of pet ownership, what August 2017 \| Volume 8 \| Article 1416 Frontiers in Psychology \| www.frontiersin.org 4 Human–Animal Interaction and Older Adults Gee et al. believe this exciting research area is ripe for growth. While initial ﬁndings in many areas of health and well-being are promising, there are numerous opportunities for improving the breadth, depth, and quality of the research on HAI and human aging. As such, the ﬁeld is at an exciting and critical turning point, with the potential for signiﬁcant growth in our understanding of how animals can impact older adults in ways that may support meaningful changes to policy and practice in the future. factors are important to consider when trying to help “match” people with pets? Are there contraindications for pet ownership, and what barriers to pet ownership might exist that are unique to the older adult population? In AAA/T, there is a need for additional rigorous, longitudinal research to assess the clinical eﬀects of these interventions. For example, is AAA/T helpful in managing or treating dementia, or sustaining cognition in older age? And are there speciﬁc barriers or potential issues related to using AAA/AAT with older patients – for people and for animals? CONCLUSION We gratefully acknowledge that the publication cost for this manuscript was supported by Scientiﬁc and Regulatory Aﬀairs, Mars Incorporated. We gratefully acknowledge that the publication cost for this manuscript was supported by Scientiﬁc and Regulatory Aﬀairs, Mars Incorporated. Although it is clear that the impact of animals on healthy human aging is complicated and fraught with challenges, we RECOMMENDATIONS FOR FUTURE RESEARCH factors are important to consider when trying to help “match” people with pets? Are there contraindications for pet ownership, and what barriers to pet ownership might exist that are unique to the older adult population? In AAA/T, there is a need for additional rigorous, longitudinal research to assess the clinical eﬀects of these interventions. For example, is AAA/T helpful in managing or treating dementia, or sustaining cognition in older age? And are there speciﬁc barriers or potential issues related to using AAA/AAT with older patients – for people and for animals? Investigating HAI in older populations provides opportunities to examine how the impact of HAI may vary developmentally and across other demographic characteristics. Research can also examine the impact of HAI on a variety of outcomes (e.g., physical and mental health) and unearth the mechanisms by which these eﬀects operate, ultimately better informing the ways in which pets may impact the lives of our rapidly growing older adult population. AUTHOR CONTRIBUTIONS All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. REFERENCES “Experimental design in research on aging.,” in Handbook of the Psychology of Aging, 2nd Edn, eds W. Schaie and S. Willis (New York, NY: Van Nostrand Reinhold Company), 35–60. Garrity, T. F., Stallones, L. F., Marx, M. B., and Johnson, T. P. (1989). Pet ownership and attachment as supportive factors in the health of the elderly. 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Psychogeriatrics 11, 125–129. doi: 10.1111/j.1479-8301.2010.00329.x Willmott, H., Greenheld, N., and Goddard, R. (2012). Beware of the dog? Copyright © 2017 Gee, Mueller and Curl. 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. interactions and sense of community. Soc. Anim. 15, 43–56. doi: 10.1163/ 156853007X169333 Human–Animal Interaction and Older Adults Frontiers in Psychology \| www.frontiersin.org REFERENCES An observational study of dog-related musculoskeletal injury in the UK. Accid. Anal. Prev. 46, 52–54. doi: 10.1016/j.aap.2011.10.004 Mossello, E., Ridolﬁ, A., Mello, A. M., Lorenzini, G., Mugnai, F., Piccini, C., et al. (2011). Animal-assisted activity and emotional status of patients with Alzheimer’s disease in day care. Int. Psychogeriatr. 23, 899–905. doi: 10.1017/ S1041610211000226 Wood, L. J., Giles-Corti, B., Bulsara, M. K., and Bosch, D. A. (2007). More than a furry companion: the ripple eﬀect of companion animals on neighborhood August 2017 \| Volume 8 \| Article 1416 Frontiers in Psychology \| www.frontiersin.org 6 Human–Animal Interaction and Older Adults Gee et al. interactions and sense of community. Soc. Anim. 15, 43–56. doi: 10.1163/ 156853007X169333 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. August 2017 \| Volume 8 \| Article 1416 Frontiers in Psychology \| www.frontiersin.org 7
https://openalex.org/W4379534489	https://link.springer.com/content/pdf/10.1007/s41971-023-00153-4.pdf	German	null	Training and Continuing Medical Education in Gastroenterology and Hepatology	Journal für gastroenterologische und hepatologische erkrankungen/Journal für gastroenterologische und hepatologische Erkrankungen	2,023	cc-by	2,900	Aus- und Fortbildung Aus- und Fortbildung J. Gastroenterol. Hepatol. Erkr. 2023 · 21:55–58 https://doi.org/10.1007/s41971-023-00153-4 Angenommen: 25. April 2023 Online publiziert: 6. Juni 2023 © Der/die Autor(en) 2023 J. Gastroenterol. Hepatol. Erkr. 2023 · 21:55–58 https://doi.org/10.1007/s41971-023-00153-4 Angenommen: 25. April 2023 Online publiziert: 6. Juni 2023 © Der/die Autor(en) 2023 Heinz F. Hammer1 · Michael Gschwantler2 · Harald Hofer3 · Peter Fickert1 1Klinische Abteilung für Gastroenterologie und Hepatologie,Medizinische Universitätsklinik Graz, Graz, Österreich Heinz F. Hammer1 · Michael Gschwantler2 · Harald Hofer3 · Peter Fickert1 1Klinische Abteilung für Gastroenterologie und Hepatologie,Medizinische Universitätsklinik Graz, Graz, Österreich Heinz F. Hammer1 · Michael Gschwantler2 · Harald Hofer3 · Peter Fickert1 1Klinische Abteilung für Gastroenterologie und Hepatologie,Medizinische Universitätsklinik Graz, Graz, Österreich 2Klinik Ottakring, Wien, Österreich 3Klinikum Wels-Grieskirchen, Wels, Österreich 2Klinik Ottakring, Wien, Österreich 3Klinikum Wels-Grieskirchen, Wels, Österreich Die Ausbildung und Fortbildung von Fachärzt:innen für das Sonderfach Innere Medizin mit Schwerpunkt Gastroenterolo- gie und Hepatologie (FA IM-GEHEP) ist in einem interessanten Spannungsfeld und 2fach herausfordernd: Übersicht über die Struktur der Ausbildung zum Sonderfach Innere Medizin mit Schwerpunkt Gastroenterologie und Hepatologie (FA IM-GEHEP) in Österreich 1. Es besteht ein rasant wachsender me- dizinischer Wissensschatz bei immer größerer Vielfalt diagnostischer und therapeutischer Möglichkeiten und Angebote betreﬀend alle tubulären und parenchymatösen Organe des Verdauungstrakts sowie in weiteren Kernbereichen des Fachs wie in der endoluminal-endoskopischen Diag- nostik und Therapien, den komplexen biologischen Therapien, Ultraschallme- thoden, der Funktionsdiagnostik, bei Ernährung und Stoﬀwechsel sowie in der gastrointestinalen Onkologie und bei Infektionserkrankungen. 1. Es besteht ein rasant wachsender me- dizinischer Wissensschatz bei immer größerer Vielfalt diagnostischer und therapeutischer Möglichkeiten und Angebote betreﬀend alle tubulären und parenchymatösen Organe des Verdauungstrakts sowie in weiteren Kernbereichen des Fachs wie in der endoluminal-endoskopischen Diag- nostik und Therapien, den komplexen biologischen Therapien, Ultraschallme- thoden, der Funktionsdiagnostik, bei Ernährung und Stoﬀwechsel sowie in der gastrointestinalen Onkologie und bei Infektionserkrankungen. Gegenwärtiggibtes in58 österreichischen Krankenanstalten etwa 141 Ausbildungs- stellen (wobei aktuell verlässliche Zahlen durch den Übergang der Ärzteausbil- dung in die Länderkompetenz schwierig erhebbar sind). Die aktuelle Ausbildungs- richtlinie ist unter www.aerztekammer. at/ausbildung abrufbar. Der vorgese- hene Ausbildungsweg ist in . Abb. 1 dar- gestellt und setzt sich aus 9 Monaten Basisausbildung (BA) und 27 Monaten Sonderfachgrundausbildung (SFGA) in innerer Medizin, gefolgt von 36 Monaten Ausbildung im Sonderfach Schwerpunkt Gastroenterologieund Hepatologie(SFSA) zusammen. Die Ausbildungsinhalte dazu ﬁnden sich in der Anlage 12.4 S5–7 und sollten von den Auszubildenden und Aus- bildungsverantwortlichen entsprechend beachtet werden. Die geforderten Kennt- nisse und Erfahrungen sind breit und weitreichend und sollten wie die Richt- zahlen für diagnostische/therapeutische Eingriﬀe fortlaufend diskutiert, verbes- sert und angepasst werden. Wichtig ist festzuhalten, dass es für die SFSA in Gas- troenterologie und Hepatologie aktuell in Österreich noch kein einheitliches Cur- riculum gibt (i.e. ein auf einer Theorie des Lehrens und Lernens aufbauender Lehrplan). Wir müssen uns daher der Fra- 2. Journal für Gastroenterologische und Hepatologische Erkrankungen 2 · 2023 55 Heinz F. Hammer1 · Michael Gschwantler2 · Harald Hofer3 · Peter Fickert1 1Klinische Abteilung für Gastroenterologie und Hepatologie,Medizinische Universitätsklinik Graz, Graz, Österreich Die Inhalte des aktuellen Rasterzeug- nisses, die zeitlichen Rahmenbedin- gungen einer gesetzeskonformen Ausbildung ebenso wie die Vorhaltung von Kapazitäten (z.B. Untersuchungs- frequenzen und Trainingsmöglichkei- ten) begrenzen die im Rahmen der Ausbildung tatsächlich vermittelbaren Inhalte. 2. Die Inhalte des aktuellen Rasterzeug- nisses, die zeitlichen Rahmenbedin- gungen einer gesetzeskonformen Ausbildung ebenso wie die Vorhaltung von Kapazitäten (z.B. Untersuchungs- frequenzen und Trainingsmöglichkei- ten) begrenzen die im Rahmen der Ausbildung tatsächlich vermittelbaren Inhalte. QR-Codescannen&Beitragonlinelesen 55 Journal für Gastroenterologische und Hepatologische Erkrankungen 2 · 2023 5 Aus- und Fortbildung BA 9 Monate SFGA: 27 Monate SFSA: 36 Monate Prüfung Abb. 1 8 AusbildungSonderfachInnereMedizinSchwerpunktGE/HE.BABasisausbildung,SFGASon- derfach Grundausbildung, SFSA Sonderfach Schwerpunktausbildung Entwicklung des Curriculums für die Ausbildung im Sonderfach Schwerpunkt Gastroenterologie (SFSA) Vor der Konkretisierung von Ausbildungs- inhalten und der Zuordnung dieser Aus- bildungsinhalte zur verfügbaren Ausbil- dungszeit gilt es, die allgemeinen Grund- sätze der Ausbildung von Fachärzt:innen für das Sonderfach Innere Medizin mit Schwerpunkt Gastroenterologie und He- patologie(FAIM-GEHEP)zudeﬁnieren.Was dürfen sich Patient:innen, Zuweiser:innen, KrankenhausträgerundSozialversicherun- gen von einem/einer FA IM-GEHEP erwar- ten? Abb. 1 8 AusbildungSonderfachInnereMedizinSchwerpunktGE/HE.BABasisausbildung,SFGASon- derfach Grundausbildung, SFSA Sonderfach Schwerpunktausbildung ge stellen, was, wie und wo in welcher Form (i.e. Lerntiefe und Kompetenzlevel) erlernbar und ausbildungsrelevant ist. (www.oeggh.at), die UEG (www.ueg.eu) oder die DGVS (www.dgvs.de), allgemein bekannt und erweitert werden. Verwie- sen wird dabei auch auf verfügbare Apps, wie z.B. die ÖGGH-App und die UEG- Guideline-App. Schließlich soll auch ein Fokus auf die Möglichkeiten zur ﬁnanzi- ellen Unterstützung von Auszubildenden gelegt werden. So übernimmt z.B. die ÖGGH nach erfolgreichem Abschluss der gastroenterologischen Facharztprüfung für die Prüﬂinge die Kurskosten für die Teilnahme am DGVS-Intensivkurs für die Vorbereitung auf die gastroenterologische Facharztprüfung. 1. Fachliche Kompetenz: FA IM-GEHEP verfügen über spezialisierte fachliche Kompetenzen, Kenntnisse und Fer- tigkeiten, die für die Betreuung von Patient:innen notwendig sind, die an Krankheiten oder Symptomen leiden, die aufgrund z.B. Inzidenz und Präva- lenz, Komplexität oder Schweregrad über dem Ausbildungs- und Tätig- keitsspektrum von Allgemeinmedizi- ner:innen und anderen Fachärzt:innen im Sonderfach innere Medizin liegen. 1. Fachliche Kompetenz: FA IM-GEHEP verfügen über spezialisierte fachliche Kompetenzen, Kenntnisse und Fer- tigkeiten, die für die Betreuung von Patient:innen notwendig sind, die an Krankheiten oder Symptomen leiden, die aufgrund z.B. Inzidenz und Präva- lenz, Komplexität oder Schweregrad über dem Ausbildungs- und Tätig- keitsspektrum von Allgemeinmedizi- ner:innen und anderen Fachärzt:innen im Sonderfach innere Medizin liegen. Arbeitsgruppe für Ausbildung innerhalb der ÖGGH Der Vorstand der ÖGGH hat daher im Vor- jahr beschlossen, dem Thema Ausbildung und Fortbildung vertieftes Augenmerk zu schenken, und hat dafür eine eigene Ar- beitsgruppe für Ausbildung installiert. Die Aufgabenbereiche dieser Arbeitsgruppe sind in . Tab. 1 zusammengefasst. Diese Arbeitsgruppe soll als Service- plattform der ÖGGH für Ausbildungs- verantwortliche und Ausbildungsassis- tent:innen dienen. Ziele sind eine gegen- seitige Hilfestellung und Informationsaus- tausch mit Fokus auf das „wer, was, wann, wo und wie“ der Ausbildung. Dafür not- wendig wird ein Informationsabgleich mit Darstellung des aktuellen Stands sowie ein realistisches Ausloten zukünftiger Not- wendigkeiten und Möglichkeiten sein. Ge- meinsam sollen neue Ausbildungsformate entwickelt werden sowie ein Curriculum für 36 Monate Sonderfachschwerpunkt- ausbildung ausformuliert werden. Weitere Aktivitäten sollen Train-the-Trainer-Kurse mit Fokus auf Ausbildung in Endoskopie, Sonographie und Funktionsdiagnostik (i.e. „Clinical Skills und Kompetenzen) sowie eine Austauschbörse für Assistent:innen in Ausbildung sein. Durch Abgleich mit Fortbildungsangeboten anderer nationa- ler und internationaler Fachgesellschaf- ten, z.B. für Ultraschall (ÖGUM, DGUM, EFSUMB) oder Ernährung (AKE, DGEM, ESPEN), sollen die klassischen Fortbil- dungsangebote, wie z.B. durch die ÖGGH 2. Kommunikative Kompetenz: FA IM- GEHEP können mit dem Wissens- und Informationsstand des Gegenübers (Patient:innen, Zuweiser:innen, Kran- kenhausträger, Sozialversicherungen) in angepassten und verständlichen Worten die Ursachen, Abklärungs- und Behandlungsmöglichkeiten von Symptomen und Krankheiten des Verdauungstrakts erklären. 2. Ausbildung und Forschung von den Jungen für die Jungen – die neue „young ÖGGH“ Die Zielsetzung für eine Spezialisierung wird der Erwerb von Kenntnissen und Fertigkeiten für die Betreuung bei seltenen und/oder hochkomplexen GEHEP Erkrankungen sein, die eine Betreuung in hochspezi- alisierten Zentren erforderlich macht (z.B. spezialisierte GI-Endoskopie, Ultraschall, Funktionsdiagnostik, Be- treuung von LTX-Patient:innen, ...). Weiters muss deﬁniert werden, welche zusätzlichen Ausbildungsmodule nach der Sonderfachschwerpunktausbil- dung (SFSA) GE/HEP notwendig sein werden. Grundsätzlich muss in einem solchen Ausbildungsstadium bei einer hochspezialisierten ärztlichen Tätigkeit davon ausgegangen werden, dass es mit hoher Wahrscheinlichkeit notwen- dig oder unumgänglich sein wird, dass die Tiefe der beruﬂichen Tätigkeit die Breite der GEHEP-Kompetenz ersetzt. . Außerhalb des Aufgabenbereichs der ÖGGH-Arbeitsgruppe aber wird der Kompetenzlevel in der Ausbildung für das Sonderfach innere Medizin („all- gemeine innere Medizin“) und andere Sonderfächer der inneren Medizin (36 Monate, nach SFGA) zu berück- sichtigen sein. Es braucht hier einen sorgfältigen Diskussionsprozess, wel- che Ausbildungsinhalte mit welchem Kompetenzlevel angestrebt werden sollen. 3. Spezialisierungen: Die Breite und Tiefe der ärztlichen Aufgaben und Tätigkei- ten im Bereich der Gastroenterologie, Hepatologie, Endoskopie, Ultraschall und assoziierter Themenbereiche bringt es mit sich, dass nicht alle dieser Bereiche im Rahmen der SFSA in der Tiefe gelernt und praktiziert werden können. Es muss geklärt werden, welche GEHEP-Kenntnisse und -Fer- tigkeiten vor allem oder nur in dafür spezialisiertenZentrenangeboten wer- den können oder sollen. Im Sinne einer für die Betreuungsqualität gewünsch- ten Spezialisierung sind umgekehrt abgestufte Versorgungskonzepte notwendig. Die Zielsetzung für eine Spezialisierung wird der Erwerb von Kenntnissen und Fertigkeiten für die Betreuung bei seltenen und/oder hochkomplexen GEHEP Erkrankungen sein, die eine Betreuung in hochspezi- alisierten Zentren erforderlich macht (z.B. spezialisierte GI-Endoskopie, Ult h ll F kti di tik B die verfügbaren hochspezialisierten Betreuungsmöglichkeiten haben. GEHEP auf den S. 5–7 beschrieben. Der allgemeine Grundsatz in der Aus- bildung zum/zur FA IM-GEHEP muss dabei sein, dass in dieser Phase der Ausbildung die Tiefe der Ausbildung in GEHEP nicht die Breite ersetzen darf. Es gilt zu klären, in welcher Zeit die Inhalte und Kompetenzlevel erlernt werden können. Der gegenwärtige Ausbildungsplan sieht vor, dass die Ausbildung zum/zur FA IM-GEHEP in mindestens 36 Monaten durchgeführt wird. Realistisch erscheint dies nur, wenn ein hoher Anteil der Arbeitszeit für Ausbildung aufgewendet werden kann. Wenn dagegen nur ein geringer Teil der Arbeitskraft/zeit für die Aus- bildung aufgewendet werden kann (z.B. Erledigung allgemeininternisti- scher Aufgaben oder Forschung und Lehre), ist davon auszugehen, dass die notwendige Ausbildungszeit länger sein kann. Journal für Gastroenterologische und Hepatologische Erkrankungen 2 · 2023 57 die verfügbaren hochspezialisierten Betreuungsmöglichkeiten haben. GEHEP auf den S. 5–7 beschrieben. Der allgemeine Grundsatz in der Aus- bildung zum/zur FA IM-GEHEP muss dabei sein, dass in dieser Phase der Ausbildung die Tiefe der Ausbildung in GEHEP nicht die Breite ersetzen darf. Es gilt zu klären, in welcher Zeit die Inhalte und Kompetenzlevel erlernt werden können. Der gegenwärtige Ausbildungsplan sieht vor, dass die Ausbildung zum/zur FA IM-GEHEP in mindestens 36 Monaten durchgeführt wird. Realistisch erscheint dies nur, wenn ein hoher Anteil der Arbeitszeit für Ausbildung aufgewendet werden kann. Wenn dagegen nur ein geringer Teil der Arbeitskraft/zeit für die Aus- bildung aufgewendet werden kann (z.B. Erledigung allgemeininternisti- scher Aufgaben oder Forschung und Lehre), ist davon auszugehen, dass die notwendige Ausbildungszeit länger sein kann. Eine weitere Verkürzung oder Verdichtung scheint für uns ausgeschlossen zu sein und ein „FA IM-GEHEP light“ muss im Sinne der uns anvertrauten Patient:innen und einer weiteren qualitativ positiven Entwicklung unseres Fachs sowie un- ter Berücksichtigung der vom EBGH koordinierten Entwicklung des Fachs Ultraschall, Funktionsdiagnostik, Be- treuung von LTX-Patient:innen, ...). Weiters muss deﬁniert werden, welche zusätzlichen Ausbildungsmodule nach der Sonderfachschwerpunktausbil- dung (SFSA) GE/HEP notwendig sein werden. Grundsätzlich muss in einem solchen Ausbildungsstadium bei einer hochspezialisierten ärztlichen Tätigkeit davon ausgegangen werden, dass es mit hoher Wahrscheinlichkeit notwen- dig oder unumgänglich sein wird, dass die Tiefe der beruﬂichen Tätigkeit die Breite der GEHEP-Kompetenz ersetzt. In weiterer Folge muss die Zuordnung von Ausbildungsinhalten und Kompetenzlevel erfolgen. Ausgehend von den im Ausbil- dungskatalog FA IM-GEHEP dargestellten Ausbildungsinhalten ist eine detaillierte Liste von Ausbildungsinhalten mit zuge- ordnetem Kompetenzlevel zu entwickeln. Dies kann angelehnt an die detaillierte Auﬂistung der Ausbildungsinhalte im vom European Board of Gastroenterology and Hepatology(EBGH)entwickelten,„training curriculum“ (Blue Book) erfolgen. 4. 1. Für die Sonderfachgrundausbildung (SFGA) innere Medizin sind im Aus- bildungskatalog FA IM GEHEP (www. aerztekammer.at/ausbildung), S. 1–4, die Inhalte beschrieben. Diese Inhalte müssen dahingehend konkretisiert werden, dass geklärt werden muss, welcher Kompetenzlevel von Kennt- nissen und Fertigkeiten aus der GEHEP in der Ausbildung zum FA IM-GEHEP grundlegend in den ersten 27 Monaten der Ausbildung vermittelt werden soll und kann. 4. Außerhalb des Aufgabenbereichs der ÖGGH-Arbeitsgruppe aber wird der Kompetenzlevel in der Ausbildung für das Sonderfach innere Medizin („all- gemeine innere Medizin“) und andere Sonderfächer der inneren Medizin (36 Monate, nach SFGA) zu berück- sichtigen sein. Es braucht hier einen sorgfältigen Diskussionsprozess, wel- che Ausbildungsinhalte mit welchem Kompetenzlevel angestrebt werden sollen. 2. Ausbildung und Forschung von den Jungen für die Jungen – die neue „young ÖGGH“ Eine weitere Verkürzung oder Verdichtung scheint für uns ausgeschlossen zu sein und ein „FA IM-GEHEP light“ muss im Sinne der uns anvertrauten Patient:innen und einer weiteren qualitativ positiven Entwicklung unseres Fachs sowie un- ter Berücksichtigung der vom EBGH koordinierten Entwicklung des Fachs Ausbildung und Forschung von den Jungen für die Jungen – die neue „young ÖGGH“ Zudem wurde durch Vorstandsbeschluss die young ÖGGH als weitere neue Arbeits- gruppe installiert, um den fachärztlichen und wissenschaftlichen Nachwuchs in un- serem Fach bestmöglich zu fördern. Neue Fortbildungsaktivitäten wie ein „Pre“- Symposium bei unserer Jahrestagung (14.–17. Juni 2023 im Congress Graz; oeg- gh.at/veranstaltungen/liste.html) für die jungen Mitglieder unserer ÖGGH sollen die angehenden Fachärzt:innen praxisori- entiert durch fallbasiertes Lernen an ihre Aufgaben heranführen und forschungs- geleitete Lehre fördern. Zudem soll dieser Kurs Orientierung und Hilfe in der Vorbe- reitung auf unsere mündlich strukturierte, fallbasierte Fachärzt:innenprüfung bieten. 3. Entwicklungs- und Fortbildungskom- petenz: FAIM-GEHEPverfügenüber die Möglichkeiten und Fähigkeiten sowie über den Willen, sich auf dem neuesten Stand des Wissens (Kenntnisse und Fer- tigkeiten) auf dem Gebiet der GEHEP zu halten, und dokumentieren diese ständige Fortbildung. 3. 4. Kompetenz für abgestufte Betreu- ung: FA IM-GEHEP können für die Betreuung bei GEHEP-Spezialproble- men ihre Patient:innen an die fachlich kompetenten (hoch-)spezialisierten Zentren zuweisen, weil sie den dafür notwendigen Überblick über den ak- tuellen Stand der diagnostischen und therapeutischen Möglichkeiten und 56 Journal für Gastroenterologische und Hepatologische Erkrankungen 2 · 2023 Tab. 1 Die Aufgabenbereiche derArbeitsgruppefürAusbildung derÖGGH Deﬁnition von Ausbildungszielen Lerninhalte, Lerntiefe und Kompetenzlevel Deﬁnition von Grundwissen und -kompetenzen für alle vs. Spezialwissen und -kompetenzen Abgleich mit internationalen Empfehlungen (Blue Book des EBGH) Curriculum Abstimmung mit verfügbarer Ausbildungszeit Gap-Analysen und Schließen von Lücken Austauschprogramme Ausbildungsverbunde Vernetzung der Ausbildungs-OÄ Vernetzung der Auszubildenden – young ÖGGH Informationsplattform für Ausbildungsprogramme internationalerFachge- sellschaften Standardisiertes Training – Evaluation des Trainings Ausbildung Train-the-Trainer-Programme (Sonographie, Endoskopie, Funktionsdiagnos- tik, ...) Praxisnähe der Facharztprüfung weiter verbessern (Endovideos, hochqualita- tive Bilder, ...) Prüfung Abgleich mit internationalen Modellen (z.B. Schweizer Modell der mehrteili- gen Prüfung) EBGH European Board of Gastroenterology and Hepatology, OÄ Oberärzt:innen, ÖGGH Österreichi- sche Gesellschaft für Gastroenterologie und Hepatologie in anderen europäischen Ländern vermieden werden. in anderen europäischen Ländern vermieden werden. . Spezialisierungen: Die Breite und Tiefe der ärztlichen Aufgaben und Tätigkei- ten im Bereich der Gastroenterologie, Hepatologie, Endoskopie, Ultraschall und assoziierter Themenbereiche bringt es mit sich, dass nicht alle dieser Bereiche im Rahmen der SFSA in der Tiefe gelernt und praktiziert werden können. Es muss geklärt werden, welche GEHEP-Kenntnisse und -Fer- tigkeiten vor allem oder nur in dafür spezialisiertenZentrenangeboten wer- den können oder sollen. Im Sinne einer für die Betreuungsqualität gewünsch- ten Spezialisierung sind umgekehrt abgestufte Versorgungskonzepte notwendig. Kontinuierliche Weiterbildung in der Gastroenterologie: Gegenwart und Zukunft DieindiesemArtikelenthaltenenBilderundsonstiges Drittmaterialunterliegenebenfallsdergenannten CreativeCommonsLizenz,sofernsichausderAbbil- dungslegendenichtsanderesergibt.Soferndasbe- treﬀendeMaterialnichtunterdergenanntenCreative CommonsLizenz stehtunddiebetreﬀendeHandlung nichtnachgesetzlichenVorschriftenerlaubtist,istfür dieobenaufgeführtenWeiterverwendungendesMa- terialsdieEinwilligungdesjeweiligenRechteinhabers einzuholen. Kontinuierliche Weiterbildung in der Gastroenterologie: Gegenwart und Zukunft Ausbildung und Forschungsausbildung wie z.B. zentralisierte praktische Kurse im Vergleich zu dezentralisierten Lehrzen- tren. Schließlich erfordert das Verhältnis zwischen Kosten und Bildungsgewinn eine kontinuierliche Überwachung und kritische Bewertung zum Erreichen eines maximalen Wirkungsgrads. Die ärztliche Fortbildung und Weiterbil- dung ist eine Verpﬂichtung, die Ärzt:innen im Besonderen ihren Patient:innen und den Kostenträgern schulden sowie dem Gesundheitswesen und der Gesellschaft im Allgemeinen zum Zweck des größ- ten Gemeinwohls. Ein allgemeines Ziel der Fortbildung ist das Schließen von Bildungs- und Kompetenzlücken (i.e. Wis- sen und Fertigkeiten). Kontinuierliche Weiterbildung muss Ärzt:innen mit den notwendigen Werkzeugen ausstatten, um den Patient:innen die bestmöglichen Gesundheitsversorgungsoptionen unter Berücksichtigung der variablen Einschrän- kungen der Gesundheitsversorgung in verschiedenen Regionen anzubieten, die Herausforderungen der Zukunft zu verste- hen und die Forschung voranzutreiben. Fortbildung muss Werkzeuge, Wissen und Verhaltensweisen zur Verfügung stel- len, die Ärzt:innen ﬁt machen, um die öﬀentliche Diskussion über zukünftige Ausrichtungen und Entwicklungen der Gesundheitsversorgung in unserem Fach im Sinne unserer Patient:innen zu steuern. g g Ein weiterer Schwerpunkt muss auf al- ternativeUnterrichtsstrategiengelegtwer- den, darunter „train the hand“, d.h. Schu- lungen vor Ort in Zentren, oder „train the brain“, d.h. Problemlösungskurse, die auf die Verbesserung der analytischen Fähig- keiten abzielen. Training und Lehre sollen den Fokus auf die „Lerntreppe“ legen, also vom „Hören“ über das „Sehen“ bis hin zum „Tun“. Wir müssen auch selbstständig und nicht erst nach Auﬀorderung einen Dis- kurs über Rezertiﬁzierung in unserem Fach beginnen. Es wird Mut, Tatkraft und Zu- versicht brauchen, die modernen Heraus- forderungen in unserem Fach zu meistern. Gemeinsam können wir es schaﬀen, also packen wir es gemeinsam an! Wir laden alle ein, die dabei mithelfen wollen. WeitereDetailszurLizenz entnehmenSiebitteder Lizenzinformationaufhttp://creativecommons.org/ licenses/by/4.0/deed.de. Hinweis des Verlags. Der Verlag bleibt in Hinblick auf geograﬁsche Zuordnungen und Gebietsbezeich- nungen in veröﬀentlichten Karten und Instituts- adressen neutral. Hinweis des Verlags. Der Verlag bleibt in Hinblick auf geograﬁsche Zuordnungen und Gebietsbezeich- nungen in veröﬀentlichten Karten und Instituts- adressen neutral. Korrespondenzadresse Ao.Univ.-Prof. Dr.med.univ. Heinz F. Hammer Klinische Abteilung für Gastroenterologie und Hepatologie, Medizinische Universitätsklinik Graz Auenbruggerplatz 15, 8036 Graz, Österreich heinz.hammer@medunigraz.at In der Zukunft wird angesichts der Ver- änderungen in den Fortbildungsansprü- chen der nachkommenden Ärzt:innen- generationen ein vermehrtes Augenmerk auf virtuelle Fortbildungsangebote gelegt werden müssen. Gleichzeitig wird auch ein verstärktes Augenmerk auf die Eﬃzi- enz der Ausbildung gelegt werden müs- sen. die verfügbaren hochspezialisierten Betreuungsmöglichkeiten haben. Für die Schwerpunktausbildung Sonderfach GEHEP (SFSA) sind die Inhalte im Ausbildungskatalog FA IM- Journal für Gastroenterologische und Hepatologische Erkrankungen 2 · 2023 57 Aus- und Fortbildung Kontinuierliche Weiterbildung in der Gastroenterologie: Gegenwart und Zukunft Die Einbindung moderner digitaler Informationsplattformen und Trainings- möglichkeiten (z.B. virtuelle Endoskopie oder Sonographie) in die Ausbildung, klinische Arbeit und Fortbildung wird Standard werden. Fortbildungsangebo- te müssen sich einer neuen Bewertung stellen. Kursbewertungen müssen da- hingehend reformiert werden, dass mehr Gewicht auf die mittelfristigen Auswir- kungen von Kursen und Ausbildungen auf Wissen, Kompetenz, Leistung, Fähig- keiten und berufliches Verhalten gelegt wird. Praktische Ausbildung und Ver- mittlung von Fähigkeiten müssen neu bewertet werden, einschließlich der Be- wertung der geeignetsten Möglichkeiten zur Bereitstellung der besten klinischen Funding. Open access funding provided by Medical University of Graz. Hinweis des Verlags. Der Verlag bleibt in Hinblick auf geograﬁsche Zuordnungen und Gebietsbezeich- nungen in veröﬀentlichten Karten und Instituts- adressen neutral. Einhaltung ethischer Richtlinien Interessenkonﬂikt. H.F.Hammer,M.Gschwantler, H.HoferundP.Fickertgebenan,dasskeinInteressen- konﬂiktbesteht. FürdiesenBeitragwurdenvondenAutor/-innen keineStudienanMenschenoderTierendurchgeführt. FürdieaufgeführtenStudiengeltendiejeweilsdort angegebenenethischenRichtlinien. Open Access.DieserArtikelwirdunterderCreative CommonsNamensnennung4.0InternationalLizenz veröﬀentlicht,welchedieNutzung,Vervielfältigung, Bearbeitung,VerbreitungundWiedergabeinjegli- chemMediumundFormaterlaubt,sofernSieden/die ursprünglichenAutor(en)unddieQuelleordnungsge- mäßnennen,einenLinkzurCreativeCommonsLizenz beifügenundangeben,obÄnderungenvorgenom- menwurden. 58 Journal für Gastroenterologische und Hepatologische Erkrankungen 2 · 2023
https://openalex.org/W2117513401	https://bmcurol.biomedcentral.com/counter/pdf/10.1186/1471-2490-6-17	English	null	Testicular infarction secondary to protein S deficiency: a case report	BMC urology	2,006	cc-by	1,936	Abstract Background: Protein S deficiency is an inherited cause of thrombophilia. We present the second reported case in the literature of a man developing testicular infarction secondary to protein S deficiency. Case presentation: A 63 year old man presented with sudden onset of pain in his left hemi- scrotum. Despite oral warfarin therapy the plasma INR was only 1.4 at presentation. Doppler ultrasound scan of the scrotum confirmed absent blood flow to the left testis with increased echogenicity. Orchidectomy was performed to remove the necrotic testis. Post-operatively the patient did well and was referred to the Regional Haemophilia and Thrombosis Centre for further management. Conclusion: The case highlights a rare but potential complication of protein S deficiency and demonstrates the importance of adequate anticoagulation in these patients. BioMed Central BioMed Central BioMed Central Received: 01 August 2005 Accepted: 07 July 2006 © 2006 McKay 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. Page 1 of 3 (page number not for citation purposes) p Testicular infarction secondary to protein S deficiency: a case report ddress: 1The Department of Surgery, Daisy Hill Hospital, Newry, Co Down, BT35 8DR, N. Ireland, UK and 2C/O The nstitute of Clinical Science, The Royal Victoria Hospital, Belfast, BT12 6BA, N. Ireland, UK Email: Damian McKay* - dam.mckay@virgin.net; Conor Marron - cdmarron@dsl.pipex.com; Robin Brown - robin@robinandjoan.co.uk * Corresponding author * Corresponding author Published: 07 July 2006 BMC Urology 2006, 6:17 doi:10.1186/1471-2490-6-17 Received: 01 August 2005 Accepted: 07 July 2006 This article is available from: http://www.biomedcentral.com/1471-2490/6/17 © 2006 McKay 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. Received: 01 August 2005 Accepted: 07 July 2006 Op Case report Testicular infarction secondary to protein S deficiency: a case report Damian McKay*1,2, Conor Marron1 and Robin Brown1 Open Access Discussion We believe this to be only the second such case of testicu- lar infarction secondary to protein S deficiency reported in the world literature [1]. http://www.biomedcentral.com/1471-2490/6/17 http://www.biomedcentral.com/1471-2490/6/17 daily. The patient also self administered 1 mg/kg of enox- aparin since diagnosed in 1993. The patient had neither previous cardiovascular history nor clinical evidence of atrial fibrillation. occurred despite subcutaneous enoxaparin injection. It may be that patient compliance with both oral warfarin and subcutaneous enoxaparin was poor. Warfarin- induced skin necrosis may occur in these patients on ini- tiation of warfarin therapy. This reflects a reduction in the level of protein S before the level of prothrombin is low- ered into the therapeutic range. In this condition pro- thrombin levels stimulate the formation of venous thrombi by an unclear mechanism. These patients usually require a change to heparin therapy [3,4]. Ultrasonography of the left testis demonstrated increased echogenicity around the head of the epididymus in partic- ular, suggesting haematoma. The echogenicity of the testis was abnormal and no blood flow was demonstrated on Doppler interrogation. At surgery a left groin incision was made with the cord was clamped near the internal ring. The testis and epididymus were haemorrhagic and necrotic and there was no evidence of torsion. Protein S deficiency has been associated with arterial as well as venous occlusion [5,6] and it is now suggested to be an independent risk factor for peripheral arterial insuf- ficiency [6]. Histological examination revealed a necrotic testis. The blood vessels were congested with evidence of thrombus. No obvious emboli were present. There was no evidence of carcinoma. Post surgery the patient recovered well and was commenced on clopidogrel 75 mg and enoxaparin 100 mg daily. He was referred to the Regional Haemo- philia and Thrombosis Centre for further management. He has since been reviewed and is well. Fluctuations in protein S levels are also common includ- ing in healthy members of the population [7]. It may be that individuals are more likely to have a thrombotic event during a dip in protein S levels. Other conditions, related to thrombophilia, that have been reported as aeti- ological factors in cases of testicular infarction are sickle cell disease, polycythemia, trauma and epididymo-orchi- tis [2,8,9]. Learning points 1. Testicular infarction is a rare complication of inade- quate anticoagulation in patients with pro-thrombotic conditions. Testicular infarction secondary to Protein S deficiency is exceedingly rare. The patient presents with symptoms of testicular pain. On examination, the scrotum may be red, swollen and tender and may mimic the clinical examina- tion of a patient with testicular torsion. The pathophysiol- ogy of this clinical picture is congestion and thrombosis of the venous and arterial blood supply to the testis. Infarc- tion of other organs such as brain, heart, spleen and more commonly recognised as sequelae of the condition. 2. Ultrasound scanning is a useful modality in confirming the diagnosis in these patients. 3. A dilemma arises in the case of a young man with thrombilia and possible testicular torsion. The history and ultrasound findings could be identical but if the aetiology was due to thrombosis would systemic anticoagulation with low molecular weight heparins be more suitable than surgery? And how would one differentiate between the two? Protein S deficiencies are associated with superficial and deep vein thrombosis and pulmonary embolism [2]. Pro- tein S deficiency may be hereditary or acquired. The acquired state is usually due to hepatic diseases or a vita- min K deficiency. Congenital protein S deficiency is an autosomal dominant disease, and the heterozygous state occurs in approximately 2% of unselected patients with venous thromboembolism. 4. Testicular infarction due to causes other than torsion or trauma is very rare. It would be useful to screen these patients for Protein S deficiency and other pro-clotting conditions. Background g Protein S deficiency is a rare blood disorder which is a risk factor for thrombophilia. It affects around 1 in 20000 of the population [1]. It may be hereditary or acquired. Hereditary protein s deficiency is divided into three types: Type 1, Type 2a and Type 2b. p A 63 year old man presented to his General Practitioner with a history of left sided testicular pain for 24 hours. The pain had increased with time. He was referred to the Acci- dent and Emergency Department of our hospital where he was examined and an ultrasound scan arranged. He had no urinary symptoms. He was currently under the care of the haematology department with protein S deficiency. He had a history of DVT and PE in 1965 and received anti- coagulation therapy with warfarin for a year. The diagno- sis of protein S deficiency was made in 1993, after investigation for recurrent leg ulceration, and therapy with warfarin and subsequently low molecular weight heparin commenced. At presentation his International Normalised Ratio (INR) was sub-therapeutic at 1.4, despite daily oral administration of 6 mg of Warfarin The most common cause of acquired protein S deficiency is pregnancy. Acquired protein S deficiency may be seen in some patients who have developed disseminated intravas- cular coagulation (DIC), deep vein thrombosis (DVT) or pulmonary embolism (PE). The treatment of protein s deficiency is systemic anticoagulation with warfarin or a similar agent. Testicular infarction is a rare but recognised complication of thrombophilia. Page 1 of 3 (page number not for citation purposes) Page 1 of 3 (page number not for citation purposes) BMC Urology 2006, 6:17 http://www.biomedcentral.com/1471-2490/6/17 References 1. Lee YI, Huang CN, Huang CH: Testicular infarction associated with protein S deficiency. J Urol 2001, 165:1220-1221. 1. Lee YI, Huang CN, Huang CH: Testicular infarction associated with protein S deficiency. J Urol 2001, 165:1220-1221. p y J 2. Medline Plus: [http://www.nlm.nih.gov/medlineplus/ency/article/ 000559.htm]. p y J 2. Medline Plus: [http://www.nlm.nih.gov/medlineplus/ency/article/ 000559.htm]. 3. Lee YI, Huang CN, Huang CH: Testicular infarction associated with protein S deficiency. J Urol 2001, 165:1220-1221. 3. Lee YI, Huang CN, Huang CH: Testicular infarction associated with protein S deficiency. J Urol 2001, 165:1220-1221. 4. Horn JR, Danziger LH, Davis RJ: Warfarin Induced Skin Necrosis: report of four cases. American Journal of Hospital Pharmacy 1981, 38(11):1763-1768. ( ) 5. Yang Y, Algazy KM: Warfarin Induced Necrosis in a patient with a mutation of the prothrombin gene. NEJM 1999, 340:735. 6. Greiner K, Hafner G, Dick B, Peetz D, Prellwitz W, Pfeiffer N: Reti- nal vascular occlusion and deficiencies in the protein C path- way. Am J Ophthalmol 1999, 128(1):69-74. y J p ( ) 7. Cho YP, Kwon TW, Ahn JH, Kang GH, Han MS, Kim YH, Kwak JH, Lee SG: Protein C and/or S deficiency presenting as periph- eral arterial insufficiency. Br J Radiol 2005, 78(931):601-5. 8. Undar L, Ertugrul C, Altunbas H, Akca S: Circadian variations in natural coagulation inhibitors protein C, protein S and anti- thrombin in healthy men: a possible association with inter- leukin-6. Thromb Haemost 1999, 81(4):571-5. ( ) 9. Gofrit ON, Rund D, Shapiro A, Pappo O, Landau EH, Pode D: Seg- mental testicular infarction due to sickle cell disease. J Urol 1998, 160:835-836. 10. Urwin GH, Kehoe N, Dundas S, Fox M: Testicular infarction in a patient with sickle cell trait. Br J Urol 1986, 58:340-341. Competing interests The author(s) declare that they have no competing inter- ests. Abbreviations INR – International Normalised Ratio. The management of Protein S deficiency involves initial heparinisation at the time of the initial presentation fol- lowed by commencement on warfarin. In this case the patient's INR was sub therapeutic at 1.4. Levels greater than 2.0 are required to provide adequate anticoagula- tion. It is surprising that his INR was low as he attended the haematology clinic every fortnight for monitoring of his INR. What is more surprising is that this event DIC – Disseminated Intra-vascular Coagulation. DIC – Disseminated Intra-vascular Coagulation. DIC – Disseminated Intra-vascular Coagulation. DVT – Deep Vein Thrombosis. DVT – Deep Vein Thrombosis. P.E – Pulmonary Embolism. P.E – Pulmonary Embolism. Page 2 of 3 (page number not for citation purposes) BMC Urology 2006, 6:17 http://www.biomedcentral.com/1471-2490/6/17 Authors' contributions Damian McKay and Conor Marron reviewed the case notes, carried out the literature review and prepared the initial manuscript. Mr R Brown was the consultant sur- geon looking after the patient and identified the case for publication and reviewed the manuscript prior to submis- sion. Acknowledgements Written consent was obtained from the patient for publication of the case report. Pre-publication history The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2490/6/17/prepub 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 3 of 3 (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 Publish with BioMed Central and every scientist can read your work free of charge
https://openalex.org/W3041441913	https://www.cambridge.org/core/services/aop-cambridge-core/content/view/D69C5AE92341D14A6F3F500B43E0B216/S193578932000230Xa.pdf/div-class-title-extensively-drug-resistant-tuberculosis-in-the-time-of-covid-19-how-has-the-landscape-changed-for-pakistan-div.pdf	English	null	Extensively Drug-Resistant Tuberculosis in the Time of COVID-19—How has the Landscape Changed for Pakistan?	Disaster medicine and public health preparedness	2,020	cc-by	967	ABSTRACT The incidence of extensively drug-resistant tuberculosis (XDR-TB) has been rising consistently in Pakistan, and the country is likely to experience another surge of cases in the midst of the COVID-19 crisis. It is imperative to consider how the rising proportion of XDR-TB is best tackled during the pandemic; this includes finding a solution to the problem of non-adherence at the level of community-based healthcare, the utility and practicality of simultaneous testing for COVID-19 and TB, and reconciliation of the World Health Organization’s recommendation of home-based treatment with the need for frequent monitoring of anti-tubercular therapy in XDR-TB. Operational research is needed expeditiously to bypass these limitations. Keywords: COVID-19, tuberculosis, XDR-TB Keywords: COVID-19, tuberculosis, XDR-TB T d T uberculosis (TB) has established itself as the world’s leading infectious-disease killer, caus- ing approximately 1.5 million deaths annually around the globe. Notably, a disproportionate 95% of these deaths are restricted to middle-income/ low-income countries, one of which is Pakistan. Ongoing efforts to control the spread of TB, although consistent, have been hindered by a lack of patient awareness, unemployment or underpaid labor resulting in financial constraints, and the social stigma of tuber- culosis itself, all of which collectively discourage patients from adhering to treatment.1 This inadequate/ incomplete treatment is thought to give rise to exten- sively drug-resistant forms of tuberculosis (XDR-TB), the incidence of which has been periodically rising each year.2 resistance to take root in the gaps of incomplete therapy. At the institutional level, the information note by the World Health Organization (WHO) has raised certain concerns regarding how healthcare facilities may alter their approach to TB in the COVID-19 crisis.4 The fifth recommendation of the note addresses the utility of simultaneous testing for both conditions, wherein WHO suggests an individualized patient assessment based on prognostic factors, history of exposure, and local burden of disease. Generally, investigating TB in COVID-19 patients after a week of observation for alarming symptoms (or earlier, for patients with co-morbidities) is reasonable; however, in Pakistan, where national TB burden is high and rising despite control efforts, ruling out concomitant TB in patients confirmed to have COVID-19 may be doubly important to prevent TB transmission to other patients in COVID-19 isolation wards. The questions arises, would it be advisable to immediately test for TB (including latent) even in patients without co-morbidities, if they report risk factors? LETTER TO THE EDITOR LETTER TO THE EDITOR Disaster Medicine and Public Health Preparedness e9 Copyright © 2020 Society for Disaster Medicine and Public Health, Inc. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. DOI: 10.1017/dmp.2020.230 tps://doi.org/10.1017/dmp.2020.230 Published online by Cambridge University Press Extensively Drug-Resistant Tuberculosis in the Time of COVID-19—How has the Landscape Changed for Pakistan? Mahnoor Islam REFERENCES To combat these problems; community-based health care must be optimized, with emphasis on structured counseling and family support. Operational research is needed expeditiously to find ways to bypass limitations. Furthermore, government-level action to prevent export bans and stockpiling may help to maintain a continuous flow of healthcare resources, especially with the aid of international partners for resource generation. 1. Soomro MH, Qadeer E, Mørkve O. Barriers in the management of tuberculosis in Rawalpindi, Pakistan: a qualitative study. Tanaffos. 2013; 12(4):28-34. 2. Hasan R, Jabeen K, Ali A, et al. Extensively drug-resistant tuberculosis, Pakistan. Emerg Infect Dis. 2010;16(9):1473-1475. 3. Stop TB Partnership. The potential impact of the COVID-19 response on tuberculosis in high-burden countries: a modelling analysis. http:// www.stoptb.org/assets/documents/news/Modeling%20Report_1%20May% 202020_FINAL.pdf. Accessed May 25, 2020. 4. World Health Organization (WHO). Information Note. COVID-19: considerations for tuberculosis (TB) care. https://www.who.int/docs/ default-source/documents/tuberculosis/infonote-tb-covid-19.pdf. Accessed May 25, 2020. 4. World Health Organization (WHO). Information Note. COVID-19: considerations for tuberculosis (TB) care. https://www.who.int/docs/ default-source/documents/tuberculosis/infonote-tb-covid-19.pdf. Accessed May 25, 2020. Extensively drug-resistant tuberculosis in the time of COVID-19 Correspondence and reprint requests to: Mahnoor Islam, Apartment 102, Crystal View, 62/3, Dr. Mehmood Hussain Road, D.C.H.S., Karachi, Pakistan. (e-mail: mahnoorislam95@gmail.com). of surveillance to ensure adherence and safety. While Web-based methods have been suggested for this purpose, they may not be entirely practical in low socioeconomic areas of Pakistan (which contribute the most to TB prevalence) due to a scarcity of digital resources. Correspondence and reprint requests to: Mahnoor Islam, Apartment 102, Crystal View, 62/3, Dr. Mehmood Hussain Road, D.C.H.S., Karachi, Pakistan. (e-mail: mahnoorislam95@gmail.com). ABSTRACT A recent report by the Stop TB Partnership suggested that efforts against TB may be set back even further during the coronavirus disease 2019 (COVID-19) cri- sis, with 6.3 million new cases and 1.4 million deaths expected worldwide between now and 20253; for Pakistan, it is imperative to consider how the rising proportion of XDR-TB may require a unique approach to this situation. For one, the stigma of respiratory symptoms faced by patients with TB is now com- pounded by the added threat of COVID-19, making presentation/follow-up to healthcare professionals even less likely and providing opportunity for drug Second, WHO recommends home-based TB treatment during this time; however, the treatment of XDR-TB is often associated with considerable toxicity and requires frequent evaluations over several months. In these patients, it is imperative to develop home-based models e9 Disaster Medicine and Public Health Preparedness e9 Copyright © 2020 Society for Disaster Medicine and Public Health, Inc. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. DOI: 10.1017/dmp.2020.230 https://doi.org/10.1017/dmp.2020.230 Published online by Cambridge University Press https://doi.org/10.1017/dmp.2020.230 Published online by Cambridge University Press About the Author Dow University of Health Sciences, Karachi, Pakistan. Dow University of Health Sciences, Karachi, Pakistan. VOL. 14/NO. 4 e10 Disaster Medicine and Public Health Preparedness https://doi.org/10.1017/dmp.2020.230 Published online by Cambridge University Press
https://openalex.org/W2064192968	https://ijpam.eu/contents/2014-96-2/6/6.pdf	English	null	ANALYTIC SOLUTION OF A NONLINEAR BLACK-SCHOLES EQUATION WITH PRICE SLIPPAGE	International Journal of Pure and Applied Mathematics	2,014	cc-by	2,070	PA ijpam.eu PA ijpam.eu ISSN: 1311-8080 (printed version); ISSN: 1314-3395 (on-line version) url: http://www.ijpam.eu doi: http://dx.doi.org/10.12732/ijpam.v96i2.6 ANALYTIC SOLUTION OF A NONLINEAR BLACK-SCHOLES EQUATION WITH PRICE SLIPPAGE Joseph Eyang’an Esekon Department of Statistics and Actuarial Science Maseno University P.O. Box 333-40105, Maseno, KENYA Abstract: We study a nonlinear Black-Scholes partial diﬀerential equation whose nonlinearity is as a result of transaction cost and a price slippage impact that lead to market illiquidity with feedback eﬀects. After reducing the equation into a second-order nonlinear partial diﬀerential equation, we ﬁnd that the assumption of a traveling wave proﬁle to the second-order equation reduces it further to ordinary diﬀerential equations. Solutions to all these transformed equations facilitate an analytic solution to the nonlinear Black-Scholes equation. We ﬁnally show that the option is always more volatile compared to the stock when 1∓√ 1−(1−α)2 (1−α)2 < S0 S ert. AMS Subject Classiﬁcation: 35A09, 35A20, 62P05 AMS Subject Classiﬁcation: 35A09, 35A20, 62P05 Key Words: analytic solution, feedback eﬀects, illiquid markets, transaction cost price slippage j 35 09, 35 0, 6 05 Key Words: analytic solution, feedback eﬀects, illiquid markets, transaction cost, price slippage c⃝2014 Academic Publications, Ltd. url: www.acadpubl.eu 1. Introduction Two primary assumptions are used in formulating classical arbitrage pricing theory: frictionless and competitive markets. There are no transaction costs and restrictions on trade in a frictionless market. In a competitive market, a trader can buy or sell any quantity of a security without changing its price. c⃝2014 Academic Publications, Ltd. url: www.acadpubl.eu c⃝2014 Academic Publications, Ltd. url: www.acadpubl.eu Received: April 15, 2014 230 J.E. Esekon Restrictions on trade are imposed when we have extreme market conditions. In particular, purchases/short sales are not permitted when the market has a surplus/shortage. The notion of liquidity risk is introduced on relaxing the assumptions above. The purpose of this paper is to obtain an analytic solution of the nonlin- ear Black-Scholes equation arising from the combination of transaction cost and a price slippage impact by Bakstein and Howison in [1]. This is done by substitutions and transformations, which give a second-order nonlinear partial diﬀerential equation. Assuming a traveling wave solution to the second-order equation reduces it further to ordinary diﬀerential equations (ODEs). All these transformed equations are solved to obtain an analytic solution to the nonlinear Black-Scholes equation. This paper is organized as follows. Section 2 describes the nonlinear Black- Scholes PDE used for modelling illiquid markets with a price slippage impact. The smooth solution to the nonlinear Black-Scholes equation is presented in Section 3. Section 4 concludes the paper. ANALYTIC SOLUTION OF A NONLINEAR... 231 u(S, t) ∼S −Ke−r(T−t) as S →∞. We take the last condition to mean that lim S→∞ u(S,t) S−Ke−r(T −t) = 1 uniformly for 0 ≤t ≤T with the constraint u(S, t) ≥0. uniformly for 0 ≤t ≤T with the constraint u(S, t) ≥0. Liquidity in (2.1) has been deﬁned through a combination of transaction cost and a price slippage impact. Due to ρ, bid-ask spreads dominate the price elasticity eﬀect. When α = 1, this corresponds to no slippage and equation (2.1) reduces to the PDE given by ut + 1 2σ2S2uSS(1 + 2ρSuSS) + rSuS −ru = 0. (2.2) (2.2) The solution to equation (2.2) is found in Theorem 3.0.2 of [3] for r > 0, and in Theorem 3.2 of [4] and Theorem 4.1 of [5] for r = 0. The magnitude of the market impact is determined by ρS. Large ρ implies a big market impact of hedging. If ρ = 0, the asset’s price in equation (2.1) follows the standard Black-Scholes model in [2] with constant volatility σ. 2. Bakstein and Howison (2003) Equation In this section, we will consider the continuous-time feedback eﬀects equation for illiquid markets by Bakstein and Howison in [1]. Two assets are used in the model: a bond (or a risk-free money market account with spot rate of interest r ≥0) whose value at time t is Bt ≡1, and a stock. The stock is assumed to be risky and illiquid while the bond is assumed to be riskless and liquid. This equation (see Theorem 3.1 of [1]) is given by ut + 1 2σ2S2uSS(1 + 2ρSuSS) + 1 2ρ2(1 −α)2σ2S4u3 SS + rSuS −ru = 0, (2.1) (2.1) where S is the price of the stock, ρ ≥0 is a measure of the liquidity of the market, σ is volatility, u(S, t) is the option price and α is a measure of the price slippage impact of a trade felt by all participants of a market (see Theorem 3.1 of [1]). For instance the terminal condition for a European call option is given by h(ST ) = u(S, T) = max {S −K, 0} for S ≥0, where K > 0 is the striking price and h(ST ) is a terminal claim whose hedge cost, u(St, t), is the solution to (2.1). The boundary conditions for the option are as follows: where K > 0 is the striking price and h(ST ) is a terminal claim whose hedge cost, u(St, t), is the solution to (2.1). The boundary conditions for the option are as follows: u(0, t) = 0 for 0 ≤t ≤T, ANALYTIC SOLUTION OF A NONLINEAR... 3. Smooth Solution to the Bakstein and Howison (2003) Equation Lemma 3.1. If ν(ξ) is a twice continuously diﬀerentiable function, and x and t are the spatial and time variables respectively, then there exists a traveling wave solution to the equation, Vt + 1 2σ2(Vxx +Vx) (1 + 2(Vxx + Vx)) + 1 2(1−α)2σ2(Vxx +Vx)3 +rVx = 0 (3.1) in R × (0 ∞) of the form Vt + 1 2σ2(Vxx +Vx) (1 + 2(Vxx + Vx)) + 1 2(1−α)2σ2(Vxx +Vx)3 +rVx = 0 in R × (0, ∞) of the form V (x, t) = ν(ξ) where ξ = x −ct, ξ ∈R (3.2) (3.2) for 0 ≤α < 1, 1 < α ≤2, r, σ, t > 0 and x ∈R such that V (x, t) is a traveling wave of permanent form which translates to the right with constant speed c > 0. for 0 ≤α < 1, 1 < α ≤2, r, σ, t > 0 and x ∈R such that V (x, t) is a traveling wave of permanent form which translates to the right with constant speed c > 0. Proof. Applying the chain rule to (3.2) gives Vt = −cν′(ξ), Vx = ν′(ξ), and Vxx = ν′′(ξ), where the prime denotes d dξ. Substituting these expressions into (3.1), we con- clude that ν(ξ) must satisfy the nonlinear second order ODE where the prime denotes d dξ. Substituting these expressions into (3.1), we con- clude that ν(ξ) must satisfy the nonlinear second order ODE −cν′ + 1 2σ2(ν′′ + ν′) 1 + 2(ν′′ + ν′) + 1 2(1 −α)2σ2(ν′′ + ν′)3 + rν′ = 0 (3.3) 232 J.E. Esekon in R and hence V (x, t) solves (3.1). By setting c = r, the equation resulting from (3.3) can now be solved in a closed-form by ﬁrst writing it as (1 −α)2(ν′′ + ν′)2 + 2(ν′′ + ν′) + 1 = 0 in R, where (1 −α)2 ̸= 0. The quadratic equation above is solved to get The quadratic equation above is solved to get ν′′ + ν′ = −1±√ 1−(1−α)2 (1−α)2 , 0 ≤α < 1, 1 < α ≤2. Upon integration we get the variable separable standard form (see [6]) Upon integration we get the variable separable standard form (see [6]) ν′ = eξ0−ξ + −1±√ 1−(1−α)2 (1−α)2 , 0 ≤α < 1, 1 < α ≤2, ξ0, ξ ∈R, where ξ0 is a constant of integration. 3. Smooth Solution to the Bakstein and Howison (2003) Equation This is the ﬁrst order linear autonomous and separable ODE whose solution upon integration is given by where ξ0 is a constant of integration. This is the ﬁrst order linear autonomous and separable ODE whose solution upon integration is given by ν(ξ) = −eξ0−ξ + −1±√ 1−(1−α)2 (1−α)2 ξ + δ for 0 ≤α < 1, 1 < α ≤2, ξ0, ξ ∈R, where δ is another constant of integration. Applying the initial condition for 0 ≤α < 1, 1 < α ≤2, ξ0, ξ ∈R, where δ is another constant of integration. Applying the initial condition ν(0) = 0 ν(0) = 0 to the equation above gives to the equation above gives δ = eξ0. δ = eξ0. Hence Hence ν(ξ) = −eξ0−ξ + −1±√ 1−(1−α)2 (1−α)2 ξ + eξ0 ( ) for 0 ≤α < 1, 1 < α ≤2, ξ0, ξ ∈R. Substitution gives for 0 ≤α < 1, 1 < α ≤2, ξ0, ξ ∈R. Substitution gives V (x, t) = −ex0−(x−rt) + −1±√ 1−(1−α)2 (1−α)2 (x −rt) + ex0, (3.4) for 0 ≤α < 1, 1 < α ≤2, x0, x ∈R, r > 0, t ≥0 since ξ0 = x0 −c · 0 = x0 and c = r. V (x, t) = −ex0−(x−rt) + −1±√ 1−(1−α)2 (1−α)2 (x −rt) + ex0, (3.4) for 0 ≤α < 1, 1 < α ≤2, x0, x ∈R, r > 0, t ≥0 since ξ0 = x0 −c · 0 = x0 and c = r. (3.4) Theorem 3.2. If V (x, t) is any positive solution to the nonlinear equation Vt + 1 2σ2(Vxx + Vx) (1 + 2(Vxx + Vx)) + 1 2(1 −α)2σ2(Vxx + Vx)3 + rVx = 0 ANALYTIC SOLUTION OF A NONLINEAR... ANALYTIC SOLUTION OF A NONLINEAR... 233 in R × [0, ∞) then in R × [0, ∞) then in R × [0, ∞) then in R × [0, ∞) then u(S, t) = 1 ρ −1±√ 1−(1−α)2 (1−α)2 ln S K −rt + S0 K S −S0ert (3.5) (3.5) in R × [0, ∞) solves the nonlinear Black-Scholes equation ut + 1 2σ2S2uSS(1 + 2ρSuSS) + 1 2ρ2(1 −α)2σ2S4u3 SS + rSuS −ru = 0 (3.6) for r, K, S, S0, σ, ρ > 0, t ≥0, 0 ≤α < 1, and 1 < α ≤2, where S0 is the initial stock price. (3.6) Proof. To obtain the solution to equation (3.6) we apply the transforma- tions x = ln S K and V (x, t) = ρ u(S,t) Kex to get ut = S ρ Vt, uS = 1 ρ(Vx + V ), uSS = 1 ρS (Vxx + Vx) . Substituting these expressions into (3.6) gives (3.1). Hence, applying the trans- formations above into (3.4) gives (3.5). Remark 3.3. From equation (3.5), it is clear that when 1∓√ 1−(1−α)2 (1−α)2 < S0 S ert then SuS u is always greater than one as in [2, 3, 4]. This shows that the option is always more volatile than the stock when this inequality holds. 4. Conclusion We have studied the hedging of derivatives in the presence of transaction cost and a price slippage impact that lead to market illiquidity with feedback eﬀects. Assuming the solution of a forward wave, a classical solution for the nonlinear Black-Scholes equation was found. The solution obtained can be used for pricing a European call option at time t ≥0. It is clear that when 1∓√ 1−(1−α)2 (1−α)2 < S0 S ert this solution supports the comments in [2, 3, 4] that the option is always more volatile than the stock. In conclusion, further research needs to be done to ﬁnd out the solution to the equation when c ̸= r. Another part of our interest is the use of the put-call-parity to obtain the solution of a put option. 234 J.E. Esekon References [1] D. Bakstein, S. Howison, A non-arbitrage liquidity model with observable parameters for derivatives, Working paper, (2003). [2] F. Black, M. Scholes, The pricing of options and corporate liabilities, The Journal of Political Economy, 81, No. 3 (1973), 637-654. [3] J. E. Esekon, Analytic solution of a nonlinear Black-Scholes equation, In- ternational Journal of Pure and Applied Mathematics, 82, No. 4 (2013), 547-555. [4] J. E. Esekon, A particular solution of a nonlinear Black-Scholes partial dif- ferential equation, International Journal of Pure and Applied Mathematics, 81, No. 5 (2012), 715-721. [5] J. Esekon, S. Onyango, N.O. Ongati, Analytic solution of a nonlinear Black-Scholes partial diﬀerential equation, International Journal of Pure and Applied Mathematics, 61, No. 2 (2010), 219-230. [6] J. D. Logan, An Introduction to Nonlinear Partial Diﬀerential Equations, Second Edition, John Wiley and Sons, Inc., Hoboken, New Jersey, USA (2008).
https://openalex.org/W4296213143	https://online.unisc.br/seer/index.php/psi/article/download/17330/10576	Portuguese	null	“Estamos sendo submetidos a uma escravidão disfarçada”: análise de relatos de motoristas de aplicativo à luz da Psicodinâmica do Trabalho	Psi Unisc	2,022	cc-by	9,495	Resumo Resumo O estudo teve por objetivo investigar e compreender as vivências de prazer e sofrimento, e as estratégias de mediação no trabalho a partir de publicações feitas por motoristas de aplicativos em um grupo numa rede social entre dezembro de 2019 e fevereiro de 2020. Foram selecionadas 282 laudas que passaram por uma análise temática e foram definidas em três categorias sob a luz da abordagem da Psicodinâmica do Trabalho: 1) as características do trabalho de motoristas por aplicativos; 2) as vivências de prazer e sofrimento no trabalho; 3) estratégias de mediação elaboradas pelos motoristas por aplicativos. O extrato das publicações realizadas por motoristas permitiu evidenciar que as inserções nas plataformas digitais surgem como alternativa ao desemprego ou complemento de renda. Os trabalhadores indicaram o reconhecimento dos passageiros e a gratificação como suas vivências de prazer. As vivências de sofrimento apontaram para características de servidão do trabalho e descontentamento, por razão da submissão por critérios de avaliação de desempenho, controle e vigilância pelos algoritmos digitais, e a imposição dos trabalhadores à uma intensa produtividade, longas jornadas de trabalho, baixa remuneração e falta de segurança. Foi possível notar estratégias de enfrentamento como a criação de clientelas particulares para aumentar seus rendimentos, a adoção de recursos tecnológicos para cuidados entre si, cooperação e compartilhamento de informações no ambiente virtual. Palavras-chaves: Saúde do Trabalhador; Condições de trabalho; Tecnologia; Subemprego. ISSN: 2527-1288 Recebido em: 14/02/2022 Aceito em: 03/07/2022 Como citar: Santos, J. J., Kerber, G. B. & Rissi, V. (2022). “Estamos sendo submetidos a uma escravidão disfarçada”: análise de relatos de motoristas de aplicativo à luz da Psicodinâmica do Trabalho. PSI UNISC, 6(2), 107- 123. doi: 10.17058/psiunisc.v6i2.17330 “Estamos sendo submetidos a uma escravidão disfarçada”: análise de relatos de motoristas de aplicativo à luz da Psicodinâmica do Trabalho “Estamos siendo sometidos a uma esclavitud disfrazada”: análisis de informes de conductores de aplicaciones a la luz de la Psicodinámica del Trabajo “We are being subjected to a disguised slavery”: analysis of app driver reports in the light of the Psychodynamics of Work Jefferson João dos Santos Faculdade Meridional (IMED), - RS/Brasil ORCID: 0000-0002-2025-7685 E-mail: jeffe.j.s@unochapeco.edu.br Gabriel Bacarol Kerber Faculdade Meridional (IMED), - RS/Brasil ORCID: 0000-0002-5091-1764 E-mail: gabrielkerber@outlook.com Vanessa Rissi Faculdade Meridional (IMED), - RS/Brasil ORCID: 0000-0002-1895-9354 E-mail: vanessa.rissi@imed.edu.com Resumo O estudo teve por objetivo investigar e compreender as vivências de prazer e sofrimento, e as estratégias de mediação no trabalho a partir de publicações feitas por motoristas de aplicativos em um grupo numa rede social entre dezembro de 2019 e fevereiro de 2020. Foram selecionadas 282 laudas que passaram por uma análise temática e foram definidas em três categorias sob a luz da abordagem da Psicodinâmica do Trabalho: 1) as características do trabalho de motoristas por aplicativos; 2) as vivências de prazer e sofrimento no trabalho; 3) estratégias de mediação elaboradas pelos motoristas por aplicativos O extrato das publicações realizadas por motoristas permitiu evidenciar que as inserções nas plataformas Jefferson João dos Santos Faculdade Meridional (IMED), - RS/Brasil ORCID: 0000-0002-2025-7685 E-mail: jeffe.j.s@unochapeco.edu.br Gabriel Bacarol Kerber Faculdade Meridional (IMED), - RS/Brasil ORCID: 0000-0002-5091-1764 E-mail: gabrielkerber@outlook.com Gabriel Bacarol Kerber Faculdade Meridional (IMED), - RS/Brasil ORCID: 0000-0002-5091-1764 E-mail: gabrielkerber@outlook.com Vanessa Rissi Faculdade Meridional (IMED), - RS/Brasil ORCID: 0000-0002-1895-9354 E-mail: vanessa.rissi@imed.edu.com Resumen 7 de 2020. Se seleccionaron 282 páginas, que se sometieron a un análisis temático. y se definieron en tres categorías a la luz del enfoque de la Psicodinámica del Trabajo: 1) las características del trabajo de los conductores a través de apps; 2) las experiencias de placer y sufrimiento en el trabajo; 3) estrategias de El estudio tuvo como objetivo investigar y comprender las experiencias de placer y sufrimiento, y las estrategias de mediación en el trabajo a partir de las publicaciones realizadas por los conductores de aplicaciones en un grupo en una red social entre diciembre de 2019 y febrero 107 PSI UNISC, 6(1), 107-127, 2022. “Estamos sendo submetidos a uma escravidão disfarçada”: análise de relatos de motoristas de aplicativo à luz da Psicodinâmica do Trabalho in a group on a social network between December 2019 and February 2020. 282 pages were selected. which underwent a thematic analysis and were defined in three categories in the light of the Psychodynamics of Work approach: 1) the characteristics of the work of drivers through apps; 2) the experiences of pleasure and suffering at work; 3) mediation strategies developed by drivers through apps. The extract of publications made by drivers made it possible to show that insertions in digital platforms appear as an alternative to unemployment or income supplement. Workers indicated passenger recognition and gratification as their experiences of pleasure. The experiences of suffering pointed to characteristics of work servitude and discontent, due to the submission to performance evaluation criteria, control and surveillance by digital algorithms, and the imposition of workers to intense productivity, long working hours, low remuneration and lack of security. It was possible to notice coping strategies such as the creation of private clientele to increase their income, the adoption of technological resources to care for each other, cooperation and information sharing in the virtual environment. mediación desarrolladas por los conductores a través de aplicaciones. El extracto de las publicaciones realizadas por los conductores permitió evidenciar que las inserciones en las plataformas digitales aparecen como una alternativa al desempleo o al complemento de ingresos. Los trabajadores indicaron el reconocimiento y la gratificación de los pasajeros como sus experiencias de placer. Abstract Keywords: Occupational health; Working conditions; Technology; Underemployment. The study aimed to investigate and understand the experiences of pleasure and suffering, and the mediation strategies at work from publications made by app drivers A flexibilização de jornada, de contratos, dos salários e direitos, requer flexibilizar trabalhadores. Flexibilidade é uma palavra de ordem na época da mundialização e faz parte de mudanças morfológicas do trabalho e produção na era do capitalismo global (Roso, 2017). Resumen As vivências de sofrimento apontaram para características de servidão do trabalho e descontentamento, por razão da submissão por critérios de avaliação de desempenho, controle e vigilância pelos algoritmos digitais, e a imposição dos trabalhadores à uma intensa produtividade, longas jornadas de trabalho, baixa remuneração e falta de seguridad. Fue posible notar estrategias de enfrentamiento como la creación de clientela privada para aumentar sus ingresos, la adopción de recursos tecnológicos para el cuidado mutuo, la cooperación y el intercambio de información en el entorno virtual. Palabras clave: Salud laboral; Condiciones de trabajo; Tecnología; Subempleo. PSI UNISC, 6(2), 107-123, 2022. Introdução O atual cenário mundial das relações trabalhistas é constituído por sujeitos que dependem exclusivamente do trabalho para sobreviver. Nos últimos anos, o que se percebe é uma redução das formas tradicionais de empregos e trabalhos regularizados característicos do século XX. Aqueles que não amarguram o desemprego vivenciam diariamente cada vez mais situações instáveis e precárias, onde novas modalidades de trabalho informal, intermitente e precarizado, são comumente criadas e recriadas devido aos mecanismos de flexibilização (Antunes, 2018). Dentre estas novas modalidades flexíveis de trabalho, destacam-se as terceirizações e prestações de serviços. Entre as novas variantes, a literatura apresenta a morfologia contemporânea: o trabalho uberizado. De maneira sucinta, é quando as prestações dos serviços não ocorrem de modo contínuo, mas sob demanda, e mediados por aplicativos digitais (Antunes, 2018). O case de maior sucesso neste tipo de organização de trabalho é o da empresa Uber, sob o princípio básico de fornecer serviços de transportes pré- determinados em troca de recompensas, usando aplicativos ou plataformas online para conectar A flexibilização constitui um gigantesco processo de escala mundial na desconstrução do trabalho pela retirada de direitos e garantias, conquistadas com muito esforço através de lutas da classe trabalhadora. 108 108 ́́ 108 108 ́́ PSI UNISC, 6(2), 107-123, 2022. PSI UNISC, 6(2), 107-123, 2022. “Estamos sendo submetidos a uma escravidão disfarçada”: análise de relatos de motoristas de aplicativo à luz da Psicodinâmica do Trabalho saúde ou induzir ao adoecimento (Dejours & Neto, 2012). motoristas aos seus clientes que demandam por transporte/serviço de mobilidade (Serrano & Baldanza, 2017). saúde ou induzir ao adoecimento (Dejours & Neto, 2012). O interesse da PdT ultrapassa os aspectos relacionados ao sofrimento e a doenças mentais, propondo a investigação do prazer associado ao sentido do trabalho e sua potencialidade em promover saúde. Compromete-se, sobretudo, em compreender como o trabalhador mantem a normalidade psíquica frente as pressões determinadas pelo contexto do trabalho. Deste modo, para manter equilíbrio, cria estratégias de mediação, defensivas ou de enfrentamento, para suportar ou modificar situações adversas que geram sofrimento (Dejours & Neto, 2012; Moraes, 2013). O neologismo “uberização” tem sido difundido para designar o tipo de trabalho que inclui a população de motoristas como a categoria de maior representatividade. Entretanto, ela atinge diversas classes, como os entregadores, empregadas domésticas, professores, médicos e outros (Coutinho & Ferreira, 2021). compartilhamento de experiências e mobilizações. compartilhamento de experiências e mobilizações. compartilhamento de experiências e mobilizações. o estudo teve por objetivo investigar e compreender aspectos referentes aos motivos de inserção nas plataformas, condições de trabalho, vivências de sofrimento, estratégias de mediação (enfrentamento e defensiva) e vivências de prazer, apresentados e analisados de forma individual e/ou coletiva através da livre expressão presente no ambiente virtual de um grupo destinado a este público, em uma rede social. e Procedimentos éticos e coleta de dados Pesquisas que adotam como fonte de dados informações de domínio público, coletadas de materiais postados na internet, não se enquadram no que é considerado pesquisa direta com seres humanos. Contudo, optou-se por seguir as recomendações do Guia de Ética da Association of Internet Researchrs que prevê, entre outras orientações, o uso do anonimato para proteção de identidade (Markham & Buchana, 2012). O modelo de trabalho uberizado é recente no Brasil e no mundo, gerando diversas dúvidas à sociedade, críticas aos setores jurídico/legislativo e transformações nos transportes e mobilidade urbana (Antunes, 2020). Embora este estudo não traga benefícios imediatos a classe trabalhadora, pode ser considerado um importante contribuinte para as pesquisas do escopo da Psicodinâmica do Trabalho frente a nova modalidade de trabalho que é a uberização. Considerando a potencialidade do coletivo de trabalhadores disponível no ambiente das redes sociais, acredita-se que debruçar-se nas interações virtuais seja um meio também possível de identificar a expressão e mobilização do público alvo. Neste ambiente virtual o pesquisador esteve despercebido, não afetando a prática cotidiana da amostra (Sá & Polivanov, 2013). Isto demonstrou-se útil uma vez que o ator é “estranho” a algum ambiente pretendendo apreender suas dinâmicas de funcionamento, valores e regras sociais. A identificação dos autores do conteúdo no grupo foi preservada. A fim de constituir o corpus de análise, foram selecionadas todas as postagens realizadas no intervalo de dezembro (2019) à fevereiro (2020) em determinado grupo do Facebook, destinado aos motoristas por aplicativos, que segundo sua própria descrição foi criado para que “todos os trabalhadores compartilhem suas dúvidas e experiências com intuito de melhorar cada vez mais o desempenho”. O material coletado resultou em 282 laudas com 159.764 palavras oriundas da livre comunicação entre eles. Foram consideradas postagens todas as publicações realizadas naquele ambiente e também as discussões manifestadas através dos comentários dos participantes. “Estamos sendo submetidos a uma escravidão disfarçada”: análise de relatos de motoristas de aplicativo à luz da Psicodinâmica do Trabalho “Estamos sendo submetidos a uma escravidão disfarçada”: análise de relatos de motoristas de aplicativo à luz da Psicodinâmica do Trabalho Introdução Corporações como a Uber tem utilizado a ideologia da Economia do Compartilhamento para mascarar seus negócios, apoiadas na exploração dos trabalhadores e esquivas de legislações, pois declaram-se empresas de tecnologias que conectam pessoas e, portanto, não tem o dever legal/contratual de submeterem-se as leis (Slee, 2017). Por se tratar de um assunto recente, existem poucas pesquisas empíricas desenvolvidas investigando a uberização do trabalho e, em especifico, ouvindo os motoristas por aplicativos. Entretanto, nos estudos encontrados, é possível perceber que estes trabalhadores lidam diariamente com situações estressantes com o trânsito intenso e relações com passageiros (André, Silva & Nascimento, 2019). Além disso, possuem a jornada de trabalho estendida e intensificada através dos recursos motivacionais que aparecem na tela do celular, mantendo o trabalhador em atividade por mais tempo, aumentando a competição entre os motoristas por disputa de passageiros e fomentando a precarização do trabalho ao desafiar legislações e aspectos jurídicos (Uchôa-de-Oliveira 2020). Outrossim, no cenário do desemprego, a adesão facilitada pela sedutora alternativa, com promessas de autonomia, flexibilidade e renda extra, estes motoristas assumem o risco do empreendedorismo com seus próprios recursos (Slee, 2017; Antunes, 2018; Antunes; 2020; Moraes; Oliveira & Accorsi, 2019). Em síntese, de um lado estão os/as infoproletários/as, isto é, a classe-que-vive-do- trabalho, e do outro, as empresas que controlam e detém os aplicativos permitindo o acesso do/a cliente. Estes trabalhadores ficam à disposição, esperando uma chamada ou notificação. A remuneração se constitui estritamente pelo que desempenham (Antunes, 2018). O trabalho torna-se onipresente por meio de alertas, conexões e rastreamentos de modo que a (falsa) liberdade oriunda do empreendedorismo captura o sujeito em plena era da mobilidade (Roso, 2017). As abordagens teóricas que se debruçam a estudar as relações sujeito- trabalho, desejam destacar as experiências humanas neste contexto e sua complexidade. Nesta literatura, destaca-se a Psicodinâmica do Trabalho (PdT), estudos desenvolvidos por Christophe Dejours a partir dos anos de 1990. O autor baseou suas pesquisas nas relações entre a organização do trabalho e os processos de subjetivação, com ênfase nas vivências de prazer e sofrimento, capazes de promover 109 109 - , 109 109 - , 99 Utilizando o referencial teórico- metodológico da PdT (Dejours & Neto, 2012), PSI UNISC, 6(2), 107-123, 2022. PSI UNISC, 6(2), 107-123, 2022. Procedimentos de análise de dados A análise dos dados aconteceu por meio da análise temática (AT) de Braun e Clarke (2006), seguindo orientações, sugestões e adaptações propostas por Souza (2019). Esta análise seguiu procedimentos como a familiarização dos dados através de transcrição, leitura e releitura do banco, e geração de códigos de maneira sistemática reunindo extratos relevantes definidos por temas a priori: 1) As características do trabalho de motoristas por aplicativos 2) As vivências de prazer e sofrimento no trabalho de motoristas por aplicativos; e 3) As estratégias de mediação do sofrimento elaboradas pelos motoristas por aplicativos. É fato que, diante dos preceitos do capitalismo neoliberalista, são comuns os discursos que levam os trabalhadores, em geral, a perseguirem a ilusão do negócio próprio e do empreendedorismo de si mesmo (Bianchi et al., 2020). Entretanto, a aparente liberdade decorrente da ausência de hierarquia é desmascarada por motoristas que denunciam uma divisão de trabalho oposta ao discurso predominante, onde evidenciam-se as características de servidão: “[...] Nós, motoristas de aplicativo, estamos sendo submetidos a uma escravidão disfarçada. Somos forçados a prestar um bom serviço”. “Estamos sendo submetidos a uma escravidão disfarçada”: análise de relatos de motoristas de aplicativo à luz da Psicodinâmica do Trabalho “Estamos sendo submetidos a uma escravidão disfarçada”: análise de relatos de motoristas de aplicativo à luz da Psicodinâmica do Trabalho “Estamos sendo submetidos a uma escravidão disfarçada”: análise de relatos de motoristas de aplicativo à luz da Psicodinâmica do Trabalho tema do grupo. Destaca-se, ainda, que o período escolhido foi anterior à pandemia causada pelo COVID-19 (Coronavírus). como um serviço de tecnologia que apenas realiza o intermédio entre o cliente e o prestador do serviço. Desse modo, o motorista constituiria um negócio próprio, a partir do qual poderia deliberar livremente sobre as questões que envolvem a sua rotina e processos de trabalho. Esses trabalhadores são confundidos pelo discurso ideologizante que os leva à crença de que participam de um trabalho liberto e autônomo, conforme referenciado por Bianchi, Macedo e Pacheco (2020). Delineamento Trata-se de um estudo exploratório e descritivo, pois teve como principal finalidade desenvolver e esclarecer conceitos/ideias do problema de pesquisa (Flick, 2009) em consonância com a abordagem da Psicodinâmica do Trabalho (Dejours & Neto, 2012). Utilizou-se dos pressupostos metodológicos da análise documental (Gil, 2008) para compor a amostra, que foi extraída de postagens e comentários em uma rede social, a partir da percepção e expressões de motoristas de aplicativo em um grupo destinado ao O grupo foi escolhido levando em consideração o número de membros (aproximadamente 120.000 membros), sendo excluídas postagens de anúncio/venda de serviços e veículos, e outras, que fugiam do 110 110 PSI UNISC, 6(2), 107-123, 2022. PSI UNISC, 6(2), 107-123, 2022. [...] A cada recusa, a taxa de aceitação cai e assim vem outras consequências. [...] A cada recusa, a taxa de aceitação cai e assim vem outras consequências. [...] Você tem sempre o direito de cancelar por motivo de segurança e não vai ser descontado nenhum valor, porém a taxa de cancelamento sobe! E na 99, com quatro ou cinco cancelamentos seguidos você é banido. [...] Comigo é assim, Uber é tempo online! Estou fazendo em média 12, 13 horas por dia; [...] O problema dessas empresas é que elas são sua sócia, com 50% do seu lucro você tem que rodar muito para não empatar com eles. Eu dirigia de 12h a 16h por dia. [...] O problema dessas empresas é que elas são sua sócia, com 50% do seu lucro você tem que rodar muito para não empatar com eles. Eu dirigia de 12h a 16h por dia. Em linhas gerais, constatou-se que a autonomia previamente anunciada pelos motoristas é apenas aparente, pois, na prática, são obrigados a trabalhar mais e sob os critérios impostos, para garantir sua sobrevivência e manutenção dos instrumentos de trabalho. Em relação a esses achados, adicionam-se as ideias de Abílio (2019) e Antunes (2020), ao discutirem que este trabalho constantemente vigiado pelo panóptico digital dos algoritmos expõe a vulnerabilidade a que estão submetidos estes trabalhadores. As escalas de reputação classificam os subcontratados independente de seu empenho e dedicação, podendo levar à perda da renda adicional ou o próprio “ganha pão” (Linhart, 2014; Abílio, 2019; Antunes, 2020). Outro elemento a ser compreendido quanto à adoção de jornadas de trabalho intensas pelos motoristas, é o fato de que as plataformas digitais utilizam recursos persuasivos para a manutenção da disponibilidade destes trabalhadores pelo maior número de horas possíveis. Para tanto, as empresas programam a interface dos aplicativos adotando técnicas de gamificação que pressionam os trabalhadores a ficarem mais tempo à disposição, mediante o uso de incentivos e reforços. Recompensas e promoções surgem na tela do celular incitando aos motoristas trabalharem por mais tempo, como indica (Slee, 2017) e pode ser visualizado na sequência, conforme descrito por um motorista: “[...] Às vezes viciamos em receber chamadas que passam 13, 14, 15 horas dirigindo e não percebemos”. Em relação à produtividade, evidenciou-se que há auto estabelecimento de metas, assim podendo constar: “[...] Pra bater a meta tem que rodar sábado e domingo!” e “[...] Tem que ter meta! Às vezes nos matamos de tanto trabalhar, mas só assim pra tirar lucro”. Contexto do Trabalho Essa categoria corresponde às questões que englobam informações sobre a organização/condição de trabalho, bem como as relações sócioprofissionais nele estabelecidas. A seguir serão discutidos separadamente os conteúdos explícitos através de relatos expressos pelos motoristas por aplicativos no grupo de rede social. Não obstante, as plataformas digitais demonstram suas estruturas arbitrárias de controle sobre o trabalho dos motoristas por meio de diversos mecanismos. São elas quem determinam quem pode ou não trabalhar, por meio de critérios e exigências que já são estabelecidos no momento da adesão ao aplicativo (Farias & Rached, 2017; Uber, 2022). As manifestações predominantes dos motoristas por aplicativos apontam para a internalização da ideia de que a sua atividade laborativa é autônoma, especialmente do ponto de vista da ausência de um superior hierárquico, a quem se devesse obediência: “[...] A Uber é um trabalho normal para você sobreviver sem um patrão para te dar ordens” – como aponta o comentário deste motorista. A compreensão do trabalho nos termos indicados pela frase anterior, é influenciada pelo discurso das empresas que se apresentam à sociedade Houve relatos de que motoristas foram bloqueados do sistema, impossibilitando suas atividades: “[...] O único problema que é um trabalho totalmente incerto, a qualquer momento você pode ser bloqueado”. Sobretudo, evidenciou-se que há critérios a serem seguidos com vistas à prestação de um “bom serviço”, expressos por pelo menos dois mecanismos: as notas de avaliação que os PSI UNISC, 6(2), 107-123, 2022. PSI UNISC, 6(2), 107-123, 2022. “Estamos sendo submetidos a uma escravidão disfarçada”: análise de relatos de motoristas de aplicativo à luz da Psicodinâmica do Trabalho destes relatos, é que os motoristas impõem a si próprio os ritmos – intensos – de trabalho. Essa questão é explicada por Antunes (2018) ao afirmar que no modelo just-in-time, típico do trabalho uberizado, a remuneração dos trabalhadores está atrelada à quantidade de produção e, que há, portanto, um processo de transferência da responsabilidade de gestão da produtividade aos motoristas por aplicativos. Por consequência, ao assumir a responsabilidade pela produtividade e pelos seus ganhos, haverá uma jornada de trabalho prolongada e intensificada, denunciando a auto exploração que gera mais-valia e maior produção: passageiros aferem aos motoristas após a utilização do serviço e as taxas de aceitação e cancelamento das corridas solicitadas pelos clientes na plataforma. Estes mecanismos avaliativos exercem a função de controlar, fiscalizar e punir o comportamento indesejável. Os seguintes trechos de postagens demonstram tais evidências: [...] Mas com certeza a tarifa está bem abaixo do que seria necessário. A maioria também vive na grande ilusão de ganhar muito baseado no faturamento. [...] Mas com certeza a tarifa está bem abaixo do que seria necessário. A maioria também vive na grande ilusão de ganhar muito baseado no faturamento. [...] Não estamos seguros em lugar algum, mas ser motorista de aplicativo é se colocar em perigo a cada corrida. Nota-se, por conseguinte, a presença de um problema social de segurança pública. Entretanto, a organização do trabalho mediada por plataformas digitais potencializa o risco de sofrer atos violentos e não oferece mecanismos de proteção. As notificações recebidas pelos motoristas no aplicativo não mostram uma foto do solicitador, nem o destino da corrida, apenas um nome do demandante do serviço. Portanto, os trabalhadores devem aceitar ou recusar a chamada da corrida sem que possuam informações que poderiam proteger sua integridade ou patrimônio: [...] Rodar na Uber tem que fazer cálculos. Incluíndo combustível, alimentação, documentação, manutenção e seguro, eu já teria uma dívida de 200 reais diária. Ou seja, teria que fazer partir de 200 reais para ter lucro. Por isso muita gente quebra, não conta as manutenções preventivas e possíveis corretivas que virão aliadas aos possíveis dias fracos. Há que se considerar, ainda, que a remuneração de um motorista por aplicativo não prevê qualquer garantia trabalhista, já que a modalidade não se enquadra nos parâmetros celetistas (Abílio, 2019; Abílio, 2021). O seguinte trecho expressa esta condição: [...] Entrei em contato com a Uber e perguntei o porquê que me pediram três vezes o reconhecimento facial no mesmo dia. Após alguns minutos recebi um chamado de uma mulher, assim dizia o nome no aplicativo. Cheguei no local eram dois caras, consegui sair, mas eles iam me roubar, com certeza. Moral da história: a gente tem que fazer reconhecimento facial, mostram nossa foto e a gente não tem nem uma referência do passageiro (...) a Uber não se preocupa com a nossa segurança. [...] Única coisa de fato que me incomoda nesse tipo de serviço é não ter férias remuneradas e nem folga remunerada, não tem 13º, nem FGTS, nem INSS, auxílio passagem, podia ser até combustível, nem planos pra saúde tem se ficar doente também. [...] Única coisa de fato que me incomoda nesse tipo de serviço é não ter férias remuneradas e nem folga remunerada, não tem 13º, nem FGTS, nem INSS, auxílio passagem, podia ser até combustível, nem planos pra saúde tem se ficar doente também. “Estamos sendo submetidos a uma escravidão disfarçada”: análise de relatos de motoristas de aplicativo à luz da Psicodinâmica do Trabalho estão à deriva, uma vez que as plataformas nem mesmo assumem responsabilidades por quaisquer eventos que ocorram durante o tempo em atividade. Quanto à remuneração, os motoristas por aplicativos indicam precariedade salarial e sugerem que os seus ganhos, com frequência, sequer permitem liquidez, conforme ilustrado nos trechos postados: O tema das condições de trabalho também foi discutido nas postagens do grupo de motoristas por aplicativos. Além das intempéries causadas pelo clima e trânsito, destaca-se a exposição à violência nos grandes centros urbanos: [...] Fui assaltado através da plataforma, no meu caso era impossível desconfiar do assalto. [...] A cada recusa, a taxa de aceitação cai e assim vem outras consequências. O que se pode compreender a partir 112 112 s 112 112 s PSI UNISC, 6(2), 107-123, 2022. “Estamos sendo submetidos a uma escravidão disfarçada”: análise de relatos de motoristas de aplicativo à luz da Psicodinâmica do Trabalho PSI UNISC, 6(2), 107-123, 2022. [...] Mas com certeza a tarifa está bem abaixo do que seria necessário. A maioria também vive na grande ilusão de ganhar muito baseado no faturamento. 33 Sobre isso, os estudos de Roso (2017), Valente, Patrus e Córdova Guimarães (2019), e Filgueiras e Antunes (2020), indicam que o modelo uberizado de trabalho constituiu-se sob a tríade destrutiva do capital frente ao trabalho: 113 113 o b : Conforme apontam Filgueiras e Antunes (2020), os motoristas por aplicativos PSI UNISC, 6(2), 107-123, 2022. “Estamos sendo submetidos a uma escravidão disfarçada”: análise de relatos de motoristas de aplicativo à luz da Psicodinâmica do Trabalho “Estamos sendo submetidos a uma escravidão disfarçada”: análise de relatos de motoristas de aplicativo à luz da Psicodinâmica do Trabalho Não obstante, isto implica em desgaste psicológico pelo estado de tensão emocional: “[...] O medo nunca abandona a gente, cara! Eu trabalho com medo todos os dias”. Segundo Sousa e Santos (2017), o medo originado pela degradação do organismo psíquico do trabalhador está ligado diretamente às más condições de trabalho que estão submetidos. a flexibilidade, a informalidade e a intermitência, que, por ora, são características de um trabalho precarizado. Alguns motoristas por aplicativos estão cientes das condições precarizantes e ainda denunciam esta disposição mundial à classe trabalhadora: “[...] bem-vindos à nova onda do trabalho precarizado”. Neste contexto, o trabalho precário se caracteriza como um trabalho incerto e imprevisível, no qual os riscos empregatícios são assumidos pelo trabalhador. Diretrizes históricas apontam este como um resultado do crescimento da globalização (interdependência econômica e movimento acelerado do capital/produção), bem como da expansão do neoliberalismo, ideologia que implica na desregulação e remoção de proteções sociais, corroendo a identidade dos trabalhadores e promovendo a anomia da classe (Antunes, 2018). Em referência ao medo, percebe-se, ainda, uma tendência à banalização deste sentimento. Os motoristas se conformam com a ideia de que não há o que fazer para modificar as condições que originam o medo, como demonstrado a seguir: “[...] O segredo é ir para rua. Segue os conselhos dos amigos que estão na plataforma há mais tempo que não tem erro. Estou há 6 meses tenho medo a cada corrida, isso não muda”. Quantos às frustrações, os motoristas por aplicativos tem seus relatos relacionados à falta de valorização e reconhecimento: “[...] Uma pena que nós motoristas não somos valorizados por tais empresas de aplicativos.” Na visão de Dejours (2001), o desejo do trabalhador é apenas que seu investimento no trabalho não seja fracassado ou que não seja considerado apenas como um cumpridor e executante de ordens com obediência passiva. [...] Mas com certeza a tarifa está bem abaixo do que seria necessário. A maioria também vive na grande ilusão de ganhar muito baseado no faturamento. Embora o reconhecimento seja constituído por forte expectativa por parte do trabalhador, ele raramente é conferido de forma satisfatória. Vivências de sofrimento e prazer Esta categoria temática propõe identificar as fontes geradoras de sofrimento e prazer no trabalho dos motoristas por aplicativos, identificadas através de opiniões, relatos e experiências compartilhadas em um grupo de rede social pelos próprios trabalhadores. De acordo com Dejours (2011), prazer e sofrimento são categorias indissociáveis, ao passo em que trabalhar é deparar-se inevitavelmente com o sofrimento, sentimento decorrente de um embate central entre a organização do trabalho e o funcionamento psíquico do sujeito. Para Merlo, Traesel e Baierle (2011), o reconhecimento é considerado fundamental porque possibilita que o sofrimento no trabalho seja transformado em realização e prazer. Entende-se, então, que estes trabalhadores, motoristas por aplicativos, estão expostos a um contexto de trabalho nocivo à sua saúde mental. O sofrimento dos motoristas foi evidenciado por sentimentos de medo (insegurança), estresse e frustração pela falta de reconhecimento, e, também, por desgastes físicos, indicados por cansaços e dores. Conforme já relatado pelos motoristas, uma característica presente nas condições de trabalho é a exposição constante à violência. Além da imaterialidade do seu labor, a frustação em relação aos valores recebidos é notável: 114 114 114 114 PSI UNISC, 6(2), 107-123, 2022. “Estamos sendo submetidos a uma escravidão disfarçada”: análise de relatos de motoristas de aplicativo à luz da Psicodinâmica do Trabalho [...] Uber é um quebra galho até arrumar algo fixo, ou como uma renda extra... fora isso é uma falsa ilusão. Ganha agora e gasta tudo amanhã. saudável. É como se trabalhar correspondesse ao oposto de ter saúde, pois ao final ela deverá ser recuperada (Uchôa-de-Oliveira, 2020). Há uma falsa ideia de flexibilidade no trabalho dos motoristas por aplicativos que logo é traduzida por uma jornada de trabalho extensa, a partir da qual, segundo Abílio (2021), o trabalhador a inicia sem qualquer garantia sobre qual deverá ser a carga de trabalho necessária para obter alguma remuneração importante: “[...] Fazer só Uber nos dias de hoje não é mais vida, mesmo com a flexibilidade de horários, pois se torna muito cansativo e você acaba não tendo tempo para si e muito menos para sua família.” [...] Você ganha, mas se for para ser realista você paga para trabalhar. Gasolina, pneu, amortecedor, elétrica, limpeza de ar condicionado. Tudo isso conta. É muito triste pois tem muitos iludidos assim como eu fui. A frustação por terem sido enganados pelas plataformas digitais aparece com constância entre as publicações por motoristas da amostra. Vivências de sofrimento e prazer [...] Acho que ninguém é motorista de aplicativo porque gosta, e sim porque nosso Brasil não tem emprego, (...) orem muito porque estamos sem saída, infelizmente. oportunidade de conhecer novas pessoas diariamente é considerada vantagem e fator positivo para estes trabalhadores. Outra fonte geradora de prazer relatada pelos motoristas por aplicativos foi a gratificação. Esta pode ser compreendida como bonificação adicional fornecida voluntariamente em mãos aos trabalhadores pelos passageiros no término da corrida. Entende-se esta remuneração complementar não apenas como um meio material para sobrevivência e consumo, mas reflete também uma recompensa oferecida pelo cliente ao trabalho realizado. Para Dejours (2014), a capacidade de ser remunerado através do trabalho digno, remete a sensação de utilidade, vivência de prazer que ressignifica experiências por meio da valorização. Evidencia-se no relato a seguir: Além do desemprego, a inserção dos trabalhadores nas plataformas digitais é facilitada pelo fato de que o trabalho não requer habilidades específicas ou determinado grau de escolaridade (Slee, 2017). Para iniciar as atividades, pelas empresas, não são exigidas qualificações, a não ser possuir a Carteira Nacional de Habilitação (CNH): [...] O desemprego está aí e os aplicativos sabem disso. Muitos motoristas têm só a habilitação pra apresentar ao mercado de trabalho e outros que tem curso ou até faculdade estão sem vagas na área. Os aplicativos se aproveitam (...) ou você fica em casa sem ganhar nada. [...] Nessa corrida eu ganhei 245 reais, viagem em cartão. Tranquilo, em forma de agradecimento o casal me deu 100 reais de gorjeta e falou ‘Coloca de combustível para não ter prejuízo, e Deus te abençõe’. Cara, foi uma das melhores corridas que eu já fiz. [...] Nessa corrida eu ganhei 245 reais, viagem em cartão. Tranquilo, em forma de agradecimento o casal me deu 100 reais de gorjeta e falou ‘Coloca de combustível para não ter prejuízo, e Deus te abençõe’. Cara, foi uma das melhores corridas que eu já fiz. Trata-se, portanto, de uma atividade concebida como alternativa num contexto social onde a competitividade e exigências do mercado de trabalho para obter-se um emprego são avidamente disputadas, independente do nível de instrução ou experiências na carreira (André et. al., 2019). Entretanto surge a reflexão: se as vivências de sofrimento são mais frequentes que as vivências de prazer, por que há tantos motoristas por aplicativos em atividade? Vivências de sofrimento e prazer Neste sentido, foi possível compreender que os trabalhadores, na maioria das vezes, cadastraram-se nos aplicativos motivados pelo desemprego. Foram evidentes as manifestações de que trabalhar nas plataformas digitais está distante de ser uma escolha ou uma opção do trabalhador, mas sim, uma única alternativa diante à necessidade de sobrevivência: Vivências de sofrimento e prazer De acordo com o estudo de Slee (2017), a remuneração jamais aproximou-se da renda média anunciada pelas plataformas. Quando as empresas se estabelecem em uma nova localidade, elas fornecem subsídios e ofertas especiais (para motoristas e clientes) com objetivo de ampliar e divulgar o serviço mais rapidamente. Ao consolidar-se nas novas áreas de atuação, os aplicativos digitais subtraem uma quantidade maior a cada cifra monetária, e desde modo, o valor repassado aos trabalhadores vai diminuindo a cada corrida (Slee, 2017; Filgueiras & Antunes, 2020). O fato é que os horários flexíveis podem intensificar o labor. Para Roso (2017), as distribuições flexíveis das horas laborais alteraram as fronteiras/barreiras que separam o tempo de trabalho e tempo de não-trabalho, praticamente anulando esta distinção de relevância fundamental para os trabalhadores. As vivências de prazer, embora reveladas com menor frequência, também foram manifestadas pelos motoristas por aplicativos da amostra de publicações. Ficou evidenciado na divisão: a) reconhecimento, sinalizado pela consideração dos passageiros pelo seu trabalho; e b) gratificação, objetivada pela avaliação positiva do serviço e bonificação/gorjeta. Notabilizou-se a presença de vivências de sofrimentos originadas pelos desgastes físicos ocasionados por intensas jornadas de trabalho: “Já rodei até 16 horas no final de semana. Parei, chega, o corpo está cobrando. Infelizmente as tarifas estão deixando a desejar, mas do que adianta se matar fazendo corrida e depois gastar tudo recuperando a saúde”. 55 Os clientes prestigiam e reconhecem o trabalho dos motoristas: “[...] O reconhecimento e carinho de um monte de gente que eu nunca sequer vi na vida. E isso não tem dinheiro que pague”. Sendo assim, apresenta-se a importância das relações socioprofissonais no contexto do trabalho, afinal, o reconhecimento do outro torna-se em fonte de prazer (Mendes, 2007). Conforme corroborado pelos estudos de Moraes et al. (2019), para os motoristas por aplicativos a A suposta autonomia abordada anteriormente, com ausência de qualquer garantia ou compromisso por parte das empresas digitais, vem incentivando os motoristas por aplicativos a exercer jornadas mais longas, abolindo assim dias de descanso, fundamentais para a manutenção de uma vida 115 115 . a PSI UNISC, 6(2), 107-123, 2022. “Estamos sendo submetidos a uma escravidão disfarçada”: análise de relatos de motoristas de aplicativo à luz da Psicodinâmica do Trabalho [...] Acho que ninguém é motorista de aplicativo porque gosta, e sim porque nosso Brasil não tem emprego, (...) orem muito porque estamos sem saída, infelizmente. “Estamos sendo submetidos a uma escravidão disfarçada”: análise de relatos de motoristas de aplicativo à luz da Psicodinâmica do Trabalho “Estamos sendo submetidos a uma escravidão disfarçada”: análise de relatos de motoristas de aplicativo à luz da Psicodinâmica do Trabalho Para lidar com o sentimento de medo e a insegurança identificados como vivências de sofrimento, o trabalhador cria mecanismos protetivos ao aparelho psíquico (Dejours, 2011). Alguns motoristas por aplicativos, mesmo que em menor frequência de relatos, negam os riscos de assaltos e homicídios causados pela exposição potencializadas pelo modelo de trabalho adotado nas plataformas digitais: “[...] Não importa se você é Uber ou não, a violência está para todos. Fui assaltado uma vez como Uber e duas vezes antes de conhecer os aplicativos”. Dissimilar a situação da violência presente nos grandes centros urbanos de sua atuação de trabalho é um sinal de estratégia defensiva de negação, uma vez que o motorista atua no contexto destes grandes centros urbanos colocando-se em risco ao servir uma empresa que não lhe oferece o mínimo de garantia. Para Moraes (2013a), estas estratégias de defesa são recursos psicológicos que visam negar e fantasiar a percepção do próprio sofrimento, dissimulando o contexto do trabalho com causas. negação, a fim de minimizar a percepção do sofrimento, criando motivos lógicos e racionais para os acontecimentos que afligem o trabalhador (Moraes, 2013b). Entretanto, as estratégias defensivas são construídas de maneira reativa, e embora protejam o psiquismo e possibilitem evitar a descompensação, não estimulam e nem criam alternativas na organização do trabalho que dá origem ao sofrimento. Por favorecerem a adaptação, não possibilitam ou priorizam a ação transformadora, correndo o risco de perder sua efetividade e conduzir-se à alienação (Moraes, 2013b). Nesse sentido, outro modo é possível para mediação do sofrimento: a criação e estruturação de estratégias de enfrentamento, que é o conceito que corresponde às táticas e recursos criadas pelos trabalhadores para o enfrentamento das imposições do trabalho real (Dejours, 2011). A criatividade movida pelo sofrimento fundamenta a mobilização para a ação, propondo alternativas para subverter a insatisfação em prazer (Moraes, 2013c). Do mesmo modo, para lidar com o sofrimento originado pela falta de reconhecimento por parte das plataformas digitais, os motoristas adotam estratégias defensivas de racionalização: 77 Para lidar com a frustração pelos baixos rendimentos, a criação de uma rede de passageiros particulares vem se mostrando alternativa para alguns motoristas por aplicativos: “[...] Já fiz muito isso de trabalhar 15h, 16 horas. Estratégias de mediação do sofrimento no trabalho Conforme discutido na categoria anterior, o sofrimento está inerente ao trabalho. Se é inevitável deparar-se com estas vivências, o que fazem os trabalhadores para mediá-las? O objetivo desta categoria temática é justamente conhecer as estratégias de mediação do sofrimento (defensiva e/ou de enfrentamento), elaboradas pelos próprios trabalhadores motoristas por aplicativos na amostra de publicações. [...] Pular fora como se não tem emprego? Acho que ninguém tá nas ruas arriscando a vida porque gosta. 116 116 116 116 PSI UNISC, 6(2), 107-123, 2022. “Estamos sendo submetidos a uma escravidão disfarçada”: análise de relatos de motoristas de aplicativo à luz da Psicodinâmica do Trabalho Hoje saio às 5h00 com um particular, faço os particulares até às 9h00 e ligo o aplicativo”. Essa inventividade é proibida pelas empresas que impedem este tipo de negociação (Uber, 2022). Entretanto, as estratégias de enfrentamento possuem esta dimensão transgressora, além de experimentar e criar novas formas de realizar o trabalho. Diante de clientes particulares, com o aplicativo desligado, o trabalhador não necessita dividir seu ganho com a plataforma, aumentando assim sua renda por produção: “[...] se você for fazer particular, faz sem falar para ninguém, fica só entre você e seu passageiro. E outra: você não tá roubando de [...] Acho errado gente que fica doido com a empresa por causa de corrida de 6 reais, por corridas com preço injusto, etc. Eu também fico chateado, mas não adianta (...) não adianta a galera ficar chorando. [...] Sei que os aplicativos não estão nem aí para os motoristas. Mas qual empresa está? Toda empresa visa lucros, somos só um número entre muitos. A racionalização é uma estratégia de defesa que foi identificada tanto quanto a PSI UNISC, 6(2), 107-123, 2022. PSI UNISC, 6(2), 107-123, 2022. “Estamos sendo submetidos a uma escravidão disfarçada”: análise de relatos de motoristas de aplicativo à luz da Psicodinâmica do Trabalho ninguém, você tá garantindo seu dinheiro de forma honesta”. ninguém, você tá garantindo seu dinheiro de forma honesta”. [...] Minha sugestão é trabalhar em grupo, e quando for a lugares ou passageiros suspeitos colocar a localização no whats e combinar com o grupo quando ter "localização aberta" para que todos os motoristas fiquem cuidando o colega. Aqui em Porto Alegre faço assim que diminui os problemas. [...] Minha sugestão é trabalhar em grupo, e quando for a lugares ou passageiros suspeitos colocar a localização no whats e combinar com o grupo quando ter "localização aberta" para que todos os motoristas fiquem cuidando o colega. Aqui em Porto Alegre faço assim que diminui os problemas. A exposição aos assaltos já foi referida como uma condição desestabilizadora para os motoristas por aplicativos. Por um lado, há os que mediam este sofrimento por mecanismos defensivos, por outro, moldam estratégias de enfrentamento que são compartilhadas na rede social entre o grupo de motoristas: “[...] evite trabalhar com dinheiro na noite, recicla um pouco” e “[...] desabilita a opção ‘Dinheiro’ que você já evitará 80% do risco”. Considerações finais A Uber, empresa que surgiu com o avanço das tecnologias de informação e comunicação (TICs), cresceu e consolidou-se no mercado oferecendo prestações de serviços de entrega e mobilidade, e atualmente, vem impactando a vida das pessoas e cidades. Sob a tutela deste case, surge a uberização do trabalho, um fenômeno mundial que vem transformando o mercado que não se restringe apenas aos motoristas por aplicativos, mas atinge diversas classes trabalhadora, adotando semelhantes mecanismos de gerenciamento, organização e produção no trabalho. Esta nova morfologia é considerada pela literatura crítica o sinônimo da precarização do trabalho, pois dentre tantas características destaca-se a ausência de garantias, direitos e benefícios, conquistas históricas da classe trabalhadora, bem como a falta de suporte nos custos de manutenção nas plataformas acarretando em ganhos ainda menores aos trabalhadores. Correspondente ao contexto de trabalho, pode-se considerar que liberdade, autonomia e flexibilidade, são apenas aparências, pois motoristas relatam uma divisão de trabalho com características de servidão. As normas são estabelecidas pelas empresas que utilizam taxas de aceitação/cancelamento de corridas e notas/avaliações de passageiros, como critérios avaliação de desempenho. A constante vigilância imposta pelos algoritmos digitais, sob ameaças de punição ou bloqueios, impõe aos trabalhadores uma intensa produtividade caracterizada por longas jornadas de trabalho repletas de incertezas, baixa remuneração e falta de segurança. As vivências de prazer foram identificadas pelo reconhecimento, sinalizado pela consideração dos passageiros pelo seu trabalho e a gratificação, objetivada pela avaliação positiva do serviço, bonificação e gorjeta. Por sua vez as vivências de sofrimento ficaram evidenciadas pelo sentimento de medo causado pela insegurança, estresse pelo trânsito e alguns passageiros, frustração, falta de reconhecimento das plataformas digitais, bem como desgastes físicos atribuídos as dores e cansaços. Para lidar com estas situações vivenciadas, os trabalhadores utilizam estratégias de mediação defensivas, que negam o sentimento de medo e insegurança recusando os riscos de exposição causados pelo trabalho, e racionalizam, minimizando a frustração e falta de reconhecimento. Já as estratégias de enfrentamento, movidas pela criatividade, correspondem a criação de clientela fora dos aplicativos e recursos tecnológicos de Utilizando-se do referencial teórico- metodológico da Psicodinâmica do Trabalho (PdT), foi possível atingir o objetivo deste estudo ao compreender a singularidade das formas de vivenciar e realizar o trabalho dos motoristas por aplicativos. “Estamos sendo submetidos a uma escravidão disfarçada”: análise de relatos de motoristas de aplicativo à luz da Psicodinâmica do Trabalho “Estamos sendo submetidos a uma escravidão disfarçada”: análise de relatos de motoristas de aplicativo à luz da Psicodinâmica do Trabalho “Estamos sendo submetidos a uma escravidão disfarçada”: análise de relatos de motoristas de aplicativo à luz da Psicodinâmica do Trabalho mobilização coletiva e ascender a saúde mental no trabalho. ganhos e empreendedorismo, aliadas a agenda neoliberal que incentiva estratégias de flexibilização dos contratos de trabalho. Os trabalhadores “parceiros” acreditam na inserção às plataformas digitais como uma via ao desemprego ou complemento de renda. “Estamos sendo submetidos a uma escravidão disfarçada”: análise de relatos de motoristas de aplicativo à luz da Psicodinâmica do Trabalho A orientação para ajustar as condições de pagamento disponível no próprio aplicativo tem-se mostrado uma alternativa. Dividindo os problemas diários, os trabalhadores encontram soluções coletivamente. Ao compartilhar a inteligência individual, a informação torna-se sabedoria prática, pois está implicada no coletivo, pressupondo relações fundamentadas na confiança, cooperação e espaço público de fala (Bianchi et al., 2020; Ghizoni, 2013; Lima, 2013; Mendes, 2007; Valente et al., 2019). Dentre tantas implicações do modelo uberizado de trabalho, destaca-se o esvaziamento dos espaços de construção coletiva do trabalho que enfraquece a consciência de classe. Segundo Alves (2018), o sentido de classe se caracteriza por um mecanismo importante para o enfrentamento do sofrimento e do adoecimento dos trabalhadores. A postagem de um motorista relaciona-se a esta questão: [...] A gente não sabe a força que tem! Se fôssemos unidos pararíamos com data e hora marcada. Iríamos ter atenção e poderíamos reivindicar alguns direitos. Mas infelizmente o Brasil é um país onde a massa trabalhadora vende o almoço para comprar a janta. Enquanto for assim não vamos conseguir nada e a Uber sabe disso, e se aproveita dessa situação para benefício próprio! Entretanto, alterar a forma de remuneração no aplicativo não é suficiente para lidar com as situações de tensão vivenciadas pelos motoristas. O apoio e cuidado entre os trabalhadores é encontrado por meio das redes de trabalho em comunidades, utilizando recursos tecnológicos disponíveis no próprio celular. Mecanismos de geolocalização em tempo real e aplicativos de rápida comunicação contribuem nesta estratégia de enfrentamento. A criação de grupos nas redes sociais, como é o caso deste que foi analisado na pesquisa, pode ser considerada como um importante sinal de resistência do coletivo de trabalhadores, porque é um ambiente de compartilhamento da inteligência plural e pactos para encontrar soluções dos problemas advindos da experiência no trabalho. Para Merlo, Bottega e Magnus (2013) o espaço de discussão é um local genuíno que resgata os vínculos afetivos, a solidariedade e a cooperação, podendo avançar em destino à [...] Participe de grupos de Whatsapp com outros ‘drivers’ que compartilham localização. Se iniciar a corrida e ver que é área de risco, imediatamente mande um áudio no grupo falando para ficarem de olho em você e informe o horário de chegada na frente do pax (passageiro) mesmo. 118 118 ̀ PSI UNISC, 6(2), 107-123, 2022. Considerações finais Foram extraídos fatores como: os motivos de inserção nas plataformas digitais, contexto de trabalho, vivências de sofrimento e vivências de prazer, estratégias de mediação (enfrentamento e defensiva); publicados em um grupo de uma rede social de aproximadamente 120 mil membros, destinado ao compartilhamento de experiência entre os motoristas por aplicativo. No Brasil, empresas que utilizam esta gestão de negócio vêm encontrando facilidade para ampliar seu mercado, fomentado principalmente pelo discurso fetichista de altos 119 119 s e PSI UNISC, 6(2), 107-123, 2022. “Estamos sendo submetidos a uma escravidão disfarçada”: análise de relatos de motoristas de aplicativo à luz da Psicodinâmica do Trabalho “Estamos sendo submetidos a uma escravidão disfarçada”: análise de relatos de motoristas de aplicativo à luz da Psicodinâmica do Trabalho compartilhamento da geolocalização entre coletividade. compartilhamento da geolocalização entre coletividade. O estudo também detém informações que podem ser utilizadas pelas plataformas digitais para gerar melhorias nas condições de trabalho dos motoristas por aplicativos e aprimoramentos sugeridos por eles, estreitando os laços de comunicação entre empresa e trabalhadores. Sugere-se ainda que as plataformas digitais busquem construir uma relação de diálogo mais próxima e acessível aos trabalhadores, e sobretudo, mais transparência nos mecanismos de gestão e informações. O estudo contribuiu cientificamente com o cenário de discussões empíricas sobre a uberização do trabalho no Brasil, juntamente com outras pesquisas desenvolvidas no tema. Acredita-se no potencial de esforços para subsidiar políticas de enfrentamento e regulamentação ao problema da precarização e flexibilização do trabalho em geral, bem como do adoecimento dos trabalhadores. Nesta pesquisa, evidenciou-se a importância da legislação trabalhista e políticas de regulamentação e fiscalização, pois com sua ausência e desmonte possibilitam atuações de plataformas digitais de serviços que estão colocando em risco a saúde física e mental dos trabalhadores através da insatisfação e frustração profissional, da falta de reconhecimento e valorização por parte das empresas-aplicativos. Considerando o contexto brasileiro e mundial precarização do trabalho e o desemprego um desafio a ser enfrentado, sugere-se intensificar estudos que contribuam para o desenvolvimento da temática. Pesquisas com caráter quantitativo de levantamento podem fornecer dados atualizados sobre os números e as características sociodemograficas de trabalhadores ao traçar perfis e evidenciar perspectivas mais consistentes com a realidade brasileira. Referências Ciência Administrativa, 18(1). doi: 10.21529/RECADM.2019001 Ciência Administrativa, 18(1). doi: 10.21529/RECADM.2019001 Abílio, L.C. (2019). Uberização: do empreendedorismo para o autogerenciamento subordinado. Psicoperspectivas. 18(3):41-51. doi: 10.5027/psicoperspectivas-vol18- issue3-fulltext-1674 Alves, G. (2018). Introdução. In: Casulo, A. C., Alves, G. (Orgs.). Precarização do trabalho e saúde mental: o Brasil da Era Neoliberal. Bauru: Projeto Editorial Praxis Abílio, L. C. (2021). Uberização: a informalização e o trabalhador just- in-time. Trabalho, Educação e Saúde, v. 19. doi: 10.1590/1981- 7746-sol00314 Antunes, R. (2018). O privilégio da servidão: o novo proletariado de serviços na era digital. 1. ed., São Paulo: Boitempo André, R. G., Silva, R. O., & Nascimento, R. P. (2019). “Precário não é, mas eu acho que é escravo”: Análise do Trabalho dos Motoristas da Uber sob o Enfoque da Precarização. Revista Eletrônica de 00 Antunes, R. (2020). 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Plataformas digitais, uberização do trabalho e regulação no capitalismo contemporâneo. Trabalho de Plataforma, v. 39, n. 1. doi: 10.22409/contracampo.v39i1.38901 Braun, V., & Clarke, V. (2006). Using thematic analysis in psychology. Qualitative Research in Psychology, 3(2), 77-101. doi: 10.1191/1478088706qp063oa Coutinho, R. L., & Ferreira, M. M. V. (2021). A uberização do trabalho doméstico em tempos de pandemia: Precarização de uma categoria precarizada. Palavra Seca, 1(1), 125-147. Recuperado de: https://palavraseca.direito.ufmg.br/i ndex.php/palavraseca/article/view/3/ 10 Flick, U. (2009). Desenho da Pesquisa Qualitativa. Porto Alegre, RS: Artmed. Ghizoni, L. D. (2013). Cooperação. In: Vieira, F. O. (Org.). Dicionário crítico de gestão e psicodinâmica do trabalho. Curitiba: Juruá p. 99-102 10 Gil, A. C. (2008). Métodos e técnicas de pesquisa social. 6. ed. Editora Atlas SA. Dejours, C. (2001). A banalização da injustiça social. (4a ed.). Rio de Janeiro: FGV. Lima, S. C. 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O corpo da Psicossomática. Psicologia Revista, 14(2), 245–256. 121 121 PSI UNISC, 6(2), 107-123, 2022. “Estamos sendo submetidos a uma escravidão disfarçada”: análise de relatos de motoristas de aplicativo à luz da Psicodinâmica do Trabalho trabalho entre a saúde e a patologia. Curitiba: Editora Juruá. Recommendations from the AoIR Ethics Working Committee (Version 2.0). Disponível em: http://aoir.org/reports/ethics2.pdf Moraes, R. B. de S., Oliveira, M. A. G., & Accorsi, A. (2019). Uberizazção do trabalho: a percepção dos motoristas de transporte particular por aplicativo. Revista Brasileira de Estudos Organizacionais – v. 6, n. 3, p. 647- 681. doi: 10.21583/2447- 4851.rbeo.2019.v6n3.216 Mendes, A. M. (2007). Da psicodinâmica à psicopatologia do trabalho. In: Mendes, A. M. (Org.). Psicodinâmica do Trabalho: Teoria, método e pesquisas (pp. 16-30). Casa do Psicólogo. Pochmann, M. (2016). A uberização leva à intensificação do trabalho e da competição entre os trabalhadores. Entrevista André Antunes. EPSJV/Fiocruz. Disponível em: http://www.epsjv.fiocruz.br/noticias /entrevista/a-uberizacao-leva-a- intensificacao-do-trabalho-e- dacompeticao-entre-os Merlo, A. C., Traesel, E. S., & Baierle, T. C. (2011). Trabalho imaterial e contemporaneidade: um estudo na perspectiva da Psicodinâmica do Trabalho. Arquivos Brasileiros de Psicologia, 63, 94-104. Recuperado de: http://pepsic.bvsalud.org/scielo.php ?script=sci_arttext&pid=S1809- 52672011000300010 Roso, S. D. (2017). O ardil da flexibilidade: os trabalhadores e a teoria de valor. 1. ed. São Paulo: Boitempo; 2017 Merlo, A. C.; Bottega, C. G., Magnus, C. N. (2013). Espaço público de discussão. In. Vieira, F. O. (Org.). Dicionário crítico de gestão e psicodinâmica do trabalho. Curitiba: Juruá. p. 147- 151 Sá, S. P., & Polivanov, B. B. (2013). Presentificação, vínculo e delegação nos sites de redes sociais. Comunicação Mídia e Consumo, 9(26), 13-36. doi: 10.18568/cmc.v9i26.341 Moraes, R. D. (2013a). Sofrimento criativo e patogênico. In: Vieira, F. O. (Org.). - Jefferson João dos Santos: Graduado em Psicologia pela Universidade Comunitária da Região de Chapecó, Especialista em Gestão Estratégia em Recursos Humanos e Mestre em Psicologia pela Faculdade Meridional IMED. Atualmente é Assessor de Relacionamento na Unidade Central de Educação Faem Faculdade. Ciência Administrativa, 18(1). doi: 10.21529/RECADM.2019001 Dicionário crítico de gestão e psicodinâmica do trabalho. Curitiba: Juruá p. 415-419. Serrano, P. H. S. M., & Baldanza, R. F. (2017). Tecnologias disruptivas: o caso do Uber. Revista Pensamento Contemporâneo em Administração, 11(5), 37-48. doi: 10.12712/rpca.v11i5.1078 Moraes, R. D. (2013b). Estratégias defensivas. In: Vieira, F. O. (Org.). Dicionário crítico de gestão e psicodinâmica do trabalho. Curitiba: Juruá p. 154-157 Moraes, R. D. (2013c). Estratégias de enfrentamento do sofrimento e conquista do prazer no trabalho In: Merlo, A. R. C. (Orgs.) O sujeito no 11 Slee, T. (2017). Uberização: a nova onda do trabalho precarizado. São Paulo: Elefante. 122 122 PSI UNISC, 6(2), 107-123, 2022. “Estamos sendo submetidos a uma escravidão disfarçada”: análise de relatos de motoristas de aplicativo à luz da Psicodinâmica do Trabalho Uber. (2022). Requerimentos e Regulamento. Recuperado de: https://www.uber.com/legal/pt- br/document/?country=brazil&lang =pt-br&name=general-terms-of-use Uber. (2022). Requerimentos e Regulamento. Recuperado de: https://www.uber.com/legal/pt- br/document/?country=brazil&lang =pt-br&name=general-terms-of-use Sousa, J. C. de, & Santos, A. C. B dos. (2017). Psicodinâmica do trabalho nas fases do capitalismo: análise comparativa do taylorismo-fordismo e do Toyotismo nos contextos do capitalismo burocrático e do capitalismo flexível. Revista Ciências Administrativas, 23(1), 186-216. doi: 10.5020/2318- 0722.23.1.186-216 Sousa, J. C. de, & Santos, A. C. B dos. (2017). Psicodinâmica do trabalho nas fases do capitalismo: análise comparativa do taylorismo-fordismo e do Toyotismo nos contextos do capitalismo burocrático e do capitalismo flexível. Revista Ciências Administrativas, 23(1), 186-216. doi: 10.5020/2318- 0722.23.1.186-216 Uchôa-de-Oliveira, F. M. (2020). Saúde do trabalhador e o aprofundamento da uberização do trabalho em tempos de pandemia. Revista Brasileira de Saúde Ocupacional, 45. doi: 10.1590/2317-6369000012520 Souza, L. K. (2019). Pesquisa com análise qualitativa de dados: conhecendo a Análise Temática. Arquivos Brasileiros de Psicologia, 71(2), 51-67. doi: 10.36482/1809- 5267.ARBP2019v71i2p.51-67 Valente, E., Patrus, R., & Córdova Guimarães, R. (2019). Sharing economy: becoming an Uber driver in a developing country. Revista de Gestão, 26(2), 143-160. doi: 10.1108/REGE-08-2018-0088 Valente, E., Patrus, R., & Córdova Guimarães, R. (2019). Sharing economy: becoming an Uber driver in a developing country. Revista de Gestão, 26(2), 143-160. doi: 10.1108/REGE-08-2018-0088 Declaração de Direito Autoral A submissão de originais para este periódico implica na transferência, pelos autores, dos direitos de publicação impressa e digital. Os direitos autorais para os artigos publicados são do autor, com direitos do periódico sobre a primeira publicação. Os autores somente poderão utilizar os mesmos resultados em outras publicações indicando claramente este periódico como o meio da publicação original. Em virtude de sermos um periódico de acesso aberto, permite-se o uso gratuito dos artigos em aplicações educacionais e científicas desde que citada a fonte conforme a licença CC-BY da Creative Commons. Declaração de Direito Autoral Dados sobre os autores: Dados sobre os autores: - Jefferson João dos Santos: Graduado em Psicologia pela Universidade Comunitária da Região de Chapecó, Especialista em Gestão Estratégia em Recursos Humanos e Mestre em Psicologia pela Faculdade Meridional IMED. Atualmente é Assessor de Relacionamento na Unidade Central de Educação Faem Faculdade. - Gabriel Bacarol Kerber: Psicólogo e Mestre em Psicologia pela Faculdade Meridional IMED. - Gabriel Bacarol Kerber: Psicólogo e Mestre em Psicologia pela Faculdade Meridional IMED. - Vanessa Rissi: Psicóloga, Doutora em Psicologia pela Unisinos, Mestre em Saúde Coletiva pela Unisinos e Especialista em Gestão de Pessoas pela Universidade de Passo Fundo. Professora no Programa de Pós-Graduação em Psicologia na Faculdade Meridional IMED. - Vanessa Rissi: Psicóloga, Doutora em Psicologia pela Unisinos, Mestre em Saúde Coletiva pela Unisinos e Especialista em Gestão de Pessoas pela Universidade de Passo Fundo. Professora no Programa de Pós-Graduação em Psicologia na Faculdade Meridional IMED. Declaração de Direito Autoral A submissão de originais para este periódico implica na transferência, pelos autores, dos direitos de publicação impressa e digital. Os direitos autorais para os artigos publicados são do autor, com direitos do periódico sobre a primeira publicação. Os autores somente poderão utilizar os mesmos resultados em outras publicações indicando claramente este periódico como o meio da publicação original. Em virtude de sermos um periódico de acesso aberto, permite-se o uso gratuito dos artigos em aplicações educacionais e científicas desde que citada a fonte conforme a licença CC-BY da Creative Commons. 123 123 123 123 PSI UNISC, 6(2), 107-123, 2022.
https://openalex.org/W3164817092	https://ruj.uj.edu.pl/xmlui/bitstream/handle/item/274242/strona_et-al_morphology_and_position_of_the_right_atrioventricular_valve_2021.pdf?sequence=1&isAllowed=y	English	null	Morphology and Position of the Right Atrioventricular Valve in Relation to Right Atrial Structures	Diagnostics	2,021	cc-by	7,205	Article Morphology and Position of the Right Atrioventricular Valve in Relation to Right Atrial Structures Jakub Hołda 1, Katarzyna Słodowska 1, Karolina Malinowska 1, Marcin Strona 2, Małgorzata Mazur 1, Katarzyna A. Jasi´nska 1, Aleksandra Matuszyk 1, Mateusz Koziej 1 , Jerzy A. Walocha 1 and Mateusz K. Hołda 1,3,* Jakub Hołda 1, Katarzyna Słodowska 1, Karolina Malinowska 1, Marcin Strona 2, Małgorzata Mazur 1, Katarzyna A. Jasi´nska 1, Aleksandra Matuszyk 1, Mateusz Koziej 1 , Jerzy A. Walocha 1 and Mateusz K. Hołda 1,3,* 1 HEART—Heart Embryology and Anatomy Research Team, Department of Anatomy, Jagiellonian University Medical College, 31-007 Cracow, Poland; jakub.p.holda@gmail.com (J.H.); kslodow11@gmail.com (K.S.); karolamali@wp.pl (K.M.); mmazur@uj.edu.pl (M.M.); kjasinska@uj.edu.pl (K.A.J.); amatuszyk@uj.edu.pl (A.M.); mateusz.koziej@gmail.com (M.K.); jwalocha@poczta.onet.pl (J.A.W.) 1 HEART—Heart Embryology and Anatomy Research Team, Department of Anatomy, Jagiellonian University Medical College, 31-007 Cracow, Poland; jakub.p.holda@gmail.com (J.H.); kslodow11@gmail.com (K.S.); karolamali@wp.pl (K.M.); mmazur@uj.edu.pl (M.M.); kjasinska@uj.edu.pl (K.A.J.); amatuszyk@uj.edu.pl (A.M.); mateusz.koziej@gmail.com (M.K.); jwalocha@poczta.onet.pl (J.A.W.) y j p ( ) j g ( ) j p p ( ) 2 Department of Forensic Medicine, Jagiellonian University Medical College, 31-007 Cracow, Poland; mstrona@uj.edu.pl j p 3 Department of Cardiovascular Sciences, University of Manchester, Manchester M13 9PL, UK * Correspondence: mkh@onet.eu; Tel./Fax: +48-124-229-511 j p 3 Department of Cardiovascular Sciences, University of Manchester, Manchester M13 9PL, UK * Correspondence: mkh@onet.eu; Tel./Fax: +48-124-229-511 Department of Cardiovascular Sciences, University of Manchester, Manchester M13 9PL, UK * Correspondence: mkh@onet.eu; Tel./Fax: +48-124-229-511 * Correspondence: mkh@onet.eu; Tel./Fax: +48-124-229-511 Abstract: The right atrioventricular valve (RAV) is an important anatomical structure that prevents blood backﬂow from the right ventricle to the right atrium. The complex anatomy of the RAV has lowered the success rate of surgical and transcatheter procedures performed within the area. The aim of this study was to describe the morphology of the RAV and determine its spatial position in relation to selected structures of the right atrium. We examined 200 randomly selected human adult hearts. All leaﬂets and commissures were identiﬁed and measured. The position of the RAV was deﬁned. Notably, 3-leaﬂet conﬁgurations were present in 67.0% of cases, whereas 4-leaﬂet conﬁgurations were present in 33.0%. Septal and mural leaﬂets were both signiﬁcantly shorter and higher in 4-leaﬂet than in 3-leaﬂet RAVs. Signiﬁcant domination of the muro-septal commissure in 3-leﬂet valves was noted. The supero-septal commissure was the most stable point within RAV circumference. In 3-leaﬂet valves, the muro-septal commissure was placed within the cavo-tricuspid isthmus area in 52.2% of cases, followed by the right atrial appendage vestibule region (20.9%). Keywords: coronary sinus ostium; right atrial appendage vestibule; terminal crest; tricuspid valve Received: 25 April 2021 Accepted: 24 May 2021 Published: 26 May 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Article Morphology and Position of the Right Atrioventricular Valve in Relation to Right Atrial Structures In 4-leaﬂet RAVs, the infero-septal commissure was located predominantly in the cavo-tricuspid isthmus area and infero-mural commissure was always located within the right atrial appendage vestibule region. The RAV is a highly variable structure. The supero-septal part of the RAV is the least variable component, whereas the infero-mural is the most variable. The number of detected RAV leaﬂets signiﬁcantly inﬂuences the relative position of individual valve components in relation to right atrial structures. Citation: Hołda, J.; Słodowska, K.; Malinowska, K.; Strona, M.; Mazur, M.; Jasi´nska, K.A.; Matuszyk, A.; Koziej, M.; Walocha, J.A.; Hołda, M.K. Morphology and Position of the Right Atrioventricular Valve in Relation to Right Atrial Structures. Diagnostics 2021, 11, 960. https://doi.org/ 10.3390/diagnostics11060960 Academic Editor: Michel Noutsias Received: 25 April 2021 Accepted: 24 May 2021 Published: 26 May 2021 diagnostics diagnostics diagnostics 1. Introduction The right atrioventricular valve (RAV) is an important anatomical structure of the human heart that prevents blood backﬂow from the right ventricle to the right atrium. RAV regurgitation is common, although it is mostly asymptomatic. It is typically diagnosed incidentally or when the pathology has led to severe clinical symptoms [1]. Disorders of the RAV are mainly treated with surgical techniques [2]. However, these procedures are challenging and have a high risk of complications [2–4]. Therefore, clinicians are exploring less invasive treatment alternatives [5,6]. Several promising techniques and devices are currently in development. These include the FORMA spacer device, the Trialign device, the Caval Valve Implantation (CAVI), the TriClip device and many others [5,7–9]. It is anticipated that minimally invasive procedures will become routine options for patients with contraindications to classical cardiac surgery [6,7]. Copyright: © 2021 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/). g y The RAV is a sophisticated morphological entity. The complex anatomy of the RAV has lowered the success rate of surgical and transcatheter procedures performed within https://www.mdpi.com/journal/diagnostics Diagnostics 2021, 11, 960. https://doi.org/10.3390/diagnostics11060960 Diagnostics 2021, 11, 960 2 of 9 the area [10,11]. For instance, although the RAV is commonly referred to as the tricuspid valve, a recent functional anatomy study found that almost 40% of examined specimens had four distinct leaﬂets [10]. The infero-mural region of the valve is the most variable area of right atrioventricular valve. The RAV had a lot more diversity in the shapes and sizes of its scallops, commissures, tendinous chords and papillary muscles compared to mitral valve [10,12]. The RAV leaﬂets were found to be anatomically divided into scallops in the irregular fashion with the septal leaﬂet being the most subdivided part of the RAV. Additionally, the functional scallops may be found within the RAV leaﬂets (mainly in septal and mural leaﬂets) [10]. Furthermore, the RAV annulus was found to have an irregular and nonplanar shape [10,13]. Nevertheless, no signiﬁcant differences in the overall annular sizes between valves eliciting different leaﬂet conﬁgurations were observed in the previous study [10]. Finally, there was even signiﬁcant variability in the anatomic landmarks and locations of each leaﬂet and commissure [10]. 2. Methods This study was approved on 23 April 2020 by the Bioethical Committee of the Jagiel- lonian University in Cracow, Poland (No. 1072.6120.90.2020), and its study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki. 1. Introduction Understanding the detailed morphology of the RAV and the spatial relationships between important anatomic landmarks is crucial for the success of invasive cardiac procedures. The RAV annulus size and geometry has a crucial impact on surgical repair procedures, while leaﬂets and commissure conﬁgurations, together with their location within the RAV oriﬁce, inﬂuence transcatheter procedures, especially with coaptation devices [5,9]. Therefore, the aim of this study was to gain a better understanding of the anatomy of the RAV and describe the spatial relationships between selected structures of the right atrium. We hope that the collected data will help to enhance the quality and safety of cardiac surgeries and the minimally invasive transcatheter procedures conducted within the RAV area. 2.1. Study Population We examined 200 random human adult hearts. The specimens were collected during routine forensic autopsies. The hearts were taken from Caucasian individuals (of which 22.0% were female) ranging in age from 18 to 94 (mean age was 46.9 ± 17.9 years). The donors had a mean body mass index (BMI) of 26.6 ± 4.5 kg/m2 and an average body surface area (BSA) of 1.9 ± 0.2 m2. Their primary causes of death were suicide, murder, trafﬁc accidents and home accidents. We excluded donors with severe anatomical defects, heart trauma, heart grafts, severe macroscopic pathologies and those with signs of cadaveric decomposition. 2.2. Dissection and Measurements The hearts and the proximal parts of the main vessels were dissected from each chest cavity in a routine manner. Before being immersed in 10% paraformaldehyde solution, the hearts were weighed using a 0.5 g precision electronic laboratory scale (SATIS, BSA-L Laboratory, Poland). The right atrium was opened through an intercaval incision extending from the oriﬁce of the superior vena cava to the oriﬁce of the inferior vena cava. The detailed morphology of each valve was examined. We identiﬁed, labelled, and counted all the commissures and leaﬂets. A leaﬂet was deﬁned as a tissue fold located between two commissures. Using the attitudinally correct terminology of human anatomy, we identiﬁed 4 leaﬂets: a superior, a septal, a mural and an inferior leaﬂet (although the latter was only present in hearts with a 4-leaﬂet conﬁguration) [10,14]. Commissures were deﬁned as areas within the valve located between two adjacent leaﬂets. They had a characteristic arched appearance and were accompanied by a fan-shaped cord originating from the respective papillary muscle (or septal band) located directly below the commis- sure [10,15,16]. We observed the following commissures: the supero-septal commissure Diagnostics 2021, 11, 960 3 of 9 (found between the superior and the septal leaﬂet); the supero-mural commissure (found between the superior and the mural leaﬂet); the muro-septal commissure (found between the mural and the septal leaﬂet in the 3-leaﬂet conﬁguration); the infero-septal commissure (found between the inferior and the septal leaﬂet in the 4-leaﬂet conﬁguration) and the infero-mural commissure (found between the inferior and the mural leaﬂet in the 4-leaﬂet conﬁguration) (Figure 1) [10]. (found between the superior and the septal leaﬂet); the supero-mural commissure (found between the superior and the mural leaﬂet); the muro-septal commissure (found between the mural and the septal leaﬂet in the 3-leaﬂet conﬁguration); the infero-septal commissure (found between the inferior and the septal leaﬂet in the 4-leaﬂet conﬁguration) and the infero-mural commissure (found between the inferior and the mural leaﬂet in the 4-leaﬂet conﬁguration) (Figure 1) [10]. Figure 1. Schematic views of the RAV in 3-leaﬂet and 4-leaﬂet conﬁgurations. Performed measurements are marked (A—terminal crest to muro-septal or infero-septal commissure, B—coronary sinus ostium to supero-septal commissure, C—coronary sinus ostium to muro-septa or infero-septal commissure, D—right atrial appendage vestibule superior isthmus to the supero-septal commissure, and E—right atrial appendage vestibule superior isthmus to supero-mural commissure) with the relative proportions preserved. 3.1. The RAV Morphology supero-mural muro-septal ** Results are presented in Table 1. In 67.0% of specimens, the RAV had a 3-leaﬂet conﬁguration (Figure 2A). In the remaining 33.0% of hearts, the RAV had four leaﬂets (Figure 2B). Valves with four leaﬂets had signiﬁcantly larger perimeters than those with three leaﬂets (119.2 ± 11.1 vs. 109.3 ± 11.3 mm, p = 0.001). No difference in heart weight was observed between two conﬁgurations (407.9 ± 86.5 vs. 419.3 ± 75.8 g, p = 0.363). There was a correlation between the perimeter of the valve and the donors’ BMI in 3-leaﬂet heart valves (r = 0.31, p < 0.001), but no such association was present in the 4-leaﬂet conﬁguration (r = 0.01, p = 0.922). In hearts with a 3-leaﬂet conﬁguration, the lengths of the superior leaﬂet and of the septal leaﬂet were similar, whereas the length of the mural leaﬂet was about twice as short (Table 1, p > 0.05). The same trends were noted in 4-leaﬂet heart valves (Table 1, p > 0.05). Interestingly, the individual leaﬂet height did not differ much within the same valve conﬁguration (Table 1, p > 0.05). When present, the inferior leaﬂet was comparable in size to the mural leaﬂet (both in length and in height) (Table 1, p > 0.05). Signiﬁcant differences in leaﬂet size were observed between the two valve conﬁgurations (Table 1). The superior leaﬂet had the least variability, and its length and height were comparable in 3- and 4-leaﬂet valves. Meanwhile, the septal and mural leaﬂets were both signiﬁcantly shorter and higher in the 4-leaﬂet RAVs (Table 1). ) muro septal 14.0 ± 4.8 (4.5 25.9) infero-septal * - 7.9 ± 3.0 (2.9–18.2) - infero-mural * - 7.7 ± 2.4 (3.5–13.5) - s to muro-septal or missure (mm) † 5.8 ± 9.4 (−15.6–29.7) 13.5 ± 6.4 (0.0–27.9) <0.001 to muro-septal or missure (mm) ‡ 16.1 ± 9.0 (−12.7–33.2) 7.7 ± 6.3 (-10.5–22.6) <0.001 to supero-septal com- mm) 20.1 ± 5.6 (5.3–36.7) 20.5 ± 5.1 (9.8–29.2) 0.62 mus to supero-septal e (mm) 7.3 ± 3.8 (1.3–14.3) 6.2 ± 5.3 (1.4–14.8) 0.09 mus to supero-mural e (mm) 33.9 ± 11.2 (17.5–72.7) 31.1 ± 9.6 (11.9–51.6) 0.08 on only; *—in 3-leaflet configuration; †—negative values represent commissures located within ge vestibule; ‡—negative values represent commissures located closer to the septal region than igure 1 shows schematic sites of measurements. RAAV—right atrial appendage vestibule. Bold ignificance. Figure 2. 2.2. Dissection and Measurements CSO—coronary sinus ostium, CTI—cavo-tricuspid isthmus, InfI—inferior isthmus, IVC—inferior vena cava, LH—leaﬂet height, LL—leaﬂet length, RAA—right atrial appendage, SupI—superior isthmus. Figure 1. Schematic views of the RAV in 3-leaﬂet and 4-leaﬂet conﬁgurations. Performed measurements are marked (A—terminal crest to muro-septal or infero-septal commissure, B—coronary sinus ostium to supero-septal commissure, C—coronary sinus ostium to muro-septa or infero-septal commissure, D—right atrial appendage vestibule superior isthmus to the supero-septal commissure, and E—right atrial appendage vestibule superior isthmus to supero-mural commissure) with the relative proportions preserved. CSO—coronary sinus ostium, CTI—cavo-tricuspid isthmus, InfI—inferior isthmus, IVC—inferior vena cava, LH—leaﬂet height, LL—leaﬂet length, RAA—right atrial appendage, SupI—superior isthmus. We measured the length of each leaﬂet and commissure bordering the hinge line of the RAV annulus (Figure 1). The perimeter of the annulus was calculated as the sum of the lengths of the leaﬂets and commissures. Leaﬂet height was deﬁned as the maximum distance from the hinge line to the free edge (see Figure 1). To determine the spatial position of the RAV, we described the location of the septal leaﬂet and its adjacent commissures with respect to selected landmarks within the right atrium. We measured the distance from the terminal crest (the inferior isthmus of the right atrial appendage vestibule) to the muro-septal commissure (in the 3-leaﬂet conﬁguration) and from the terminal crest to the infero-septal commissure in the 4-leaﬂet conﬁguration (see A). We also measured the distance from the center point of the ostium of the coronary sinus to the center point of the supero-septal and the muro-septal commissures (in hearts with a 3-leaﬂet conﬁguration) or the infero-septal commissure (in hearts with a 4-leaﬂet conﬁguration) (see B and C). Lastly, we measured the distance from the superior isthmus of the right atrial appendage vestibule to the supero-septal commissure and to the supero-mural commissure (see D and E) (Figure 1). Linear measurements were obtained using 0.03 mm precision electronic calipers (YATO, YT–7201, Wrocław, Poland). All measurements were performed by two indepen- dent researchers to reduce human bias. If results obtained by the two researchers differed by more than 10%, the sample was reassessed. The mean of the two new measurements was calculated, approximated to the tenth decimal place, and reported as the new ﬁnal value. 4 of 9 Diagnostics 2021, 11, 960 2.3. Statistical Analysis 2.3. Statistical Analysis The data are presented as the mean ± the standard deviation (SD), along with the minimum and maximum values (range) for continuous variables, or as percentages (%) for categorical variables. A p-value < 0.05 was considered statistically signiﬁcant, Shapiro–Wilk tests were used to determine if the quantitative data were normally distributed. Levene’s test was performed to verify relative homogeneity of variance. Student’s t-tests and the Mann–Whitney U tests were used for statistical comparisons. Correlation coefﬁcients were calculated; to detect a simple correlation (r = 0.25) with 80% power and a 5% signiﬁcance level (two-tailed; α = 0.05; β = 0.2), the minimal sample size was set at 123 cases. The analysis of variance (ANOVA) or non-parametric Kruskal–Wallis test was used to compare values between different groups. Detailed comparisons were performed using Tukey’s post hoc analyses. Qualitative variables were compared using χ2 (chi-squared) tests of proportions with Bonferroni corrections to account for the multiple comparisons. Statistical analyses were performed using StatSoft STATISTICA 13.1 software (StatSoft Inc., Tulsa, OK, USA). 5 of 9 inferior leaflet - 14.3 ± 4.6 (8.2–26.5) - superior leaflet 12.6 ± 4.2 (3.5–23.7) 13.7 ± 5.1 (4.6–26.7) 0.11 septal leaflet 11.8 ± 3.9 (3.3–20.8) 14.3 ± 4.5 (5.2–22.8) <0.001 mural leaflet 11.9 ± 3.6 (4.0–20.7) 13.6 ± 4.0 (4.4–23.8) 0.003 inferior leaflet * - 12.1 ± 8.6 (2.3–19.6) - 3.1. The RAV Morphology supero-mural muro-septal Photographs of a cadaveric heart specimens showing examples of (A) 3-leaflet and (B) 4-leaflet right atrioventricular valve. CTI—cavo-trisuspid isthmus, RAA—right atrial appendage. C i l th i d id bl ithi th 3 l fl t h t l Th Figure 2. Photographs of a cadaveric heart specimens showing examples of (A) 3-leaﬂet and (B) 4-leaﬂet right atrioventricular valve. CTI—cavo-trisuspid isthmus, RAA—right atrial appendage. Figure 2. Photographs of a cadaveric heart specimens showing examples of (A) 3-leaflet and (B) 4-leaflet right atrioventricular valve. CTI—cavo-trisuspid isthmus, RAA—right atrial appendage. Figure 2. Photographs of a cadaveric heart specimens showing examples of (A) 3-leaﬂet and (B) 4-leaﬂet right atrioventricular valve. CTI—cavo-trisuspid isthmus, RAA—right atrial appendage. 5 of 9 Diagnostics 2021, 11, 960 Table 1. Morphometric observations of structure and position of right atrioventricular valve in 3-leaﬂet and 4-leaﬂet conﬁgurations (mean ± SD with range). 3.1. The RAV Morphology supero-mural muro-septal Parameter 3-Leaﬂet Valves (n = 134) 4-Leaﬂet Valves (n = 66) p-Value Sex (% females) 20.9 24.2 0.597 Age (years) 47.1 ± 18.2 (18–94) 46.5 ± 18.4 (21–87) 0.827 BMI (kg/m2) 26.3 ± 4.8 (15.2–39.6) 27.5 ± 4.1 (17.4–37.8) 0.083 Heart weight (g) 407.9 ± 86.5 (230.0–606.5) 419.3 ± 75.8 (274.5–620.0) 0.363 Valve perimeter (mm) 109.3 ± 11.3 (86.6–156.8) 119.2 ± 11.1 (96.5–143.4) <0.001 Leaﬂet length (mm) superior leaﬂet 30.1 ± 9.2 (12.6–71.0) 28.8 ± 9.0 (11.5–50.5) 0.09 septal leaﬂet 30.5 ± 5.7 (17.7–46.6) 27.0 ± 5.7 (17.8–39.4) 0.005 mural leaﬂet 17.5 ± 6.2 (5.9–44.5) 14.8 ± 5.5 (7.2–28.0) <0.001 inferior leaﬂet * - 14.3 ± 4.6 (8.2–26.5) - Leaﬂet height (mm) superior leaﬂet 12.6 ± 4.2 (3.5–23.7) 13.7 ± 5.1 (4.6–26.7) 0.11 septal leaﬂet 11.8 ± 3.9 (3.3–20.8) 14.3 ± 4.5 (5.2–22.8) <0.001 mural leaﬂet 11.9 ± 3.6 (4.0–20.7) 13.6 ± 4.0 (4.4–23.8) 0.003 inferior leaﬂet * - 12.1 ± 8.6 (2.3–19.6) - Commissure length (mm) supero-septal 9.6 ± 3.4 (5.1–21.5) 8.6 ± 4.7 (4.2–24.5) 0.17 supero-mural 9.9 ± 4.0 (3.5–21.4) 7.5 ± 2.2 (4.0–14.3) <0.001 muro-septal ** 14.0 ± 4.8 (4.5–25.9) - - infero-septal * - 7.9 ± 3.0 (2.9–18.2) - infero-mural * - 7.7 ± 2.4 (3.5–13.5) - A—RAAV inferior isthmus to muro-septal ** or infero-septal * commissure (mm) † 5.8 ± 9.4 (−15.6–29.7) 13.5 ± 6.4 (0.0–27.9) <0.001 B—coronary sinus ostium to muro-septal ** or infero-septal * commissure (mm) ‡ 16.1 ± 9.0 (−12.7–33.2) 7.7 ± 6.3 (-10.5–22.6) <0.001 C—coronary sinus ostium to supero-septal commissure (mm) 20.1 ± 5.6 (5.3–36.7) 20.5 ± 5.1 (9.8–29.2) 0.62 D—RAAV superior isthmus to supero-septal commissure (mm) 7.3 ± 3.8 (1.3–14.3) 6.2 ± 5.3 (1.4–14.8) 0.09 E—RAAV superior isthmus to supero-mural commissure (mm) 33.9 ± 11.2 (17.5–72.7) 31.1 ± 9.6 (11.9–51.6) 0.08 —n 4-leaﬂet conﬁguration only; —in 3-leaﬂet conﬁguration; †—negative values represent commissures located within the right atrial appendage vestibule; ‡—negative values represent commissures located closer to the septal region than coronary sinus ostium; Figure 1 shows schematic sites of measurements. RAAV—right atrial appendage vestibule. Bold values denote statistical signiﬁcance. —n 4-leaﬂet conﬁguration only; —in 3-leaﬂet conﬁguration; †—negative values represent commissures located within the right atrial appendage vestibule; ‡—negative values represent commissures located closer to the septal region than coronary sinus ostium; Figure 1 shows schematic sites of measurements. RAAV—right atrial appendage vestibule. Bold values denote statistical signiﬁcance. Commissure lengths varied considerably within the 3-leaﬂet heart valves. 3.1. The RAV Morphology supero-mural muro-septal The muro- septal commissure was substantially larger than the two remaining commissures (Table 1, p < 0.001). Such length disparities were not observed in the 4-leaﬂet conﬁguration, where all four commissures had comparable lengths (Table 1, p > 0.05). Furthermore, the supero- mural commissure was signiﬁcantly longer in the 3-leaﬂet heart valve when compared to the 4-leaﬂet heart valve (9.9 ± 4.0 vs. 7.5 ± 2.2 mm, p < 0.001). ( p ) Age, sex and heart weight variables did not inﬂuence RAV conﬁguration, leaﬂet size or commissure length (all r < 0.2 and p > 0.05). 3.2. The Spatial Position of the RAV The number of detected leaﬂets in the RAV was found to affect the relative position of the valve components. The supero-septal commissure was located on the septal side of the right atrium in proximity to the apex of Koch’s triangle, and its location was the most stable point within the RAV circumference. Its position remained unchanged when comparing Diagnostics 2021, 11, 960 6 of 9 osition side of the 3- and 4-leaﬂet heart valves (Table 1, p > 0.05). In all examined hearts, the supero-septal commissure was located on the left-superior side of the ostium of the coronary sinus (Figure 1). The supero-septal commissure was never present within the vestibule of the right atrial appendage. This contrasted with the location of the supero-mural commissure, which was always found in the vestibule’s middle sector. We observed a strong positive correlation between the length of the supero-septal commissure and the distance between the commissure and the ostium of the coronary sinus (r = 0.51, p < 0.001). No association was detected between the distance from the commissure to the ostium of the coronary sinus and the septal leaﬂet length (r = 0.12, p = 0.31). paring the 3- and 4-leaflet heart valves (Table 1, p > 0.05). In all examined hearts, the su- pero-septal commissure was located on the left-superior side of the ostium of the coronary sinus (Figure 1). The supero-septal commissure was never present within the vestibule of the right atrial appendage. This contrasted with the location of the supero-mural commis- sure, which was always found in the vestibule’s middle sector. We observed a strong pos- itive correlation between the length of the supero-septal commissure and the distance be- tween the commissure and the ostium of the coronary sinus (r = 0.51, p < 0.001). No asso- ciation was detected between the distance from the commissure to the ostium of the cor- onary sinus and the septal leaflet length (r = 0.12, p = 0.31). p g ( p ) The location of commissures found within the infero-mural aspect of the RAV annulus (the muro-septal and the infero-septal commissures) varied considerably, not only between different RAV conﬁgurations but also between individual hearts with the same number of RAV leaﬂets. In the 3-leaﬂet heart valves, the muro-septal commissure was located further away from the ostium of the coronary sinus than the infero-septal commissure in the 4-leaﬂet conﬁguration (16.1 ± 9.0 vs. 3.2. The Spatial Position of the RAV 7.7 ± 6.3 mm, p < 0.001). The opposite trend was observed for the commissure’s distance to the terminal crest (5.8 ± 9.4 vs. 13.5 ± 6.4 mm, respectively; Table 1, p < 0.001). The location of the muro-septal commissure differed signiﬁcantly: in 52.2% of specimens, it was located in the area of the cavo-tricuspid isthmus (situated between the ostium of the coronary sinus and the terminal crest); in 9.0% of hearts, it was situated at the level or to the left of the ostium of the coronary sinus; in 17.9%, it was at the level of the terminal crest; in the remaining 20.9% of cases, it was found within the vestibule of the right atrial appendage (Figure 3). Additionally, there was a positive correlation between the segment from the muro-septal commissure to the terminal crest and the superior leaﬂet length (r = 0.32, p = 0.008). Meanwhile, this same segment had a negative correlation with septal leaﬂet length (r = −0.38, p = 0.001). y p g p The location of commissures found within the infero-mural aspect of the RAV annu- lus (the muro-septal and the infero-septal commissures) varied considerably, not only be- tween different RAV configurations but also between individual hearts with the same number of RAV leaflets. In the 3-leaflet heart valves, the muro-septal commissure was located further away from the ostium of the coronary sinus than the infero-septal com- missure in the 4-leaflet configuration (16.1 ± 9.0 vs. 7.7 ± 6.3 mm, p < 0.001). The opposite trend was observed for the commissure’s distance to the terminal crest (5.8 ± 9.4 vs. 13.5 ± 6.4 mm, respectively; Table 1, p < 0.001). The location of the muro-septal commissure dif- fered significantly: in 52.2% of specimens, it was located in the area of the cavo-tricuspid isthmus (situated between the ostium of the coronary sinus and the terminal crest); in 9.0% of hearts, it was situated at the level or to the left of the ostium of the coronary sinus; in 17.9%, it was at the level of the terminal crest; in the remaining 20.9% of cases, it was found within the vestibule of the right atrial appendage (Figure 3). Additionally, there was a positive correlation between the segment from the muro-septal commissure to the terminal crest and the superior leaflet length (r = 0.32, p = 0.008). 4. Discussion Despite its important function, the RAV is often considered as the “forgotten valve”. However, our study further shows the complexity and variability of the RAV’s morphology. This valve is much more irregular than its sister valve, the mitral valve, which has a remarkably more simple and predictable structure [15]. While the mitral valve is almost always composed of a 2-leaﬂet conﬁguration, the RAV often presents with a 4-leaﬂet conﬁguration. In the scientiﬁc literature, the RAV is often referred to as the “tricuspid valve”, although we strongly suggest avoiding this nomenclature, since it may mistakenly suggest the valve’s construction [10]. Moreover, before attempting any interventions within the RAV region, it is important to determine the amount of leaﬂets using either echocardiography or cardiac computed tomography [17–19]. Finally, it is important to know that there are considerable differences in leaﬂet and commissure sizes between 3- and 4-leaﬂet RAVs (Table 1). Our study has shown that the supero/septal region of the RAV is the least variable area, whereas the infero/mural region differs signiﬁcantly between 3-and 4-leaﬂet RAVs. It seems that the formation of the additional fourth leaﬂet takes place at the expense of the size of the muro-septal commissure and of the septal leaﬂet, which also explains the shifted position of the muro-septal and infero-septal commissures (Table 1). Besides the variable morphology of the RAV’s leaﬂets and commissures, there are other causes that affect the valve’s hemodynamic characteristics. Taken together, they make RAV imaging and interventions (both surgical and percutaneous) challenging [13]. The RAV annulus is a relatively large, ﬂexible, fragile and highly irregular structure, and it is easily affected by changes in the shape and size of the right ventricle [20]. The nonplanarity of the RAV annulus and its heterogeneous regional behavior may have a strong association with leaﬂet conﬁguration. This study has shown that there is a signiﬁcant difference in the size of the annulus between 3- and 4-leaﬂet valves. Initially larger right atrioventricular oriﬁce size may force the creation of the additional leaﬂet and commissure to seal the oriﬁce that are located with the inferior/mural region of the annulus [10,19]. g When planning therapeutic procedures on the RAV, it is important to determine its morphology, position and neighboring cardiac structures [17]. 3.2. The Spatial Position of the RAV Meanwhile, this same segment had a negative correlation with septal leaflet length (r = −0.38, p = 0.001). Figure 3. Photographs of cadaveric heart specimens showing examples of (A) 3-leaflet and (B) 4- leaflet right atrioventricular valves. CSO—coronary sinus ostium, RAA—right atrial appendage, TC—terminal crest. Figure 3. Photographs of cadaveric heart specimens showing examples of (A) 3-leaﬂet and (B) 4-leaﬂet right atrioventricular valves. CSO—coronary sinus ostium, RAA—right atrial appendage, TC—terminal crest. Figure 3. Photographs of cadaveric heart specimens showing examples of (A) 3-leaflet and (B) 4- leaflet right atrioventricular valves. CSO—coronary sinus ostium, RAA—right atrial appendage, TC—terminal crest. Figure 3. Photographs of cadaveric heart specimens showing examples of (A) 3-leaﬂet and (B) 4-leaﬂet right atrioventricular valves. CSO—coronary sinus ostium, RAA—right atrial appendage, TC—terminal crest. In 4-leaﬂet RAVs, the infero-septal commissure was predominantly located in the cavo-tricuspid isthmus area (81.8% of cases). It was never located within the area of the vestibule of the right atrial appendage. In the remaining 12.1% of hearts, the commissure was located at the level of the ostium of the coronary sinus, while in 6.1% of cases, it Diagnostics 2021, 11, 960 7 of 9 pointed towards the septal direction. The spatial position of the infero-septal commissure in the 4-leaﬂet valve was inﬂuenced by the length of the septal leaﬂet (there was a positive correlation with the commissure’s distance to the ostium of the coronary sinus (r = 0.51, p = 0.03) and a negative correlation with the commissure’s distance to the terminal crest (r = −0.37, p = 0.03). The infero-mural commissure was always located within the area of the right atrial appendage vestibule. pointed towards the septal direction. The spatial position of the infero-septal commissure in the 4-leaﬂet valve was inﬂuenced by the length of the septal leaﬂet (there was a positive correlation with the commissure’s distance to the ostium of the coronary sinus (r = 0.51, p = 0.03) and a negative correlation with the commissure’s distance to the terminal crest (r = −0.37, p = 0.03). The infero-mural commissure was always located within the area of the right atrial appendage vestibule. The relative positions of the components of the RAV were not affected by the donors’ age, sex, cardiac weight or any other anthropometric features (weight, height, BMI) (all r < 0.2 and p > 0.05). 4. Discussion The RAV annulus has important anatomical relationships with the right coronary artery, the acute marginal branch, the small cardiac vein (located within the vestibule of the right atrial appendage), the terminal crest, the cavo-tricuspid isthmus, the coronary sinus (and its ostium), the atrio-ventricular node and the aortic valve [21–24]. The above-mentioned structures are subject to injury, and clinicians must factor in their respective positions before attempting any invasive procedures. Although the inferior/mural region of the RAV has the most unpredictable morphology, it should be considered the safest area for interventions due to its remoteness from crucial heart structures (the only exception being the right coronary artery) [25]. y Transcatheter RAV annulus reduction techniques involve placing special pledgeted sutures on the superior and mural leaﬂet. These procedures reduce the entire circumference of the valve annulus and seal the oriﬁce. This, in turn, improves the RAV’s hemodynamic properties [26,27]. Our observations have shown that the location of the supero-mural commissure is quite consistent. It is usually located near the middle isthmus of the vestibule of the right atrial appendage and is relatively easy to discern. However, the locations of Diagnostics 2021, 11, 960 8 of 9 the commissures and leaﬂets of the mural/inferior region of the valve are not always clear. Even RAVs with the same number of leaﬂets present with a lot of heterogeneity (see Table 1). In 3-leaﬂet valves, the larger septal leaﬂet ends much closer to the terminal crest and further away from the ostium of the coronary sinus when compared to 4-leaﬂet RAVs. The muro-septal commissure in a 3-leaﬂet valve may be located anywhere between the left side of Koch’s triangle and the right side of the middle isthmus of the right atrial appendage vestibule. In 4-leaﬂet valves, the smaller septal leaﬂet pushes the inferior leaﬂet to the left into the territory of the cavo-tricuspid isthmus but never to the region of the vestibule of the right atrial appendage. The presence of an additional inferior leaﬂet and its adjacent commissures shifts the mural leaﬂet to the right side of the valve. Therefore, we recommend determining the detailed morphology of leaﬂet valves before qualifying patients for invasive procedures. It is equally important to study the course of coronary vessels within the RAV region in order to avoid any damage to these vessels [17,25,28]. There are some limitations to this study. 4. Discussion The main one is that this study was performed on autopsied material preserved in paraformaldehyde solution. Consequently, there may have been slight changes to the size and shape of the studied hearts due to ﬁxation. Nevertheless, our earlier studies have demonstrated that using paraformaldehyde solution did not cause important changes in cardiac dimensions [29,30]. Furthermore, since we analyzed post-mortem material, we were not able to assess the behavior and dimensional changes of RAV components during the cardiac cycle. Additionally, we only investigated hearts from healthy donors and, therefore, our results may not be applicable to patients with structural valvular diseases. Despite these limitations, we consider that this study has provided insight into the morphological and topographical analyses of the RAV, as well as into the relationships between individual RAV components and right atrial structures. Author Contributions: Conceptualization, J.H.; data curation, J.H., K.S., M.M. and A.M.; formal analysis, K.S., M.M., K.M. and M.K.; investigation, J.H., K.S., M.M. and A.M.; methodology, J.H., M.S., M.M. and M.K.H.; project administration, J.A.W. and M.K.H.; resources, J.H., M.S. and M.K.H.; supervision, J.A.W. and M.K.H.; visualization, J.H., K.A.J. and K.M.; writing—original draft, J.H.; writing—review and editing, K.A.J. and M.K.H. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Funding: This research received no external funding. Institutional Review Board Statement: This study was approved on 23 April 2020 by the Bioethical Committee of the Jagiellonian University in Cracow, Poland (No 1072.6120.90.2020). Institutional Review Board Statement: This study was approved on 23 April 2020 by the Bioethical Committee of the Jagiellonian University in Cracow, Poland (No 1072.6120.90.2020). Informed Consent Statement: Consent not required under local law. Informed Consent Statement: Consent not required under local law. Data Availability Statement: Data are available from the authors upon reasonable request. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. 7. Rodes-Cabau, J.; Hahn, R.T.; Latib, A.; Laule, M.; Lauten, A.; Maisano, F.; Schofer, J.; Campelo-Parada Transcatheter Therapies for Treating Tricuspid Regurgitation. J. Am. Coll. Cardiol. 2016, 67, 1829–1845. 4. Vassileva, C.M.; Shabosky, J.; Boley, T.; Markwell, S.; Hazelrigg, S. Tricuspid valve surgery: The past 10 y Inpatient Sample (NIS) database. J. Thorac Cardiovasc. Surg. 2012, 143, 1043–1049. [CrossRef] 7. Rodes-Cabau, J.; Hahn, R.T.; Latib, A.; Laule, M.; Lauten, A.; Maisano, F.; Schofer, J.; Campelo-Parada, F.; Puri, R.; Vahanian, A. Transcatheter Therapies for Treating Tricuspid Regurgitation. J. Am. Coll. Cardiol. 2016, 67, 1829–1845. [CrossRef] 5. Asmarats, L.; Puri, R.; Latib, A.; Navia, J.L.; Rodés-Cabau, J. Transcatheter Tricuspid Valve Interventions: Landscape, Challenges and Future Directions. J. Am. Coll Cardiol. 2018, 71, 2935–2956. 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https://openalex.org/W3130081947	http://agritrop.cirad.fr/597267/7/597267-ed.pdf	English	null	Integrating end‐user preferences into breeding programmes for roots, tubers and bananas	International journal of food science & technology	2,021	cc-by	3,431	Summary “Consumers have their say: assessing preferred quality traits of roots, tubers and cooking bananas, and implications for breeding” special issue, brings together new knowledge about quality traits required for roots, tubers and bananas (RTB) varieties to successfully meet diverse user preferences and expectations, along the variety development and RTB value chains (production, processing, marketing, food prepara- tion, consumption). Key RTB crops in sub-Saharan Africa are cassava, yams, sweetpotatoes, potatoes and bananas/plantains. They are mainly consumed directly as boiled pieces or pounded in the form of smooth, not sticky, and stretchable dough. They are also stewed, steamed or fried. Cassava, the most widely grown RTB, is generally boiled, stewed or steamed in Eastern and Southern Africa, and in West and Central Africa is usually processed directly into derivative products, e.g. whole root fermentation through retting or heap fermentation; fermentation/dewatering of the mash. Biophysical and social knowl- edge presented in this issue help elaborate goals for both the processing unit operations (food scientist control) and variety traits (breeder control). In this article, we introduce the key root, tuber and banana (RTB) crops and their derived food products in sub-Saharan Africa (SSA), summarise the goals and methodologies of assessing end-user preferences aimed at breeding suitable varieties and highlight some key ﬁndings of the research reported in the special issue – Consumers have their say: assessing preferred quality traits of roots, tubers and cooking bananas, and implica- tions for breeding. This issue includes 23 original research papers, ﬁve review papers and one methodol- ogy paper. The authors are aﬃliated with national research programmes, universities, CGIAR centres (IITA (Nigeria), CIP (Peru) and the Alliance of Bioversity International and CIAT (Colombia)), NRI, CIRAD, NGOs and others. The core contributors pre- sent results from the RTBfoods project (Breeding roots tubers and banana products for end-user preferences https://rtbfoods.cirad.fr/) and/or the CGIAR Research Program on Roots, Tubers and Bananas https://www. rtb.cgiar.org/. The Bill & Melinda Gates Foundation https://www.gatesfoundation.org/ supports these devel- opments by investing in strategic RTB-related initia- tives in SSA. A call for papers by the journal editors elicited an additional six original research papers. Following a consultation with RTB experts from diﬀerent backgrounds and disciplines, 11 food prod- ucts particularly important for RTB-based staple diets in SSA were selected for the research reported in this issue. / j © 2021 CIRAD. International Journal of Food Science & Technology published by John Wiley & Sons Ltd on behalf of Institute of Food, Science and Technology (IFSTTF) 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. Dominique Dufour,1,2* Clair Hershey,3 Bruce R. Hamaker4 & Jim Lorenzen5 CIRAD, UMR Qualisud, Montpellier F-34398, France 1 CIRAD, UMR Qualisud, Montpellier F-34398, France 2 Qualisud, CIRAD, Institut Agro, Univ Montpellier, Avignon Universite, Universite de La Reunion, Montpellier, France 3 International Consultant, Flinton, PA, USA , Q , p , 2 Qualisud, CIRAD, Institut Agro, Univ Montpellier, Avignon Universite, Universite de La Reunion, Montp 3 International Consultant, Flinton, PA, USA 4 Whistler Center for Carbohydrate Research Purdue University, West Lafayette, IN, USA 5 Bill & Melinda Gates Foundation Discovery/Crop R&D Global Growth & Opportunity Agr 4 Whistler Center for Carbohydrate Research Purdue University, West Lafayette, IN, USA 5 Bill & Melinda Gates Foundation, Discovery/Crop R&D Global Growth & Opportunity, Agriculture, Seattle, WA, USA 4 Whistler Center for Carbohydrate Research Purdue University, West Lafayette, IN, USA 5 Bill & Melinda Gates Foundation, Discovery/Crop R&D Global Growth & Opportunity, Agriculture, Seattle, WA, USA doi:10.1111/ijfs.14911 International Journal of Food Science and Technology 2021, 56, 1071–1075 International Journal of Food Science and Technology 2021, 56, 1071–1075 International Journal of Food Science and Technology 2021, 56, 1071–1075 1071 Summary http://www.fao.org/faostat/en/#data/FBS RTB Rank Countries Cassava (Kg/capita/yr) Yams (Kg/capita/yr) Potato (Kg/Capita/Yr) Sweetpotato (Kg/Capita/Yr) Other Roots (aroids) (Kg/Capita/Yr) Total Starchy Roots (Kg/Capita/Yr) Bananas (All types) (Kg/Capita/Yr) Total RTB (Kg/Capita/Yr) 1 Ghana 232 155 0 4 28 419 125 544 2 Rwanda 104 4 93 84 12 297 209 505 3 Côte d'Ivoire 101 194 0 0 0 295 51 346 4 Malawi 90 0 52 134 0 277 44 320 5 Congo 269 3 2 1 10 285 31 315 6 Central African Republic 153 87 0 0 26 266 29 295 7 Benin 117 161 0 5 0 284 1 285 8 Gabon 76 64 1 2 20 163 110 273 9 Nigeria 119 101 5 14 8 246 15 261 10 Uganda 78 0 4 51 0 133 120 254 11 Sao Tome and Principe 6 10 2 0 28 46 196 243 12 Angola 158 0 15 34 0 207 35 242 13 Mozambique 193 0 9 23 0 225 16 241 14 Cameroon 99 13 6 13 24 156 77 233 15 United Republic of Tanzania 71 0 20 72 0 163 53 215 16 Togo 105 71 0 1 2 179 3 181 17 Guinea 97 9 5 15 7 133 49 181 18 Liberia 102 4 0 5 5 116 37 153 19 Namibia 0 0 19 0 127 146 2 148 20 Sierra Leone 107 0 0 23 0 130 6 136 21 Madagascar 92 0 5 20 5 122 13 135 Colour correspondence (Kg/capita/year): 200-300 150-200 100-150 50-100 0-50 Faostat, new food balance. http://www.fao.org/faostat/en/#data/FBS importance of cassava, yam and cooking bananas in SSA, with total RTB consumption well above 100 kg per capita per year in most countries. By way of com- parison, RTB consumption in Europe and North America is around 60 kg per capita per year, mainly potatoes. production and use trends (Scott, 2021), describe case studies of varietal change (replacing old varieties with new ones) (Thiele et al., 2021), summarise analytical methods for rapid quality assessment of yam and cas- sava using near-infrared spectroscopy (Alamu et al., 2021), describe gari end-user preferences (Awoyale et al., 2021) and review the literature to develop a pro- duct proﬁle for fried sweet potato in West Africa (Carey et al., 2021). p Faostat, new food balance. Summary Multidisciplinary teams of breeders, social scien- tists, food technologists and others used new methods for capturing user quality preferences (Forsythe et al., 2021; Teeken et al., 2021), through surveys and pro- duct evaluations conducted with farmers, traders, pro- cessors and consumers. It is notable that the Forsythe et al.’s paper (described further below) is the ﬁrst reg- istered report to be published by IJFST. p p y The original research papers bring together new knowledge about quality traits required for RTB vari- eties to successfully meet diverse user preferences and expectations, at each step along the variety develop- ment and RTB value chains (production, processing, marketing, food preparation, consumption). Papers address key problems in the breeding of RTB crops in SSA: inadequate understanding of requirements for diﬀerent end uses, missing information on the physico- chemical factors determining these requirements and absence of high-throughput screening protocols. The review papers provide a broad overview of RTB 1072 Editorial Table 1 RTB food supply ranking in principal producing countries of Africa (2017) (kg per capita per year) Faostat, new food balance. © 2021 CIRAD. International Journal of Food Science & Technology published by John Wiley & Sons Ltd on behalf of Institute of Food, Science and Technology (IFSTTF) International Journal of Food Science and Technology 2021 © 2021 CIRAD. International Journal of Food Science & Technology published by John Wiley & Sons Ltd on behalf of Institute of Food Science and Technology (IFSTTF) International Journal of Food Science and Technology 2021 © 2021 CIRAD. International Journal of Food Science & Technology published by John Wiley & Sons Ltd on behalf of Institute of Food, Science and Technology (IFSTTF) © 2021 CIRAD. International Journal of Food Science & Technology published by John Wiley & Sons Ltd on behalf of Institute of Food, Science and Technology (IFSTTF) International Journal of Food Science and Technology 2021 Summary http://www.fao.org/fa ostat/en/#data/FBS The ranking of countries (ﬁrst column) corresponds to the importance of total RTBs in local diets, as shown in the last column. FAO estimates that 342 mil- lion tonnes of RTBs were produced in Africa in 2018, 34% of which in Nigeria alone and 52% in West Africa (FAOSTAT, 2018). Cassava contribute 50%; yam, 21%; sweet potato, 8%; potato, 8%; cooking bananas, 6%; and dessert bananas, 4% of the African RTB production (FAOSTAT, 2018; Lescot, 2020). This major RTB consumption is distributed across the SSA tropical belt (Fig. 1). Roots, tubers and bananas play an essential role as staple foods in the tropics and subtropics, and particu- larly in Africa where they are culturally highly valued (Muimba-Kankolongo, 2018; Kennedy et al., 2019; Lebot, 2019; Petsakos et al., 2019; Scott, 2021). Key RTB crops in the African context are cassava, cooking bananas/plantains, yams, sweet potatoes and potatoes (FAO, 1990; Lebot, 2019). Due to their adaptability in diﬀerent ecosystems and high yields compared with local cereals, they are a primary and reliable source of calories for populations in SSA. However, these crops are highly perishable and diﬃcult to store or transport due to their bulkiness and high moisture content. In order to overcome these issues, and in the case of cas- sava, to reduce levels of the toxic cyanogenic com- pounds, a wide range of processes have been developed over millennia to convert fresh roots and tubers into a wide array of more stable and safe prod- ucts with preferred local traits (Hahn, 1989). Cassava, yam and plantain contribute to the devel- opment of original African cuisines without mimicking exogenous cuisine or the westernisation of African urban consumption. Bricas et al. (2016) showed the capacity of African urban markets to drive the devel- opment of RTB local food chains in relation to the preferences and needs of users. A research priority is to adapt RTB value chains to this urbanising context so that consumers can have ready access to these foods and more nutritious options (Petsakos et al., 2019; Thiele & Friedmann, 2020). p ( ) Table 1, based on FAO’s new food balance data FAOSTAT, (2018), shows the overwhelming dietary Editorial 1073 Editorial 1073 Figure 1 Map of RTB food supply in Africa, 2017 (kg per capita per year). [Colour ﬁgure can be viewed at wileyonlinelibrary.com] RTB food supply in Africa, 2017 (kg per capita per year). © 2021 CIRAD. International Journal of Food Science & Technology published by John Wiley & Sons Ltd on behalf of Institute of Food Science and Technology (IFSTTF) International Journal of Food Science and Technology 2021 International Journal of Food Science and Technology 2021 Summary [Colour ﬁgure can be viewed at wileyonlinelibrary.com] Figure 1 Map of RTB food supply in Africa, 2017 (kg per capita per year). [Colour ﬁgure can be viewed at w roasting (gari) and dough formation (eba). All these unit operations are part of the formulation of food products with quality traits strongly sought after by local consumers. Hahn (1989) showed the importance of these diﬀerent processing techniques in relation to the geographical distribution of key cassava products in SSA. Farmer processors and consumers have strong preferences in terms of raw material quality, depending on the processes developed for the formulation of each cassava-based food (Bechoﬀet al., 2018). In Africa, women play crucial roles in the produc- tion, processing and preparation of RTB crops, but their input is often under-valued in technology design that may aﬀect any of these activities (Rubin & Man- fre, 2014; Weltzien et al., 2020). Many of the studies in this special issue looked speciﬁcally at the current and potential women’s beneﬁts through their participa- tion in optimised product quality evaluation and criti- cal feedback to breeding programmes. RTBs are mainly consumed directly in SSA as boiled pieces or pounded in the form of smooth, not sticky, and stretchable dough. They are also stewed, steamed or fried and eaten with African traditional soups. Cas- sava is generally boiled, stewed or steamed in eastern and southern Africa, and in West Africa is usually pro- cessed directly after harvest into derivative products, for example whole root fermentation and softening through retting (fufu, lafun, b^aton, chikwangue); heap fermenta- tion (kwon); and fermentation/dewatering of the mash after grinding, followed by steam cooking (attieke), Breeding programmes for RTB crops initially gave priority to yield, dry matter and disease/pest resis- tance. As progress in these basic requirements has been met, they must now focus on end-product quality traits and processor and consumer preferences for quality characteristics. Processing ability and quality of end products are a common issue across improved varieties of RTBs, and this can contribute to low levels of varietal adoption and its subsequent beneﬁts (Thiele et al., 2021). International Journal of Food Science and Technology 2021 1074 Editorial Editorial To address these challenges, an interdisciplinary ﬁve- step methodology was developed for the RTBfoods project to better understand consumer preferences for quality characteristics among diverse user groups along the food chain (Forsythe et al., 2021). References Alamu, E.O., Nuwamanya, E., Cornet, D. et al. (2021). Near-in- frared spectroscopy applications for high-throughput phenotyping for cassava and yam: a review. International Journal of Food Science and Technology, Special Issue: Consumers have their say: assessing preferred quality traits of roots, tubers and cooking bana- nas, and implications for breeding 56, 1491–1501. Awoyale, W., Alamu, E.O., Chijioke, U. et al. (2021). A review of cassava semolina (Gari) end-user preferences and implications for varietal trait evaluation. International Journal of Food Science and Technology. Special Issue: Consumers have their say: assessing pre- ferred quality traits of roots, tubers and cooking bananas, and implications for breeding, 56, 1206–1222. Bechoﬀ, A., Tomlins, K.I., Fliedel, G. et al. (2018). Cassava traits and end-user preference: relating traits to consumer liking, sensory perception, and genetics. Critical Reviews in Food Science and Nutrition, 58, 547–567. Bricas, N., Tchamda, C. & Martin, P. (2016). Are the cities of West and Central Africa so dependant on food imports? Cahiers Agricul- tures, 25, 10. p According to research reported in this special issue, the main characteristics preferred by RTB consumers of boiled products are rapid softening and/or the development of friability or mealiness during cooking. For pounded products, the main objective is to obtain a smooth and stretchy dough with easy-to-swallow tex- ture, colours and taste/aroma typical of each RTB (matooke, pounded yam, eba). For cassava-derived products, colour, acidity due to fermentation, sticki- ness in the ﬁngers and texture of the dough in the mouth are key sensory traits. Additionally, the swel- ling characteristics of gari (in cold or hot water), attieke (during steaming) and fufu (in hot water) con- tribute to consumer preferences. Carey, T., Ssali, R. & Low, J. (2021). Literature review to guide development of a product proﬁle for fried sweetpotato in West Africa. International Journal of Food Science and Technology; Spe- cial Issue: Consumers have their say: assessing preferred quality traits of roots, tubers and cooking bananas, and implications for breeding, 56, 1410–1418. FAO (1990). Roots Tubers Plantains and Bananas in Human Nutri- tion. p. 182. Food and nutrition series. Rome: Food and Agricul- ture Organization of the United Nations. FAO food and nutrition series, no 24. ISBN 92-5102862-1. FAOSTAT (2018). FAO New food balance 2017, last consultation November 2020. http://www.fao.org/faostat/en/#data/QC Forsythe, L., Tufan, H., Bouniol, A., Kleih, U. & Fliedel, G. (2021). References An interdisciplinary and participatory methodology to improve user acceptability of root, tuber and banana varieties. International Jour- nal of Food Science and Technology; Special Issue: Consumers have their say: assessing preferred quality traits of roots, tubers and cooking bananas, and implications for breeding, 56, 1115–1123. In summary, this special issue deﬁnes and communi- cates an improved knowledge of the end-user preferred quality characteristics of RTB products. This knowledge helps elaborate goals for both the processing unit opera- tions (food scientist control) and variety traits (breeder control). A dynamic and close interaction among social scientists, food technologists, biochemists and plant breeders will contribute to better end-user acceptance and the successful deployment of technologies, for a pos- itive impact on RTB value chains in Africa. Hahn, S.K. (1989). An overview of African traditional cassava pro- cessing and utilization. Outlook on Agriculture, 18, 110–118. Kennedy, G., Raneri, J.E., Stoian, D. et al. (2019). Roots, tubers and bananas: contributions to food security. Encyclopedia of Food Security and Sustainability, 3, 231–255. Lebot, V. (2019). Tropical Root and Tuber Crops: Cassava, Sweet Potato, Yams and Aroids (Crop Production Science in Horticul- ture). 2nd edn, p. 544. Wallingford: CABI. ) p g ot, T. (2020). Banana genetic diversity. Fruitrop, 269, 98– Muimba-Kankolongo, A. (2018). Root and Tuber Crops. In: Food Crop Production by Smallholder Farmers in Southern Africa: Chal- lenges and Opportunities for Improvement. Chapter 9, Pp. 123–172. Amsterdam: Elsevier. Summary The methodology includes a state of knowledge review, consultations with key informants and rural communities, diagnostics of process performance (technical and socio-economic) with experienced processors, and consumer testing in urban and rural areas. Quality characteristics are then prioritised into a food product proﬁle. Through testing and analysis, individual quality characteristics are asso- ciated with biochemical and physical traits of RTB fresh materials, which can be shared with breeders to develop improved selection tools. Many of the papers in the special issue report result from the application of this methodology. Physicochemical scientists, food tech- nologists and breeders then need to jointly develop and implement high-throughput phenotyping systems to screen large numbers of potentially improved genotypes and achieve signiﬁcant genetic gains towards acceptable varieties for both producers and end users. Conceptualization (equal); Writing-review & editing (equal). Jim Lorenzen: Conceptualization (equal); Funding acquisition (lead); Project administration (supporting); Resources (lead); Supervision (support- ing); Validation (supporting). © 2021 CIRAD. International Journal of Food Science & Technology published by John Wiley & Sons Ltd on behalf of Institute of Food, Science and Technology (IFSTTF) International Journal of Food Science and Technology 2021 Author Contributions Dominique Dufour: Conceptualization (lead); Funding acquisition (lead); Investigation (supporting); Method- ology (supporting); Project administration (lead); Supervision (lead); Validation (equal); Writing-original draft (lead). Clair H Hershey: Conceptualization (sup- porting); Writing-original draft (supporting); Writing- review & editing (lead). Bruce Rankin Hamaker: Petsakos, A., Prager, S.D., Gonzalez, C.E. et al. (2019). Understanding the consequences of changes in the production frontiers for roots, tubers and bananas. Global Food Security, 20, 180–188. Rubin, D. & Manfre, C. (2014). Promoting gender-equitable agricul- tural value chains: issues, opportunities, and next steps. In: Gender in Agriculture (edited by A. Quisumbing, R. Meinzen-Dick, T. Raney, A. Kroppenstedt, J. Behrman & A. Peterman). Pp. 287–314 Dordrecht: Springer. © 2021 CIRAD. International Journal of Food Science & Technology published by John Wiley & Sons Ltd b h lf f I i f F d S i d T h l (IFSTTF) International Journal of Food Science and Technology 2021 © 2021 CIRAD. International Journal of Food Science & Technology published by John Wiley & Sons Ltd on behalf of Institute of Food, Science and Technology (IFSTTF) International Journal of Food Science and Technology 2021 1075 Editorial Consumers have their say: assessing preferred quality traits of roots, tubers and cooking bananas, and implications for breeding, 56, 1076–1092. Scott, G. (2021). A review of root, tuber and banana crops in devel- oping countries: past, present, and future. International Journal of Food Science and Technology; Special Issue: Consumers have their say: assessing preferred quality traits of roots, tubers and cooking bananas, and implications for breeding, 56, 1093–1114. Thiele, G. & Friedmann, M. (2020). The vital importance of RTB crops in the One CGIAR portfolio. RTB Research Brief 02. 4 p. Lima, Peru: CGIAR Research Program on Roots, Tubers and Bananas. https://cgspace.cgiar.org/handle/10568/109915, https:// cgspace.cgiar.org/bitstream/handle/10568/109915/The%20vital% 20importance%20of%20RTB%20crops_ResearchBrief02.pdf?se quence=3&isAllowed=y Teeken, B., Agbona, A., Abolore, B. et al. (2021). Understanding cassava varietal preferences through pairwise ranking of gari-eba and fufu prepared by local farmer-processors. Special Issue: Con- sumers have their say: assessing preferred quality traits of roots, tubers and cooking bananas, and implications for breeding, 56, 1258–1277. Weltzien, E., Rattunde, F., Christinck, A. & Ashby, J. (2020). Gender and farmer preferences for varietal traits: evidence and issues for crop improvement. Plant Breeding Reviews, 43, 243– 278. Thiele, G., Dufour, D., Vernier, P. et al. (2021). A review of varietal change in roots, tubers and bananas: consumer preferences and other drivers of adoption and implications for breeding. © 2021 CIRAD. International Journal of Food Science & Technology published by John Wiley & Sons Ltd on behalf of Institute of Food, Science and Technology (IFSTTF) International Journal of Food Science and Technology 2021 © 2021 CIRAD. International Journal of Food Science & Technology published by John Wiley & Sons Ltd on behalf of Institute of Food, Science and Technology (IFSTTF) Author Contributions Interna- tional Journal of Food Science and Technology; Special Issue:
https://openalex.org/W4246259293	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1470198/pdf/envhper00321-0016-color.pdf	English	null	Mexico Moves to Phase out DDT and Chlordane	Environmental health perspectives	1,997	public-domain	2,285	Mexico Moves to Phase out DDT and Chlordane ki h l d h Instead of audit privilege laws, the EPA has a penalty mitigation policy that the agency says offers reduced penalties for com- panies that fix violations uncovered by vol- untary audits. "We've gotten over 150 dis- closures from over 400 facilities in the coun- try," says Frey. "In the majority of the cases there has been no penalty. These are record- keeping violations." But they are not neces- sarily minor, Frey adds, because such reports involve toxic release inventories or informa- tion crucial to safe drinking water programs. "We think the EPA policy, offering signifi- cant mitigation, is very reasonable," says Woodall. Taking the lead among the countries of Latin America, the Mexican government on 16 July 1997 unveiled a program designed to phase out all uses of the pesticides DDT and chlordane within 10 years. Calling for an 80% reduction in the use of DDT over the next five years, cessation ofadditional uses by the year 2007, and elimination of the use of chlordane by December 1998, Mexico hopes its experience will provide a model for other Latin American and Caribbean countries attempting to reduce their own dependence on organochlorine pesticides. The specific elements of the program as they relate to each chemical are contained in a North American Regional Action Plan (NARAP) for each chemical. The NARAPs were drafted by the Commission for Environmental Cooperation (CEC), a Montreal-based intergovernmental organiza- tion that is jointly administered by Carol Browner, administrator of the U.S. EPA; Julia Carabias, minister of the Environment, Natural Resources, and Fisheries of Mexico; and Christine Stewart, minister of Environment Canada. The CEC was created to coordinate the agenda of the North American Agreement on Environmental Cooperation, which serves as a complement to the environmental provisions established under the North American Free Trade Agreement. But in the opinion of Nancy Newkirk, a Washington lawyer who counsels businesses and tracks audit privilege legislation, the pol- icy has weaknesses. "The [EPA] policy is quite good. But the Justice Department isn't bound by it [and] no other federal, state, or regulatory body is bound by it, so you can have a situation where a company does an audit, finds a problem, and gets treated as though the [EPA] found the problem," she says, which means the company may be pun- ished. Forum Forum Environmental Evaluation at the Mexican National Institute of Ecology in Mexico City, the NARAP for DDT will build upon the country's existing malaria control pro- gram-which has successfully reduced the annual incidence of the disease from 2.4 mil- lion cases in the 1940s and 1950s to approxi- mately 5,000 cases today-while simultane- ously reducing annual domestic DDT pro- duction and use from 25,000 tons to a pro- duction rate of approximately 600 tons. The use ofDDT is presently restricted to selected government-authorized applications in dwellings. withdraw portions of Colorado's delegated authority under the Clean Water Act on the grounds that it prohibits the state from effec- tively enforcing the federal law and from operating a program approvable under feder- al law. any sort of organization that, [although] not required to be reported under our current system, is important for people to know." Critics say that laws that grant immunity from prosecution can protect people who violate environmental laws and regulations. The EPA is opposed to the privilege and immunity laws for this reason. "We oppose them all," says Bertram Frey, deputy counsel for the EPA's Midwest region. "They block the search for truth," he says. The debate over audit privilege legisla- tion will soon move to Washington, DC. In June, senators Kay Bailey Hutchison (R- Texas) and Trent Lott (R-Mississippi) intro- duced federal audit privilege legislation. This is the second time around for such federal legislation; an earlier attempt, which appeared to protect willful criminal conduct by not specifically excluding it, died in the last congress. Moreover, Frey argues, such legislation can actually penalize companies that spend money to comply with environmental law and regulations. Companies that don't com- ply may have an economic advantage in states with such legislation because they potentially don't get penalized for failing to follow environmental regulations. g Much of the success of Mexico's malaria control program (there have been no record- ed deaths from malaria since 1982) is due to improvements in sanitation, increased disease surveillance, and integrated pest manage- ment schemes that focus pesticide applica- tions on critical habitats and stages in the mosquito's life cycle. According to de Nava, the NARAP will use these proven strategies while conducting experimental pilot studies on alternatives to DDT. Forum Such alternatives include biological control agents such as lar- val parasites and adult predators, microbial products such as Bacillus thuringiensis, and other less persistent pesticides such as pyrethroids. Additionally, the NARAP calls for increased community involvement in the malaria control program, increased enforce- ment against illegal uses of DDT, and restrictions on transborder movement of malaria-infected populations. The Health of Nations i H lth O i ti (PAHO) t Since 1902, the Pan American Health Organization (PAHO), the wdrld's oldest interna- tional agency, has been working to combat disease and improve living standards in the Americas and the Caribbean. Though wars, political schisms, language barriers, and mistrust have been formidable challenges to its mission, PAHO continues to unite diverse peoples against the common enemies of natural disaster and disease. The task facing PAHO which as te r nal office of the World Health Organization (WHO), is a fotridble one. In America and the Caribbean, 130 million p l do not have access to safe drinking water and 160 million c permanent access to basic health services. Despite such obstacles, PAHO has made huge achievements in protecting public health, siu as completely eradicat- ing polio from the region. The last w p viru in the Americas was detected on 5 September 1991 in Peru. i Use in Mexico of chlordane, which is banned from use in the United States and Canada, is limited to urban applications for the control of termites. Until recently, Mexico imported approximately 45 tons of chlordane annually from the United States (where it is legal to manufacture it). Under the NARAP for chlordane, however, Mexico intends to deplete its existing stocks and will no longer allow imports of the pesticide. Furthermore, Velsicol, the sole U.S. manu- facturer of chlordane, announced in May of this year that it had voluntarily ceased pro- duction at all of its national and internation- al facilities. Part of PAHO's success stems from its ability to adapt the resources of the most technologically advanced countries to the needs of the most isolated villages. For example, PAHO distributes relevant health information throughout the Americas via its World Wide Web site, located at http://wwwpaho.og. The site is as expansive and diverse as the region it covers, with information on everything from which countries are experiencing measles outbreaks to what public health resources are available in Barbados to the infant mliti trends in Nicaragua. g The What's New link on the PAHO home page provides links to the most current information, which in turn is linked to other related material. For example, from the What's New page users can access the latest articles written for Nui PAHO's electronic food and nutrition magazine, and then follow the Back link on the Nutil page to access the PAHO Food and Nutrition Program home page. The Health of Nations i H lth O i ti (PAHO) t (Users should be aware that links labeled "Back" always lead to more general information.) Also available under the What's New link are the contents and abstracts of the most current issues of the Pan Ameican Journa of Public Heat and information on ordering the latest books and technical papers published by PAHO. Upcoming PAHO conferences, the status of certain diseases in the Americas, and PAHO press releases are also provided under the What's New link Under the NARAP for chlordane, a multi-phase regulatory program has been initiated that focuses primarily on the devel- opment of an integrated termite control strategy, prohibition of importation, envi- ronmental monitoring and risk assessment of chlordane-exposed individuals, monitoring of existing stocks, and cancellation of the existing registration for use in Mexico. p p The majority of the public health information on the PAHO site is found through the Technical Information home page. The Technical Information page lists the seven major divisions of PAHO with links to pages covering each division's programs and projects. Information on how to obtain PAHO/WHO fellowships and how to subscribe to PAHO's AHEALTH listserv discussion group can also be found by following the Fellowships link and the Information Systems link on this page. The Disasters/Humanitarian Assistance link on the Technical Information page is connected to a list of materials to help nations prepare for and recover from turl disasters. Information is provided on how to contact relief coordinators, the contents ofa WHO emergency health kit, how to order reliefsupply man- agement software, PAHO's quarterly disaster newsletter, and a wide range of disaster train- ing materials. An extensive disaster database available on the Web is also being planned. The Health Systems and Services Development division home page includes a link to the home page of BRJDGE a newsletter on health research, and to directories of public health institu- tions throughout Latin America and the Caribbean. g g According to Andrew Hamilton, scien- tific director of the CEC, Mexico will look to agencies within the United States and Canada for both technical expertise in implementing the NARAPs and assistance in obtaining funding. "Involvement by U.S. and Canadian agencies will help to strength- en proposals geared toward international lending agencies such as the World Bank," he said. "For example, the U.S. Environmental Health Perspectives * Volume 105, Number 8, Auqust 1997 Mexico Moves to Phase out DDT and Chlordane ki h l d h Newkirk says the EPA's opposition to audit privilege laws so far has consisted of testifying against such laws in state capitals and working to modify them. The agency has testified in Texas and Utah and is dis- cussing legislation with Michigan. The EPA also opposed audit privilege legislation dur- ing debate in the last congress. I Pesticide prohibition. Mexico has launched an ambitious program to phase out the use of the dangerous pesticides DDT and chlordane. DDT, which has long been banned in both the United States and Canada, still finds limited use in Mexico as part of the country's arsenal in the war against the Anopheles mosquito, carrier of the Plasmodium parasite that causes malaria. Approximately 60% of the Mexican territo- ry, representing an area inhabited by dose to 45 million people, provides an environment suitable for malaria transmission. According to Cristina Cortinas de Nava, coordinator of the Union of Chemical Substances and The EPA has another weapon against the laws, which to date it hasn't used: the agency can remove a state's authority to enforce fed- eral environmental statutes. That would dry up federal environmental grants and also means that a state's businesses would have to deal with EPA regional headquarters for per- mits instead of state regulators, which can be far more time-consuming. In Colorado, Vincent is part of a group of environmental leaders who have petitioned the EPA to Pesticide prohibition. Mexico has launched an ambitious program to phase out the use of the dangerous pesticides DDT and chlordane. 790 Volume 105, Number 8, August 1997 * Environmental Health Perspectives Forum That Mexico has been able to successful- ly limit the spread of malaria while simulta- neously reducing the use of DDT leaves de Nava hopeful about eliminating the pesti- cide's use altogether. "I'm very optimistic; the campaign has already made great strides," she said. "The problem is one of economics-alternatives can cost up to three times as much as DDT, and we need to be sure that the alternatives don't pose risks [that are greater than] those posed by DDT." The Health of Nations i H lth O i ti (PAHO) t Agency for International Development [USAID] has had significant experience from its work in the tropics." The USAID can be of direct assistance to Mexico in terms of providing expertise in malaria control and they can also help by directly acting as co-sponsors for grant proposals to the World Bank. i ffi d i The Country Health Profiles link on the PAHO home page takes users to a list ofall the countries and colonies in the region that PAHO serves. Each item on this fist is a link to a health summary for that territory, updated every four years, with statistics such as rate of population gowth, ther age and geographic distribution of the population, ifant mortality, and life expectancy. i i Hamilton reaffirmed Mexico's commit- ment to eliminating its use of organochlo- rines, and stated that among the incentives for terminating the pesticides' use is a desire to take part in the international effort to reduce the migration of persistent chemicals to arctic environments. "The idea of a tropi- cal country taking an active role in the Arctic won't go unnoticed," he said. p y Under the Media Center link on the PAHO home page, users can dick on the PAHO Speakers' Bureau link to get details on how to bring a PAHO public health expert to their communities to meet with local professional or community groups. There are also links to the first two issues of PAHO's new Penpectives on Healt ma ne. More infomation on PAHO's many print and electronic publications is available under thhe Publications link on the home page. As with the Web site itself, most of these publications are available in both English and Spanish. 791
https://openalex.org/W2737819333	https://www.pure.ed.ac.uk/ws/files/40281855/s41598_017_06468_6.pdf	English	null	An all-in-one UniSam vector system for efficient gene activation	Scientific reports	2,017	cc-by	6,679	Publisher Rights Statement: Publisher Rights Statement: Publisher Rights Statement: 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/. Digital Object Identifier (DOI): 10.1038/s41598-017-06468-6 Link: Link to publication record in Edinburgh Research Explorer Document Version: Publisher's PDF, also known as Version of record Published In: Scientific Reports Edinburgh Research Explorer General rights C i h f h General rights Copyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. An all-in-one UniSam vector system for efficient gene activation Citation for published version: Fidanza, A, Lopez yrigoyen, M, Romano, N, Jones, R, Taylor, A & Forrester, LM 2017, 'An all-in-one UniSam vector system for efficient gene activation', Scientific Reports. https://doi.org/10.1038/s41598-017- 06468-6 Citation for published version: Fidanza, A, Lopez yrigoyen, M, Romano, N, Jones, R, Taylor, A & Forrester, LM 2017, 'An all-in-one UniSam vector system for efficient gene activation', Scientific Reports. https://doi.org/10.1038/s41598-017- 06468-6 Citation for published version: Fidanza, A, Lopez yrigoyen, M, Romano, N, Jones, R, Taylor, A & Forrester, LM 2017, 'An all-in-one UniSam vector system for efficient gene activation', Scientific Reports. https://doi.org/10.1038/s41598-017- 06468-6 An all-in-one UniSam vector system for efficient gene activation Antonella Fidanza1, Martha Lopez-Yrigoyen1, Nicola Romanò 2, Rhiannon Jones1, A. Helen Taylor1 & Lesley M. Forrester 1 OPEN Antonella Fidanza1, Martha Lopez-Yrigoyen1, Nicola Romanò 2, Rhiannon Jones1, A. Helen Taylor1 & Lesley M. Forrester 1 Received: 15 November 2016 Accepted: 13 June 2017 Published: xx xx xxxx We have generated a drug-free, all-in-one dCAS9-SAM vector that can activate endogenous gene expression with the potential to modify cell fate. We demonstrate that this strategy can be used in a number of cell lines and avoids exceptionally high levels of gene expression that are observed in standard transgenic approaches. Compared to the multi-plasmid system, this all-in-one vector activates gene expression to a comparable level but the reduced overall DNA content results in significantly higher viability of transfected cells. This allowed us to use the RUNX1C-GFP human embryonic stem cell reporter cell line to monitor gene activation in individual cells and to show that activation could occur at all stages of the cell cycle. Manipulation of transcription factor expression has been used to programme cell fate and this approach has pri- marily involved the delivery of exogenous cDNA by plasmid or viral expression vectors. The CRISPR-CAS9 system has revolutionized genome editing1 but more recently the catalytically dead CAS9 (dCAS9) system has been used to modulate endogenous gene expression via activation, repression and chromatin modification2–6. This strategy has been successfully used to program cell fate7, 8. Engineered versions of dCas9 fused to activation domains such as VP64, VPR, p65 or p300 can activate the expression of endogenous genes when directed to their regulatory regions by specific guide RNAs (gRNAs)3. Significant progress has been made in the search for the best combinatorial and synergistic approach to mediate endogenous gene expression and the Synergistic Activators Mediators (SAM) is one of the most powerful tool to date9, 10. This system combines the use of CAS9-VP64 and a specifically designed multi-domain activator (MS2-p65-HSF1) that binds to MS2 hairpins of engineered gRNAs (gRNA 2.0). The system is based on a multiple plasmid approach and consequently, the need for multiple drug selection to achieve homoge- neity. Although a significant advance, these limitations make it challenging to use in cells that are sensitive to viral transduction and/or drug selection. We describe the design and generation of a novel all-in-one strategy that can activate gene expression without drug selection in a number of cell lines including human Embryonic Stem Cells (hESCs). An all-in-one UniSam vector system for efficient gene activation Antonella Fidanza1, Martha Lopez-Yrigoyen1, Nicola Romanò 2, Rhiannon Jones1, A. Helen Taylor1 & Lesley M. Forrester 1 OPEN In a proof of principle experiment we demonstrate that the all-in-one vector containing a single gRNA directed to MyoD1 is able to mediate the trans-differentiation of mouse embryonic fibroblasts into myocytes. Take down policy Take down policy The University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorer content complies with UK legislation. If you believe that the public display of this file breaches copyright please contact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately and investigate your claim. Download date: 24. Oct. 2024 www.nature.com/scientificreports www.nature.com/scientificreports SCIEntIFIC ReporTS \| 7: 6394 \| DOI:10.1038/s41598-017-06468-6 Results and Discussionh The all-in-one vector (herein referred to as UniSAM) consists of the CAS9-VP64 and MS2-p65-HSF1 cDNAs separated by 2A peptides to ensure the generation of independent polypeptides (Fig. 1a). An mCherry tag located at the 3′ end of this cassette allows identification and/or isolation of cells that have been successfully transfected and that are expressing all preceding components (Fig. 1b). The cassette is under the control of the EF1α pro- moter and terminates with a synthetic polyadenylation signal. The vector also carries a U6 promoter driving the expression of the gRNA 2.0 backbone with a BbsI cloning site that enables cloning of the desired gRNA. This simple design means that activation plasmids for any gene of interest can be generate in a single step. All these components have been inserted into a PiggyBac backbone that can be used to mediate transient activation of gene expression or, in the presence of transposase, it can be integrated into the genome and subsequently excised allowing more precise temporal control of expression11. The smaller size of the PiggyBac vector allows for a lower total DNA to ORF ratio compared to lentiviruses, reducing the overall amount of DNA delivered and predictably increasing viability of transfected cells. g y We generated a number of UniSAM vectors designed to activate genes encoding transcription factors involved in the production and differentiation of hematopoietic cells including RUNX1c, HOXA9, HOXA5, KLF1 and BCL11a12–15. We designed 4-6 gRNAs for each gene using the online tools available at http://sam. genome-engineering.org/database/ (Supplementary Table S1), generated activation plasmids and tested their 1Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK. 2Centre for Integrative Physiology, University of Edinburgh, Edinburgh, UK. Correspondence and requests for materials should be addressed to A.F. (email: afidanza@staffmail.ed.ac.uk) or L.M.F. (email: L.Forrester@ed.ac.uk) SCIEntIFIC ReporTS \| 7: 6394 \| DOI:10.1038/s41598-017-06468-6 1 www.nature.com/scientificreports/ Figure 1. Design and assessment of all-in-one UniSAM vectors in HEK293T and HeLa cells. (a) Schematic of UniSAM vector in PiggyBac backbone. (b) Expression of mCherry in HEK293 and HeLa cells demonstrates efficient transfection and expression of the UniSAM vector. (c) Activation of five transcription factors and HBG1 following transfection of UniSAM vectors carrying single gRNAs, in HEK293T cells. (d) Activation of five transcription factors with single gRNAs, in HeLa cells. Data in (c) and (d) represent the mean activation by 4–6 single gRNAs for each gene (n = 3, Mann Whitney t-test). Results and Discussionh (e,f) Relationship between basal expression and activation levels for the different genes in HEK293T (e) and HeLa (f) (n = 72 from 3 independent experiments, linear regression with F-test). (g,h) Analyses of variance in Ct values following activation in HEK293T (g) and HeLa (h) (n = 3, F-test, p < 0.03). Figure 1. Design and assessment of all-in-one UniSAM vectors in HEK293T and HeLa cells. (a) Schematic of UniSAM vector in PiggyBac backbone. (b) Expression of mCherry in HEK293 and HeLa cells demonstrates Figure 1. Design and assessment of all-in-one UniSAM vectors in HEK293T and HeLa cells. (a) Schematic UniSAM vector in PiggyBac backbone. (b) Expression of mCherry in HEK293 and HeLa cells demonstrates efficient transfection and expression of the UniSAM vector. (c) Activation of five transcription factors and HBG1 following transfection of UniSAM vectors carrying single gRNAs, in HEK293T cells. (d) Activation of five transcription factors with single gRNAs, in HeLa cells. Data in (c) and (d) represent the mean activation by 4–6 single gRNAs for each gene (n = 3, Mann Whitney t-test). (e,f) Relationship between basal expression and activation levels for the different genes in HEK293T (e) and HeLa (f) (n = 72 from 3 independent experiments, linear regression with F-test). (g,h) Analyses of variance in Ct values following activation in HEK293T (g) and HeLa (h) (n = 3, F-test, p < 0.03). activity in HEK293 and HeLa cells (Fig. 1, Supplementary Fig. S1a). Two days after transfection significant activa- tion of RUNX1c and KLF1 expression was observed in both cell lines but there was minimal activation of HOXA5, HOXA9 and BCL11a (Fig. 1c,d). It was previously reported that inter-gene variability in transcriptional activation was linked to the basal expression9. Here we also show that gene activation was also inversely related to the basal expression level with HOXA5, HOXA9 and BCL11a being expressed at higher levels in both cell lines compared to RUNX1c and KLF1 (Fig. 1e,f). Since gene activation mediates the recruitment of the transcriptional machinery we reasoned that genes that were being actively transcribed would be more resistant to further transcriptional activation and that there could be a limiting level of expression that this strategy would permit. To further test this hypothesis we compared all raw Ct values in the absence of gRNAs (empty vector, (EV)) with the value after the addition of gRNAs. Results and Discussionh We noted that the raw Ct values of all genes after activation were comparable to the overall basal levels of activation and that there was a reduction in the variance of their distribution (Fig. 1g,h). Taken together these data indicate that the level of mRNA expression achieved for any gene using this strategy avoids exceptionally high levels of gene expression. p y g g p To demonstrate that gene expression activation was also associated with increased protein production, HEK293T were transfected with the UniSAM-RUNX1c vectors and stained by immunocytochemistry. We observed an increase in the proportion of RUNX1+ cells, thus implying an increase in RUNX1c protein, and this varied depending on the gRNA used (Fig. 2a,b). RUNX1 protein was detected in a very small number of control, non-activated HEK293T cells and this allowed us to quantify the physiological level of protein expression in indi- vidual cells. Upon activation with the UniSAM system the level of protein expression within activated cells was comparable to the level in control cells but significantly lower than cells transfected with a standard CMV-driven RUNX1c expression plasmid (Fig. 2c). This further supports our hypothesis that there is an upper limit to the level of activation achieved using this strategy and that abnormally high, non-physiological levels, observed in classical transgenic approaches, are avoided. g pp , To assess whether the UniSAM strategy could be used to alter the expression of a cell surface marker we activated SPN (which encodes for CD43) in HeLa cells and in human erythroleukaemia (HEL) cell line. CD43 is expressed in HEL but not HeLa cells so this system allowed us to test the influence of the basal expression on the level of activation and to assess the upper limits of gene activation at the protein level. We successfully activated the expression of CD43 on the cell surface of HeLa cells with a single gRNA for CD43 (Fig. 2d–f) but the addition of the same gRNAs to HEL cells, where CD43 was already expressed a relatively high level, had no significant effect (Fig. 2e,f). This experiment further confirms that the basal expression is a strong inferring factor in achiev- ing activation and that the system avoids high non-physiological levels of expression.fi g y g p y g p To assess whether our all-in-one UniSAM vector offered a significant advantage over the previously published multiplasmid strategies we compared the two systems directly. SCIEntIFIC ReporTS \| 7: 6394 \| DOI:10.1038/s41598-017-06468-6 Results and Discussionh of the coding elements of the two plasmid systems including gRNAs to the RUNX1c locus then gene activation was assessed by qPCR. We demonstrated that the UniSAM and multiplasmid approach mediated comparable activation of RUNX1c for all four gRNAs (Fig. 3a). It is interesting to note that g2 seems to be the most efficient gRNA in Hela cells whereas in HEK293, g4 was the most efficient indicating a cell line depend effect of the specific gRNAs. When we directly compared the two strategies in hESCs we noted a significant difference in cell viability. RUNX1C-GFP hESC that were transfected with the multiplasmid system exhibited a significantly higher amount of cell death one day after transfection, when compared to the UniSAM strategy (Fig. 3b,c). This reduced cell viability was confirmed by flow cytometry both at one and two day after transfection (Fig. 3d,e). Thus, our unique UniSAM strategy is apparently able to mediate comparable gene activation levels compared to the published multiplasmid strategies but the significant improvement on cell viability makes it a more attractive strategy to use, particularly in cell types that are sensitive to transfection.h p y yp This increased viability allowed us to assess the UniSAM strategy in the RUNX1C-EGFP reporter hESC line where we were able to assess the heterogeneity of activation between individual cells14. The transfection efficiency of UniSAM vectors was monitored using the mCherry reporter and the level of activation of the endogenous gene was assessed by expression of EGFP, knocked-in under the distal promoter that specifically drives RUNX1c14 (Fig. 4a). The presence of the different RUNX1c-gRNAs resulted in the emergence of varying proportions of RUNX1C-EGFP cells (Fig. 4b) and the different gRNAs mediated different levels of EGFP expression (Fig. 4c). The level of activation by the different gRNAs in hESCs showed a similar trend to that observed in HEK293 cells with g4 being the most powerful (Figs 1g, 4b–c). Flow cytometry allowed analysis at the single cell level and this identified a correlation between the level of UniSAM in each cell (reported by mCherry) and the level of gene activation (Fig. 4d) that was not possible using the less sensitive immunocytochemistry analyses. Statistical anal- ysis of flow cytometry data for the RUNX1C-g4 uncovered a bimodal trend in expression of EGFP and piecewise regression identified a threshold of UniSAM expression that was consistent between experiments (Supplementary Figure S1b,c). Results and Discussionh HeLa were transfected with equimolar quantities SCIEntIFIC ReporTS \| 7: 6394 \| DOI:10.1038/s41598-017-06468-6 2 www.nature.com/scientificreports/ Figure 2. UniSAM gene activation results physiological levels of protein expression. (a) Immunocytochemistry analyses of HEK293T cells transfected with the UniSAM carrying no gRNA (empty vector (EV)) or a single gRNAs directed to RUNX1c (n = 4 for each of the gRNAs, g1 to g4) or a CMV-driven pRUNX1c expression plasmid (scale bar, 50 μm). (b) Percentage of RUNX1 + cells following transfection of UniSAM vectors carrying gRNA 1–4) (n = 4, Kruskal Wallis One-way ANOVA, p < 0.05, p < 0.01). (c) Comparison of RUNX1 expression levels in individual cells mediated by UniSAM and CMV-driven pRUNX1c expression plasmid analysed by Columbus Image Analysis Software (n = 4, Kruskal Walis One-way ANOVA, ns p > 0.5; p < 0.03). (d) Flow cytometry analysis of HEL and HeLa cells transfected with the empty vector (EV) or a UniSAM containing a sgRNA for CD43. (e) Quantification of the proportion of CD43 + cells in (d) (n = 4; Sidak’s two-way Anova, ***p < 0.0001). (f) Quantification of CD43 expression level (assessed by mean fluorescent intensity (MFI)) in HEL and HeLa cells after activation (n = 4; Dunn’s one-way Anova; p > 0.05). Figure 2. UniSAM gene activation results physiological levels of protein expression. (a) Immunocytochemistry analyses of HEK293T cells transfected with the UniSAM carrying no gRNA (empty vector (EV)) or a single gRNAs directed to RUNX1c (n = 4 for each of the gRNAs, g1 to g4) or a CMV-driven pRUNX1c expression plasmid (scale bar, 50 μm). (b) Percentage of RUNX1 + cells following transfection of UniSAM vectors carrying gRNA 1–4) (n = 4, Kruskal Wallis One-way ANOVA, p < 0.05, p < 0.01). (c) Comparison of RUNX1 expression levels in individual cells mediated by UniSAM and CMV-driven pRUNX1c expression plasmid analysed by Columbus Image Analysis Software (n = 4, Kruskal Walis One-way ANOVA, ns p > 0.5; p < 0.03). (d) Flow cytometry analysis of HEL and HeLa cells transfected with the empty vector (EV) or a UniSAM containing a sgRNA for CD43. (e) Quantification of the proportion of CD43 + cells in (d) (n = 4; Sidak’s two-way Anova, ***p < 0.0001). (f) Quantification of CD43 expression level (assessed by mean fluorescent intensity (MFI)) in HEL and HeLa cells after activation (n = 4; Dunn’s one-way Anova; p > 0.05). SCIEntIFIC ReporTS \| 7: 6394 \| DOI:10.1038/s41598-017-06468-6 Results and Discussionh As expected the activation level above that threshold ‘breakpoint’ was significantly higher than that below (Fig. 4e). These data show that the level of activation can be tunable by modifying the level of expression of the Cas9-SAM effector and/or the amount of gRNA in agreement with previously studies that have shown that altering the amount of gRNA can mediate the activation level16. g g We observed that RUNX1C-EGFP was activated in a subpopulation of successfully transfected cells, com- parable with that previously reported in hESCs using a drug-selectable dCas9-VP64 strategy17. Here we were able to test whether gene activation was occurring preferentially in cells at specific stages of the cell cycle. Flow cytometry analysis of Hoechst-stained cells revealed that activated cells (mCherry+EGFP+) were present in cells at all stages of the cell cycle when each of the 4 gRNAs was used (Fig. 4f). Interestingly, cells activated by g1 and g2 were slightly enriched for cycling cells (G2/M) whereas the majority of cells that were activated by g4 were in SCIEntIFIC ReporTS \| 7: 6394 \| DOI:10.1038/s41598-017-06468-6 3 www.nature.com/scientificreports/ Figure 3. Direct comparison of the UniSAM system and the multiplasmid system. (a) Activation of RUNX1c using the UniSAM or multiplasmid systems with four different gRNAs (g1-g4) in HeLa (n = 3, Mann-Whitney t-Test). (b) Quantification of cell viability of hESCs one day after transfection as measured by Trypan Blue staining (n = 5; Mann-Whitney t-test, p < 0.01). (c) Visual microscopic appearance of transfected hESCs (identified by mCherry expression) one day after transfection indicating that there are more live cells using the UniSAM system (scale bar indicates 50 μm). (d) Flow cytometry analysis of hESCs samples collected two days after transfection, gate indicates cells. (e) Comparison of the viable cells at day 1 and day 2 post transfection of hESCs, as measured by flow cytometry analyses of DAPI staining (n = 3; Dunn’s one-way Anova; p > 0.01, **p < 0.0001). Figure 3. Direct comparison of the UniSAM system and the multiplasmid system. (a) Activation of RUNX1c using the UniSAM or multiplasmid systems with four different gRNAs (g1-g4) in HeLa (n = 3, Mann-Whitney t-Test). (b) Quantification of cell viability of hESCs one day after transfection as measured by Trypan Blue staining (n = 5; Mann-Whitney t-test, p < 0.01). Results and Discussionh (c) Visual microscopic appearance of transfected hESCs (identified by mCherry expression) one day after transfection indicating that there are more live cells using the UniSAM system (scale bar indicates 50 μm). (d) Flow cytometry analysis of hESCs samples collected two days after transfection, gate indicates cells. (e) Comparison of the viable cells at day 1 and day 2 post transfection of hESCs, as measured by flow cytometry analyses of DAPI staining (n = 3; Dunn’s one-way Anova; p > 0.01, *p < 0.0001). G0/G1. To determine whether this was a preferential susceptibly to activation at a particular stage or an effect of RUNX1 C activation itself we evaluated the mCherry intensity that would be diluted out as cells divide. There was an inverse correlation between the level of RUNX1C (EGFP) and mCherry, indicating that cells expressing high levels of RUNX1C were retaining mCherry expression suggesting a lower rate of proliferation in that population (Fig. 4g). Thus the higher proportion of cells in G0/G1 is more likely caused by the higher levels of RUNX1C rather than being an effect of preferential activation at this stage in the cell cycle.h gf p g y The ultimate aim of this gene activation strategy in hESC would be to modify cell fate and/or to enhance in vitro differentiation protocols. In a proof of principle experiment to demonstrate that genes activated in this way could modify cell fate we tested the strategy in a well-established transdifferentiation assay that requires activation of a single gene6, 18. We designed three gRNAs predicted to activate murine MyoD1, generated UniSAM-MyoD1 vectors and integrated these into the genome of murine fibroblast using the HyPBase Transposase7, 19. A pMyoD1 vector was used as a positive control for fibroblast-myocyte transdifferentiation. In our preliminary experiments we observed increased expression of MyoD1 and the presence of multinucleated syncytium expressing Myosin Heavy Chain I (MHCI) protein in cells that had been transfected with the UniSAM-MyoD1_g3 vector but not in cells transfected with the control empty vector indicating that a proportion of fibroblasts had transdifferentiated into myocyte by activation of MyoD1 (Supplementary Figure S1d,e). y y y y ( pp y g ) In summary we have designed and generated a UniSAM vector that can be used to activate the expression of endogenous genes with the potential to modify cell fate without the need for drug selection. SCIEntIFIC ReporTS \| 7: 6394 \| DOI:10.1038/s41598-017-06468-6 Results and Discussionh Figure 4. Functional assessment of UniSAM vectors in human pluripotent stem cells. (a) Flow cytometry analyses of RUNX1C-EGFP reporter hESCs transfected with UniSAM vectors carrying no gRNA (empty vector (EV)) or single gRNAs (g1-g4) directed to RUNX1C showing transfection efficiency (mCherry) and gene activation (EGFP) (n = 6). (b) Quantification of the number of activated cells as a proportion of transfected cells (n = 6, Kruskal Wallis One-way ANOVA, p < 0.05, p < 0.01, p < 0.0001). (c) Quantification of the level of gene activation by the different gRNAs indicated by the mean fluorescence intensity (MFI) of EGFP (n = 6, Kruskal Walis One-way ANOVA, p < 0.01, *p < 0.001). (d,e) ‘Breakpoint’ in the activation level mediated by RUNX1c_g4 (n = 6) and (quantification (e) of the fluorescence above and below that breakpoint (n = 6, one-way ANOVA, p < 0.001). (f) Proportion of RUNX1C-GFP-expressing cells in each stage of the cell cycle after activation by gRNAs g1 to g4 (n = 3, one way ANOVA). (g) Linear regression analyses of UniSAM expression level (mCherry) versus RUNX1c gene activation (EGFP) (n = 6; F-test). the direct comparison of cells in which genes have been activated with those that have not under the same exper- imental conditions and so provides a tool to understand the heterogeneity of activation at the single cell level. Importantly the increased cell viability using the UniSAM vector compared to multiplasmid strategies provides a system that can be used in transfection-sensitive cell lines. Thus the strategy can be exploited to finely tune the expression of transcription factor networks and thus to modulate the differentiation pathways of pluripotent stem cells in the quest to produce therapeutic cell types. the direct comparison of cells in which genes have been activated with those that have not under the same exper- imental conditions and so provides a tool to understand the heterogeneity of activation at the single cell level. Importantly the increased cell viability using the UniSAM vector compared to multiplasmid strategies provides a system that can be used in transfection-sensitive cell lines. Thus the strategy can be exploited to finely tune the expression of transcription factor networks and thus to modulate the differentiation pathways of pluripotent stem cells in the quest to produce therapeutic cell types. Results and Discussionh The strategy allows SCIEntIFIC ReporTS \| 7: 6394 \| DOI:10.1038/s41598-017-06468-6 4 www.nature.com/scientificreports/ Figure 4. Functional assessment of UniSAM vectors in human pluripotent stem cells. (a) Flow cytometry analyses of RUNX1C-EGFP reporter hESCs transfected with UniSAM vectors carrying no gRNA (empty vector (EV)) or single gRNAs (g1-g4) directed to RUNX1C showing transfection efficiency (mCherry) and gene activation (EGFP) (n = 6). (b) Quantification of the number of activated cells as a proportion of transfected cells (n = 6, Kruskal Wallis One-way ANOVA, p < 0.05, p < 0.01, p < 0.0001). (c) Quantification of the level of gene activation by the different gRNAs indicated by the mean fluorescence intensity (MFI) of EGFP (n = 6, Kruskal Walis One-way ANOVA, p < 0.01, *p < 0.001). (d,e) ‘Breakpoint’ in the activation level mediated by RUNX1c_g4 (n = 6) and (quantification (e) of the fluorescence above and below that breakpoint (n = 6, one-way ANOVA, p < 0.001). (f) Proportion of RUNX1C-GFP-expressing cells in each stage of the cell cycle after activation by gRNAs g1 to g4 (n = 3, one way ANOVA). (g) Linear regression analyses of UniSAM expression level (mCherry) versus RUNX1c gene activation (EGFP) (n = 6; F-test). Figure 4. Functional assessment of UniSAM vectors in human pluripotent stem cells. (a) Flow cytometry Figure 4. Functional assessment of UniSAM vectors in human pluripotent stem cells. (a) Flow cytometry analyses of RUNX1C-EGFP reporter hESCs transfected with UniSAM vectors carrying no gRNA (empty vector (EV)) or single gRNAs (g1-g4) directed to RUNX1C showing transfection efficiency (mCherry) and gene activation (EGFP) (n = 6). (b) Quantification of the number of activated cells as a proportion of transfected cells (n = 6, Kruskal Wallis One-way ANOVA, p < 0.05, p < 0.01, p < 0.0001). (c) Quantification of the level of gene activation by the different gRNAs indicated by the mean fluorescence intensity (MFI) of EGFP (n = 6, Kruskal Walis One-way ANOVA, p < 0.01, *p < 0.001). (d,e) ‘Breakpoint’ in the activation level mediated by RUNX1c_g4 (n = 6) and (quantification (e) of the fluorescence above and below that breakpoint (n = 6, one-way ANOVA, *p < 0.001). (f) Proportion of RUNX1C-GFP-expressing cells in each stage of the cell cycle after activation by gRNAs g1 to g4 (n = 3, one way ANOVA). (g) Linear regression analyses of UniSAM expression level (mCherry) versus RUNX1c gene activation (EGFP) (n = 6; F-test). Methods U iSAM UniSAM vector production. The PiggyBac backbone was prepared by NheI and PacI digestion of the PB- CAG-hCD2 (Kind gift of Kesiure Kaji). EF1α-Cas9-Vp64 (Addgene-61422) was also digested with NheI and PacI (New England Biolab) and ligated into PiggyBac backbone to generate the PB-EF1α-Cas9-VP64. The P2A- MS2-p65-HSF1 and the T2A-mCherry-PolyA-U6-gRNA2.0 backbone were synthesized as double strand DNA (Thermo Scientific and Integrated DNA technology, respectively). All BbsI sites, except in the gRNA backbone were mutated and codon optimized according to the codon usage in Homo sapiens. Overlapping regions for Gibson assembly were included in the design of synthetic dsDNA fragments. Flanking SapI sites were added to release the synthetic dsDNA fragment following subcloning into pGEMT easy vector (Promega). PB-EF1α- Cas9-VP64 was linearized by NheI digestion and assembled at 50 °C for 1 hour with Sap1-digested P2A-MS2-p65- HSF1 and T2A-mCherry-PolyA-U6-gRNA2.0 backbone. Correctly assembled UniSAM vector was confirmed by complete Sanger sequencing. gRNAs were obtained from Integrated DNA technology as two single strand oligos SCIEntIFIC ReporTS \| 7: 6394 \| DOI:10.1038/s41598-017-06468-6 5 www.nature.com/scientificreports/ and annealed in 20 μl reaction in Quick Ligation Buffer (Promega) with 9 μl of each oligos and annealedin in the thermal cycler 95 °C for 5 minutes fo llowed by cooling to 25 °C with 1 °C/minute ramp. 25 ng of purified BbsI lin- earized UniSAM or 25 ng of Purified BsmBI linearized lenti sgRNA(MS2)_puro (gift from Feng Zhang -Addgene plasmid #73797) was ligated with 1 μl of annealed gRNA diluted 1:500 using the Quick Ligation Kit (Promega). Ligation was used to transform Top10 E.Coli, correctly ligated gRNAs were confirmed by Sanger sequencing. UniSAM plasmid is available from Addgene (ID 99866). Cell culture and transfections. HEK293T were cultured in Glasgow Minimal Essential Medium (GMEM) with Non Essential Amino Acid, GlutaMax and 10% FCS (Gibco) and passaged every few days, at the ratio 1:6. HeLa cells were cultured in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) with Glutmax and 5% FCS (Gibco) and passaged every few days, at a ratio of 1:6. HEL were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM) with 10% FCS (Gibco) and passaged every few days, at a ratio of 1:4. 2 × 105 cells were plated, transfected at 6–8 hours with 0.75 μg of DNA using Xfect Transfection reagent (Clontech) and then analysed 2 days after. Cells were also transfected with 0.5 μg CMV-RUNX1C (Addgene-12426) for the overexpres- sion. Methods U iSAM RUNX1C-EGFP hESCs were cultured in StemPro hESC SFM (Gibco) with 20 ng/ml bFGF (R&D). Wells were coated with CELLstart at least 1 hour before plating and cells were passaged using the StemPro EZPassage tool (ThermoFisher Scientific). Media change was performed every day and cells passaged every 3–4 days at a ratio of 1:4. 3 × 105 cells were plated into a coated 6 well plate and reverse transfected with 2 μg of UniSAM DNA using the Xfect Transfection reagent (Clontech) and analyzed 2 days later. For the comparison with the multi- plasmid system, 0.5 μg UniSAM or equimolar ratio of the Multiplasmid and 1 μl of Lipofectamine 2000 reagent (ThermoScientifc) were used with pmCherryN1 (Clonetech) being co-transfected to monitor transfected cells by flow cytometry. The multiplasmid systems consisted of lenti sgRNA(MS2)_puro optimized backbone (Addgene plasmid #73797); EF1α-Cas9-Vp64-Blast (Addgene plasmid #61425); lenti MS2-P65-HSF1-Hygro (Addgene plasmid #61426). To achieve an equimolar ratio of all the parts of the multiplasmid vectors the mass ration of Uni SAM:Cas9-VP64:MS2-p65-HSF1:sgRNA (1:1.14:0.95:0.68) was used. p g ( ) Primary mouse embryonic fibroblasts (kindly provided by Kaji Keisuke) were cultured in GMEM with Glutamine/Pyruvate and 10% FCS (Gibco) on Gelatin-(Sigma Aldrich) coated flasks. 1.5 × 105 fibroblasts, between passage 2 and 4, were plated overnight and then transfected with 1.5 μg of UniSAM and 0.5 μg of Transposase HyPBase19 or 1.5 μg of CMV-MyoD1 (gift of Andrea Corsinotti) using 6 μl of FuGENE Transfection Reagent (Promega). Two days after transfection fibroblast were maintained under starvation condition in GMEM, Glutamine/Pyruvate and 2% Horse serum (Sigma Aldrich) and then analysed by immunocytochemistry 7.5 days later. Gene expression. Total RNA was purified using the RNAeasy Mini Kit (Qiagen) and cDNA synthesized from 500 ng of total RNA using the High Capacity cDNA synthesis Kit (Applied Biosystem). 2 ng of cDNA were amplified per reaction and each reaction was performed in triplicate using the LightCycler 384 (Roche) with SYBR Green Master Mix II (Roche). A melting curve was performed and analyzed for each gene to ensure the specificity of the amplification. For human RNA expression analyses GADPH, β-Actin and B2M were used as reference genes and the geometrical mean was used to normalize the data. For mouse RNA expression anal- ysis the Sdha reference gene was used to normalize the data. Primer sequence and efficiencies are reported in Supplementary Table 2. Immunocytochemistry. Acknowledgements g We thank Andrew Elefanty and Ed Stanley for the RUNX1C-GFP cell line, Keisuke Kaji for the PB-CAG-hCD2 plasmid, Miguel Foronda for the help in the plasmid design, Andrea Corsinotti for reagents and discussion and Fiona Rossi and Claire Cryer for flow cytometry. The work was funded by the Wellcome Trust (Grant No. 102610) and performed as part of the Novosang Consortium (www.novosang.co.uk). MLY received a PhD scholarship from CONACYT. Methods U iSAM 6 SCIEntIFIC ReporTS \| 7: 6394 \| DOI:10.1038/s41598-017-06468-6 www.nature.com/scientificreports/ Statistical and R analyses. Results are expressed as mean ± standard error mean. Statistical analyses were performed using Graph Pad Prism 6.0 with the exception of segmented fitting (Fig. 2d and Figure S1b) performed with R. Identification of the breakpoints has been performed using the R package “segmented”. Statistical and R analyses. Results are expressed as mean ± standard error mean. 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Medial HOXA genes demarcate haematopoietic stem cell fate during human development. Nature cell biology 18, 595–606, doi:10.1038/ncb3354 (2016). h 12. Dou, D. R. et al. Medial HOXA genes demarcate haematopoietic stem cell fate during human development. Methods U iSAM Cells were fixed in 4% PFA in PBS at room temperature for 10 minutes, permeabi- lized in PBS-T (Triton-X100 (0.2% for the MHCI and 0.4% for the RUNX1)) for 20 minutes and blocked in PBS-T with 1% BSA and 3% goat serum for 1 hour. Primary antibodies were incubated in blocking solution over night at 4 °C (RUNX1 1:200 - ab92336, Abcam) or room temperature for 2 hours (MHCI at 2 μg/ml, MF-20, DSHB). Cells were then washed in PBS-T and incubated with secondary antibodies for 1 hour at room temperature (for RUNX1: donkey α-rabbit 1:200 - A-11008; for MHCI goat α-mouse 1:1000- A11017 or goat α-mouse 1:1000- A10036 (Thermo Scientific). Cells were washed in PBS-T and counterstained with DAPI. Images were generated using the Zeiss Observer microscope. Specific algorithm was developed for the Operetta Imaging System to ana- lyse the numbers of RUNX1 + cells and the staining intensity (Perkin Elmer). Cell nuclei were first identified using the DAPI staining with dimension and intensity cut offs being set to minimize inaccurate nuclear identifica- tion. Using control cells the RUNX1 + gate was then set to identify the nuclei expressing RUNX1 and the intensity of Alexa Fluor 488 staining was measured for each RUNX1-positive nuclei. Flow cytometry. Single cell suspensions were obtained by Trypsin treatment (Gibco), resuspended in PBS with 1%BSA and 5 mM EDTA and analysed in the LSR Fortessa Analyser (BD). Dead cells were gated out using DAPI staining. For the cell cycle analysis cells were stained with Hoechst 33342 at the final concentration of 10 μg/ml in the culture medium 2 hours prior to harvesting. Cells were kept on ice until analysed using the LSR Fortessa Analyser (BD). For cell viability comparison between the UniSAM and the multiplasmid system, cells were acquired for 90 seconds at a medium speed to acquire the same volume of cell solution to ensure comparabil- ity in cell viability between samples. For CD43 cell surface staining, single cell suspension of HeLa and HEL cells were obtained by StemPro Accutase Cell Dissociation Reagent (Gibco), washed, blocked in PBS with 1% BSA and resuspended in staining solution (PBS with 1% BSA) with mouse anti-human CD43-APC (1:100)(eBioscience) for 10 minutes at room temperature before being washed and analysed using the LSR Fortessa Analyser (BD). FlowJo 10.1 was used for all flow cytometry data analyse. Additional Information Supplementary information accompanies this paper at doi:10.1038/s41598-017-06468-6h Supplementary information accompanies this paper at doi:10.1038/s41598-017-06468-6 Competing Interests: The authors declare that they have no competing interests. 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 an 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/. 7 SCIEntIFIC ReporTS \| 7: 6394 \| DOI:10.1038/s41598-017-06468-6
https://openalex.org/W3168580273	https://radar.brookes.ac.uk/radar/file/ecda2c8c-6085-4875-88fe-5c3e7cbf46be/1/Cumulative effects assessment in selected countries - 2019 - Olagunju et al.pdf	English	null	Cumulative effects assessment requirements in selected developed and developing countries	Edward Elgar Publishing eBooks	2,021	public-domain	13,292	Chapter 3: Cumulative effects assessment requirements in selected developed and developing countries Ayodele Olagunju, Divine Appiah, Paulina Maria Porto Silva Cavalcanti, Bridget Durning, Juan Carlos Tejeda González, Jason MacLean, Richard Morgan, and Rebecca Nelson Ayodele Olagunju, Divine Appiah, Paulina Maria Porto Silva Cavalcanti, Bridget Durning, Juan Carlos Tejeda González, Jason MacLean, Richard Morgan, and Rebecca Nelson Abstract Research in cumulative effects assessment (CEA) is advancing in many ways; however, gaps between normative expectations and regulatory approaches remain in many jurisdictions. This chapter profiles CEA requirements and practice in eight countries – Australia, Brazil, Canada, Ghana, Mexico, New Zealand, Nigeria and the United Kingdom – and discusses implications for practice effectiveness internationally. Overall, national governments are largely responsible for setting legal frameworks; however, each country differs in terms of requirements, scope, approach and capacity. Experience in developed countries is more advanced when it comes to sector-based innovations, industry-led guidance, and peer review and evaluation. Though capacity is reportedly limited, there has been a shift toward explicit legal provisions for higher-order environmental assessments such as strategic environmental assessment and regional environmental assessment for better CEA outcomes in some developing countries. A systems perspective to CEA legislation, consistent interpretation of provisions by courts and strong political leadership are required to facilitate better practice. Keywords: cumulative effects assessment, case studies; legal framework; best practice; developed countries; developing countries Chapter 3: Cumulative effects assessment requirements in selected developed and developing countries Ayodele Olagunju, Divine Appiah, Paulina Maria Porto Silva Cavalcanti, Bridget Durnin Chapter 3: Cumulative effects assessment requirements in selected developed and developing countries Ayodele Olagunju, Divine Appiah, Paulina Maria Porto Silva Cavalcanti, Bridget Durning, Juan Carlos Tejeda González, Jason MacLean, Richard Morgan, and Rebecca Nelson Background Over the past five decades, important legal and policy changes have taken place in many countries following the enactment of the United States’ National Environmental Policy Act, 1969, which made environmental impact assessment (EIA) mandatory for federal development projects and initiatives. Various conceptualizations of the appropriate context, scope and scale to assess the impacts of human interventions in the environment have emerged, many of which include emphasis on a systems approach and assessment of cumulative environmental effects. This progress has resulted in the advancement of methods, tools, techniques and processes to support cumulative effects assessment (CEA) as well as an increase in the number of countries making regulatory reforms to EIA laws to include CEA requirements. Decades of CEA research have also provided the scientific and technical knowledge base to assess cumulative effects at different scales – project, regional and transboundary (Quinn et al., 2004; Harriman & Noble, 2007), while being shaped by the growing influence of case law and the needs and concerns of civil society. While these research advances have increased awareness of the importance of and key processes in CEA, policy and regulatory contexts remain comparatively under- explored. In particular, there is still a need to understand inherent challenges involved in nudging international CEA practice toward best practice and a more holistic focus, beyond individual projects. The broader issues related to defining, implementing and managing cumulative effects, including the appropriate legal, policy, conceptual and technical approaches to support this work, continue to be at the centre of debate about CEA best practices. While many agree that CEA should be part of a rigorous EIA process, how that should proceed, who should be responsible, and what is meant by the term ‘cumulative’, are questions with mixed responses in various jurisdictions. To obtain a relatively global picture of progress in CEA practice, we profile a mix of countries from developed (Australia, Canada, New Zealand and the United Kingdom) and developing (Brazil, Ghana, Mexico and Nigeria) nations that, according to our experience and knowledge, have specific legal requirements for CEA or some evidence of progress in practice. Background To ensure consistency, for each country we address the following questions: 1) what specific legal provisions for CEA exist in the country’s EIA law?; 2) are any guidance documents or guidelines issued by the relevant government department to support CEA practice?; 3) what are the current gaps in practice specific to the country?; and 4) what are the strengths in current practice and what implications can be drawn for practice effectiveness locally and internationally? Progress and results vary according to country; for example, some countries – even those with advanced EIA systems – have difficulty linking legal requirements to practice because of the absence of robust guidelines and limited capabilities to grapple with the nuances of cumulative effects. However, it is still useful to examine these countries for the insights this provides to practice, legal and policy reforms, and international research on CEA. The chapter highlights progress and experience with CEA for the benefit of researchers, regulators and policy makers within the selected countries and in comparable jurisdictions, but more importantly, identifies foundational issues that are central to strengthening CEA practice internationally. Australia Australian EIA legislation occurs at both national and state/territory levels and generally links EIA to authorizations that are required before activities may proceed. The activities that require EIA vary by jurisdiction (Bates, 2016). Much of the legal and policy focus on CEA in Australian EIA relates to extractive resources projects, especially the cumulative effects of mining, petroleum and gas activities on the quantity and quality of water resources. Formal government and parliamentary inquiries have discussed concerns about inadequate consideration of cumulative effects in this context, such as the Australian Senate Environment and Communications References Committee, 2018 (Hunter, 2017). The EIA legislation of Australian states tends not to expressly mention or define cumulative effects, but to leave requirements for CEA to subordinate legislation (regulations) or non-binding policy documents. Regulations made under New South Wales’ Environmental Planning and Assessment Act, 1979 require consideration of ‘any cumulative environmental effect with other existing or likely future activities’ (Environmental Planning and Assessment Regulation, 2000, cl. 228(1)(o)). CEA policy guidelines for ‘State significant projects’ under these arrangements were being developed at the time of writing, while existing guidelines for wind farms refer to cumulative impacts, though not in detail (Department of Planning and Environment (NSW), 2016; 2018). State-level policy guidance on CEA in EIA tends to mention cumulative effects only briefly (see, for example, ACT Government, 2017; Environmental Protection Authority (Western Australia), 2018), or to provide very limited guidance (Department of Sustainability and Environment (Victoria), 2006); Environment Protection Authority (Tasmania), 2019). Accordingly, some industry sectors have produced their own CEA guidance (for example, see Kaveney et al., 2015) and pursued collaborative approaches to address the cumulative effects of resources extraction (Porter et al., 2013). Significant public concern about the cumulative effects of rapidly expanding coal seam gas (CSG, also known as coalbed methane) extraction recently led to Australia’s first national environmental legislative reference to cumulative effects, albeit one limited to the water resources context. Amendments to the Environment Protection and Biodiversity Conservation Act, 1999 (EPBC Act) (Commonwealth) in 2013 require assessment and approval of any activity involving CSG or a large coal mine that ‘has, or is likely to have, a significant impact on water resources … a) in its own right; or b) when considered with other developments, whether past, present or reasonably foreseeable developments’ (s. 528). Australia This requirement is accompanied by federal policy guidance (Department of the Environment (Commonwealth), 2013) and technical guidance on CEA-related information that proponents must provide in their EIA (IESC, 2018). A strength of this approach is its use of a statutory Independent Expert Scientific Committee (EPBC Act, ss. 505D, 528) to peer review proponent-produced CEA and advise the Minister for the Environment, the decision-maker under the EPBC Act. Queensland’s CSG-specific regime for CEA in relation to water resources (Water Act, 2000 (Queensland), ch. 3), which is largely industry-funded, is unusually detailed by Australian standards, though it protects groundwater-dependent ecosystems relatively weakly. Another comparative strength of Queensland’s arrangements is the relatively proactive approach to designating ‘cumulative management areas’ and the clear framework for regularly predicting and reporting impacts in these areas. The comparative clarity of this framework should help support practice effectiveness and transparency, notwithstanding the implementation challenges and gaps outlined below. However, the detail of specific arrangements would require considered modification for how they might apply to valued environmental components (VECs) outside the groundwater context. Special arrangements related to CEA apply in marine settings. The regulation of cumulative impacts on the Great Barrier Reef is being pursued under a strategic assessment under Part 10 of the EPBC Act and accompanying detailed policy guidance (Great Barrier Reef Marine Park Authority, 2014; 2018). The National Offshore Petroleum Safety and Environmental Management Authority (NOPSEMA), which has environmental approval functions under the Offshore Petroleum and Greenhouse Gas Storage Act, 2006 (Commonwealth) and Offshore Petroleum and Greenhouse Gas Storage (Environment) Regulations, 2009 (Commonwealth), considers cumulative effects of a project and issues brief guidance mentioning this (NOPSEMA, 2018). Australian CEA practice faces important challenges in the contentious context of resources extraction, especially in relation to data availability and sharing (particularly baseline groundwater data), availability of appropriate models, and uncertainty about allocating responsibility for dealing with cumulative impacts among project proponents (Nelson, 2019). CEAs for CSG tend to perform particularly poorly in relation to clearly identifying the temporal scope of assessment and considering the probable effects of climate change. These are crucial issues given the long durations of likely effects (Nelson, 2019). Assessing cumulative effects on Aboriginal cultural heritage, including culturally significant VECs, is a further notable gap in current policy and practice. Canada Cumulative effects are conceptualized in the Canadian research and practice context as adverse effects on VECs caused by the interaction of multiple human activities and natural processes across time and space (typically a region) (Olszynski et al., 2017). Despite the considerable scholarly attention devoted to CEA in Canada and many other countries, CEA understanding remains weak, CEA practices are wanting, and overall progress on promoting CEA is slow (Sinclair et al., 2017). CEA is listed as one of the purposes of the Canadian Environmental Assessment Act (CEAA), 2012 and was previously a requirement of the former CEAA, 1995. The purposes of CEAA, 2012 are, among others, ‘to encourage the study of the cumulative effects of physical activities in a region and the consideration of those study results in environmental assessments’ (s. 52). CEA is also an element of the first mandatory factor to be considered under CEAA, 2012. Pursuant to subsection 19(1)(a) of CEAA, 2012, a project EIA must take into account the environmental effects of the project, including ‘any cumulative environmental effects that are likely to result from the designated project in combination with other physical activities that have been or will be carried out’ (ss. 19(1)(a)). of CEAA, 2012 are, among others, ‘to encourage the study of the cumulative effects of physical activities in a region and the consideration of those study results in environmental assessments’ (s. 52). CEA is also an element of the first mandatory factor to be considered under CEAA, 2012. Pursuant to subsection 19(1)(a) of CEAA, 2012, a project EIA must take into account the environmental effects of the project, including ‘any cumulative environmental effects that are likely to result from the designated project in combination with other physical activities that have been or will be carried out’ (ss. 19(1)(a)). CEA is also sometimes carried out in conjunction with other federal regulatory processes, including species at risk management plans under the Species at Risk Act (SARA, ss. 65-70). CEA can also be conducted as part of provincial and territorial EIAs, including complementary regional environmental assessment (REA) and strategic environmental assessment (SEA) processes, and cumulative effects, when assessed, may also be considered as part of the decision-making process under the new Canadian Navigable Waters Act (ss. 7(1)(7)(e) and 29(1)(g)). Notably, however, none of these processes requires CEA. 1 Refer to the “significant adverse environmental effect” guidelines issued by the Canadian Environmental Assessment Agency (2016). Canada The Operational Policy Statement (OPS) on Assessing Cumulative Environmental Effects under CEAA, 2012 seeks to clarify the requirements under CEAA, 2012 relating to CEA (Government of Canada, 2012). The OPS stipulates that all CEAs should include the following five steps: 1) scoping (including the temporal and spatial identification of VECs in relation to other past, present, and future biophysical activities); 2) data analysis and modeling (including the incorporation of local community and Indigenous traditional ecological knowledge); 3) mitigation (assessing the technical and economic feasibility of mitigation measures); 4) significance (predicted in relation to the Canadian Environmental Assessment Agency’s significant adverse environmental effects guidelines1); and 5) follow-up (including the verification of EA accuracy and the determination of the effectiveness of implemented mitigation measures). Additional CEA guidance is provided by the Expert Panel for the Review of Environmental Assessment Processes convened by the federal government. In its report Building Common Ground: A New Vision for Impact Assessment in Canada, the Expert Panel defines cumulative effects as changes to the five pillars of sustainability – environment, economy, social, cultural, and health – caused by multiple interactions among human activities and natural processes that accumulate across space and time (Government of Canada, 2017). The Expert Panel strongly recommended the use of REAs in cases of cumulative effects on federal lands and marine areas, and emphasized the importance of identifying and evaluating alternative development scenarios as part of CEA. These recommendations, however, were not carried through in the new federal Impact Assessment Act (IAA) enacted in June 2019. Instead, the IAA, like CEAA, 2012 before it, encourages the assessment of cumulative effects on a regional basis (s 6(1)(m)), and includes “any cumulative effects that are likely to result from the designated project” among the many mandatory factors to be considered when conducting an EIA (s 22(1)(a)(ii)), tracking the very same language quoted above that was used in CEAA, 2012. This weak requirement, however, has been consistently honoured more in the breach than in compliance. Accordingly, CEA practice in Canada remains at best deficient, and at worst entirely absent from EIA processes and economic decision-making; much the same can be said of CEA’s conceptually-close cousin, adaptive management (Olszynski, 2017). However, the gap between academic research on the potential of CEA, which is considerable, and the actual practice of CEA, which is negligible, is only partly due to the lack of robust CEA legislation. 2 The new IAA, which repeals and replaces CEAA, 2012, provides for the possibility of regional and strategic assessments at the sole discretion of the Minister of the Environment (whether on the Minister’s own initiative or in response to a public request: ss. 92-103). Otherwise, the new legislation provides neither a mechanism for triggering and mandating regional or strategic assessments nor any criteria for how to conduct such assessments. The new legislation is likewise silent with respect to the outcome of regional and strategic assessments; while an assessment report is to be filed with the Minister and made public, there is no provision 3 For example, in Canada major oil pipeline projects are assessed on a pipeline-by-pipeline basis, precluding a broader assessment of regional, national, and international energy and economic strategies and their trade-offs with VEC conditions and related dimensions of sustainability (Palen et al., 2014). for decision-making, and no guidance on how the results of regional and strategic assessments are to be utilized – if at all – in future project assessments. Canada While more firmly legislating CEA – along with REA and SEA2 – is certainly important, equally important is mainstreaming what Sinclair et al. (2017) characterize as the ‘CEA mindset.’ They argue that rather than dismissing CEA as a thorny distraction from the usual business of project-level EIAs, CEA should be at the core of every assessment of VEC conditions affected by human activity. Ideally, CEA should be conducted at the REA and SEA levels, but whatever the given level of assessment – project, sectoral, regional, or strategic – it is essential to adopt a holistic approach capable of considering cumulative effects across all levels (Harriman & Noble, 2008). Crucially, this means abandoning the predominant major-project approach to EIA in Canada. Designating only “major” projects – while exempting a great many others (Government of Canada, 2015) – for what is a siloed, project-by-project EIA process3 significantly hinders the ability to assess cumulative effects and utilize CEA to facilitate the transition toward greater sustainability. Canadian CEA scholars recognize that there is a continuum of CEA practice, and that aspects of meaningful CEA do sometimes occur, albeit in a limited number of EIAs (Creasey & Ross, 2009). Counter-intuitively, the limitations of CEA understanding and practice suggest options for improvement. This counter-intuition is strengthened by the absence of serious debate in Canada over the importance of effective CEA. Perhaps the most important lesson, arising out of both the limitations of Canadian CEA practice and the various proposals for CEA reform, is the need to be far more realistic about the CEA capacity of private-sector project proponents. It is long past time for government agencies to curtail their reliance on private-sector proponents and assume responsibility for ensuring both effective CEA practice and more effective EIA overall (MacLean, 2016; MacLean et al., 2016; MacLean, 2019). While CEA practice is particularly and unjustifiably weak in Canada, growing attention to “Anthropocene risks” – that is, cross-scale systemic environmental risks with global effects – is generating increased interest in CEA-like thinking (Keys et al., 2019). Case studies from China and India focusing on sustainable water management in historical perspective, for example, illustrate how environmental for decision-making, and no guidance on how the results of regional and strategic assessments are to be utilized – if at all – in future project assessments. governance is by dint of sheer necessity moving toward a more holistic CEA mindset (Liu et al., 2019). New Zealand The main EIA law in New Zealand is the Resource Management Act, 1991 (RMA) which governs the management of natural and physical resources over most of the country, except the land managed by the Department of Conservation, and offshore resource development (both of these have their own EIA requirements, under separate legislation). The whole approach of the RMA is to ensure adverse environmental effects of resource management activities are recognised during decision-making processes, at the policy and plan levels as well as at the project level. The definition of “effect” in the RMA (s. 3) includes cumulative effects: ‘. . . (d) any cumulative effect which arises over time or in combination with other effects . . .’ (RMA, s. 3(d)). This is taken as the mandate for assessing cumulative effects, and ensures it is integrated into the broader impact assessment processes. In relation to proposed projects, resource consent applicants must include an “assessment of environmental effects” (AEE) (that is, an EIA) and that should, when appropriate, include an assessment of cumulative effects along with other impact information. The bulk of the applications nationwide are for small, local activities, so the AEEs for these are generally brief, scoping-style assessments, and would not be expected to contain any treatment of cumulative effects. Major projects have full impact reports and some treatment of cumulative effects would be expected if relevant to the proposal. In relation to policies and plans, the functions of regional and territorial authorities are directly related to the need to recognise and manage effects of resource use, including cumulative effects. For example, at the regional level, regional councils are required to: ‘[prepare] objectives and policies in relation to any actual or potential effects of the use, development, or protection of land which are of regional significance . . .’ (RMA, s. 30(1)(b)). Similarly, at the district level, the main functions of territorial authorities (district and city councils) include: (a) the establishment, implementation, and review of objectives, policies, and methods to achieve integrated management of the effects of the use, development, or protection of land and associated natural and physical resources of the district: (b) the control of any actual or potential effects of the use, development, or protection of land . . . (RMA, s. 31(1)). New Zealand Regional and territorial authorities prepare policies and plans to meet these requirements, and that framework (together with any National Policy Statements and/or National Environmental Standards produced by central government) provides the context for decisions about proposed activities in the resource consent process. In theory, therefore, the assessment of cumulative effects should occur during policy and plan preparation and as part of the AEEs produced by resource consent applicants. The Ministry for the Environment produced a guide to AEE (that is, EIA) production, last updated in 2006 (NZ Ministry for the Environment, 2006). Given the integrated nature of the process, cumulative effects are mentioned but no specific guidance is given on how to carry out such assessments. More recently the New Zealand Environmental Protection Authority produced a guide on EIA preparation under the Exclusive Economic Zone (EEZ) and Continental Shelf (Environmental Effects) Act, 2012 (for major offshore proposals in the EEZ). This briefly described two broad types of cumulative effect (intra-project and inter-project) but again provided no substantive advice on approaches. Case law strongly influences EIA practice in New Zealand and the case law around the interpretation of the definition of cumulative effect has been confused, so there has been a lack of clear direction for practitioners. An influential decision by the Court of Appeal (Dye vs Auckland Regional Council [2002] 1 NZLR 337 (CA)) applied a narrow definition of cumulative effects (among other things, that cumulative effects did not include effects of other activities, only those of the activity for which resource consent is being sought). Environment Court Judge Jon Jackson, in a recent paper discussing the assessment of effects under the RMA, believes Dye ‘has caused ongoing problems when it is applied more widely than its facts justify’ (Jackson, 2016, p. 26), and an Environment Court decision in 2017 (Wellington Fish and Game Council v Manawatu-Wanganui Regional Council [2017] NZEnvC 37) noted that the relevance of Dye, beyond the immediate context of the original case, had been placed in doubt by subsequent legal decisions. Apart from Judge Jackson’s commentary in 2017, a discussion paper on the Quality Planning website in 2009 is still one of the few substantive treatments of cumulative effects under the RMA, but also shows the almost total separation of the legal discourse from the impact assessment literature on CEA. New Zealand These suggest that judicial interpretation of cumulative effects is finally moving closer to the internationally recognised meaning. Although official guidance has not yet improved, a guide to EIA for major offshore projects by a government research institute (Clark et al., 2017) includes a section on cumulative effects that draws on impact assessment literature, which provides a clear signal for how future practice could evolve. Quality (CEQ) definition. Moreover, another recent decision of the Environment Court upheld an appeal from local residents near Christchurch, New Zealand, and refused consent for a new gravel extraction quarry (Yaldhurst v. Christchurch City Council & Harewood Gravels Limited, 2017). This was mainly on the grounds that the cumulative impacts of the existing quarries plus the proposed quarry would be significant for the local people. These suggest that judicial interpretation of cumulative effects is finally moving closer to the internationally recognised meaning. Although official guidance has not yet improved, a guide to EIA for major offshore projects by a government research institute (Clark et al., 2017) includes a section on cumulative effects that draws on impact assessment literature, which provides a clear signal for how future practice could evolve. New Zealand Practice overall at the project level is weak: EIAs for large projects are more likely to address cumulative effects but not always; smaller projects will often lack any consideration. When cumulative effects are addressed, in many cases assessors just look at the totality of effects from a project (that is, the integrated view of project impacts). In other cases, assessors are looking at likely effects added to those from existing activities in a locality (space crowding) and sometimes at possible interactions, but that is less frequent. Many of the more obvious cumulative effects in New Zealand are associated with the expansion of rural land uses such as dairying, marine farming, and horticulture, or with urban growth around major cities. Ideally these should be addressed through CEA provisions as part of an SEA process linked to regional planning. In New Zealand regional policies and plans are required under the RMA, but there is no clear mandate for SEA and little evidence of SEA-type assessments being applied at the regional level (Wilson & Ward, 2011). Cumulative effects are usually noted in regional plans, especially in their objectives, but with the implication that project level decisions will be sufficient to deal with regional level cumulative impacts. Recent moves to introduce spatial plans at the regional level may encourage fresh thinking on CEA practice. The main strength of the New Zealand system is the way the consideration of cumulative effects is integrated with the rest of the impact assessment process, and the fact that those processes, in theory, apply at the different levels of decision-making from local to national. However, practice has not lived up to the potential of the system, especially in the absence of strong guidance from the Ministry for the Environment. There are signs that things could be changing for the better. The Environment Court decision NZEnvC 37, cited above, noted with apparent acceptance the applicant’s use of a definition of cumulative effects from a NOAA publication, itself based on the US Council for Environmental Quality (CEQ) definition. Moreover, another recent decision of the Environment Court upheld an appeal from local residents near Christchurch, New Zealand, and refused consent for a new gravel extraction quarry (Yaldhurst v. Christchurch City Council & Harewood Gravels Limited, 2017). This was mainly on the grounds that the cumulative impacts of the existing quarries plus the proposed quarry would be significant for the local people. United Kingdom The regulatory requirement to address EIA, including cumulative effects at the project level, was introduced into UK law through the implementation of EC Directive 85/337. This included the requirement to describe the ‘direct effects and any indirect, secondary, cumulative. . .effects of the project’. The most recent revision to the EU Directive (2014/52/EU) was transposed into UK law in 2017. The current key ‘generic’ legislation for developments subject to the Town and Country Planning Act (1990) is the Town and Country Planning (EIA) Regulations, 2017 (as amended). For major projects in England that are subject to the Planning Act, 2008 (National Infrastructure Planning, n.d.) the relevant legislation is the Infrastructure Planning (EIA) Regulations (2017). Other authorization systems also require EIA and are subject to separate legislation (Marine Works (EIA) (Amended) Regulations, 2017, for example). Glasson and Therivel (2019, Table 3.6) list a total of 28 key UK EIA regulations. The regulations transpose the requirement EU 2014 EIA directive into law in England and the devolved administrations within the UK (Wales, Scotland and Northern Ireland). This has strengthened the requirement to consider cumulative effects which now must be taken into account when deciding whether a development should be subject to EIA (Annex III of Directive 2014/52/EU) as well as being one of the key areas of assessment (Annex IV): Annex IV—Information for the environmental impact assessment report: Annex IV—Information for the environmental impact assessment report: 5. A description of the likely significant effects of the project on the environment resulting from, inter alia: 5. A description of the likely significant effects of the project on the environment resulting from, inter alia: (e) the cumulation of effects with other existing and/or approved projects, taking into account any existing environmental problems relating to areas of particular environmental importance likely to be affected or the use of natural resources. The legislation does not define cumulative effects. The legislation does not define cumulative effects. The Planning Inspectorate, the government agency responsible for applications submitted for consent through the Planning Act, 2008, has published an advice note on CEA (PINS, 2019) to support those undertaking assessment for nationally significant infrastructure. This guidance drew heavily on research into current practice by Oxford Brookes University (Durning & Broderick, 2015). There is no other published government guidance specifically to support CEA practice in EIA. Industry-specific guidance has been published (for example, see RUK, 2013; MMO, 2014; Natural England, 2014; BSI, 2015). Broderick, Durning and Sanchez (2018) set out a generic methodology for CEA which is based on a review of guidance and best current UK and international practice. The current gaps in CEA practice in the UK include a lack of transparency and robustness in the methodology used to assess the effects and a lack of consistency and understanding of definitions and terminology. This is not unique to the UK and has long been cited as an ongoing challenge (for example, see Cooper & Sheate, 2002; IEMA, 2011; Durning & Broderick, 2015; Mitchell & Wright, 2019). There is a general lack of ‘process knowledge’ among practitioners on how to do effective CEA (Durning & Broderick, 2019) including issues such as defining significance and uncertainty in what to include in the assessment (Mitchell & Wright, 2019). Reflecting the current state of economic cycles, one of the suggested potential barriers to practice proposed by Mitchell and Wright (2019) is ‘confidentiality and impartiality’. They suggest that in ‘challenging’ commercial circumstances there may be a lack of willingness for developers to share information that is not in the public domain which limits the effectiveness of the assessment. Assessments of practice (Durning & Broderick, 2015; 2019; MMO, 2013 Assessments of practice (Durning & Broderick, 2015; 2019; MMO, 2013) demonstrate there are some areas of good practice, although it is notable that this good practice occurs when there is robust guidance which is followed, for example, in offshore renewable energy. The RUK (2013) guidelines suggested cumulative effects could be categorised as either ‘additive’ or ‘synergistic/in-combination’ effects and this definition has been also taken up by other guidance (Natural England, 2014; BSI, 2015; Broderick et al., 2018). An example of good practice identified by Broderick, Durning and Sanchez (2018, p. The legislation does not define cumulative effects. 667) from the Hornsea One Offshore Wind Farm as due to: • the baseline assessment sets out what is included and how the ‘long list’ of plans, projects and activities has been devised; • explains how spatial and temporal ranges have been identified; • the methodology for ‘screening’ projects in or out is clearly set out and the results are presented in a transparent format. There is also evidence of good practice emerging in major infrastructure projects which are subject to consenting through the Planning Act, 2008, such as the Port of Tilbury 2 cumulative effects assessment (Tilbury2, n.d.) which was subject to external peer review and evaluation using the evaluation framework set out in Durning and Broderick (2015). This is an example of undertaking a qualitative, high level CEA when insufficient information was available to undertake a quantitative assessment. The Examining Authority, in their recommendation report on whether the development be recommended for approval by the Secretary of State, considered this to be a reasonable and proportionate approach (PINS, 2018). Brazil In Brazil, federal law 6.938/81, regulated by federal decree 99.274/90, delimited objectives, principles and instruments of the National Environmental Policy (PNMA), instituted the National Environmental System (SISNAMA) and established EIA as one of its instruments. It also created the National Council for the Environment (CONAMA) as a consultative body chaired by the Minister of Environment and composed of Plenary and Technical Chambers and civil society. EIA was regulated in CONAMA Resolution 01/86 and its application has been linked to environmental licensing. From 1988, it became a mandatory constitutional requirement for project proponents to submit an EIA report, according to the text of the Federal Constitution and, later, in the States Constitutions. The requirement recently became entrenched in complementary federal law 140/2011: ‘environmental licensing is the administrative procedure for activities or undertakings that use environmental resources, effectively or potentially polluting or capable, in any way, of causing environmental degradation’. CONAMA Resolution 01/86, in its Article 6, item II, establishes that an environmental impact study must have: CONAMA Resolution 01/86, in its Article 6, item II, establishes that an environmental impact study must have: the analysis of the environmental impacts of a project and its alternatives, through identification, prediction of the magnitude and interpretation of the importance of the probable relevant impacts, discriminating: positive and negative impacts (beneficial and adverse), direct and indirect, immediate and medium and long term, temporary and permanent; their degree of reversibility; its cumulative and synergistic properties; and the distribution of social burdens and benefits (CONAMA, 1986). However, there is no explicit reference to CEA procedures, nor does it provide guidelines for its implementation. Although projects with potential to generate a high level of pollution are subject to an EIA before environmental licenses are issued, many of these projects are often defined by the boundaries of a river basin as an environmental management unit. EIA studies are usually focused on the environmental impact of the project, without the broader analysis of other existing or planned activities impacting the region or ecosystem. In other words, there is no effective treatment of cumulative impacts in Brazilian practice. Some progress has been recorded in the context of SEA, which, despite being an instrument still under development in Brazil, is gradually directing focus to cumulative impacts of large-scale projects. Brazil This is more evident in sectoral policies, such as the energy, oil and gas, transport, mining and tourism sectors, and are largely driven by individual private project developers. There has not been any legal framework or technical guidelines to support practice. An exception is the oil sector, where Ministerial Ordinance No. 198/12 of the Ministry of Mining and Energy, together with the Ministry of Environment, instituted the Environmental Assessment of Sedimentary Areas. The Ordinance makes CEA consideration a condition for granting environmental licensing for exploratory oil and gas blocs located in sedimentary basins, and other related undertakings and activities. In this case, SEA (including consideration of cumulative effects) is helping to shape decisions on areas of relevant mining-energy interest in accordance with the provisions of federal decree 6.678/2008, which approved the VII Sectorial Plan for Sea Resources. Some Brazilian states, such Minas Gerais and Bahia, are using SEA as a legal tool to understand the environmental impacts of state actions in all its aspects, thereby serving as a decision-making instrument that provides information about the possible environmental consequences of government actions (Valera, 2012). The concept of Integrated Environmental Assessment has also been applied to electric power generation programs to establish minimum environmental thresholds for key environmental indicators in river basins. In both SEA and Integrated Environmental Assessment (IEA), the analysis of socio-environmental effects, including the discussion of the cumulative and synergistic effects of co-located undertakings in river basins are being integrated to planning and decision-making. CEA practice in Brazil has some gross limitations, most of which have been documented by Sanchez (2006) and continue to be evident in current practice. These include outdated licensing process and requirements (no changes since the 1980s); lack of definitions and clear procedures in the legislation; scarce technical and methodological knowledge on cumulative impact assessment; shifting of environmental impact responsibility to private entities that have limited access to environmental information and whose focus is economic gains; limiting CEA consideration to project level, where the analysis is limited to an undertaking’s area or a project’s direct and indirect influence areas; disregard of the river basin in the delimitation of the project’s influence area; proliferation of competing standards and regulations leading to a conflict of institutional competences; lack of information exchange and integration between environmental agencies; and the unregulated practice of the SEA or the IEA. Brazil These are issues that require urgent intervention for CEA practice in Brazil to align with international best practice. Ghana Environmental impact assessment, according to Ghana’s Environmental Protection Agency (EPA, 1996), is a planning and decision making tool applied in Ghana to proposed undertakings (any activity, project, structure, investment, plan program, and so on) whose implementation or development may have a significant biophysical and social impact. The Environmental Protection Agency Act 490 Section 12(1) is further operative in tandem with the Environmental Assessment Regulations, LI 1652 of 1999, which delineates project undertakings for which EIA is mandatory and those for which EIA process is not a strict requirement. Following a systematic EIA process by a project proponent, an environmental permit is issued as an evidence of the proponent’s compliance with the Legislative Instrument (LI) 1652 of 1999. Currently, there is no explicit provision for CEA in Ghanaian environmental laws, including its Environmental Assessment Regulation document which stipulates the process for EIA practice. However, the scope of environmental impact statement (EIS) mandated by the Agency intrinsically implies a CEA consideration. While the EIA law is mute on CEA – both procedurally and methodologically – individual as well as institutional proponents including government ministries, agencies and departments often take the responsibility to include a CEA section that captures the effects of their projects in combination with other projects. The LI 1652 states that an EIS ‘shall also address possible direct and indirect impacts of the undertaking on the environment at the pre-construction, construction and operation, decommissioning and post-decommissioning phases’ (LI 1652, 1999, p. 6). The LI 1652 requirements for assessment of ‘indirect impacts’ is often interpreted by proponents to mean a requirement for CEA. For example, the proposed Abidjan-Lagos highway corridor project, a flagship project of the Economic Community of West African States (ECOWAS), is a large-scale regional infrastructure development that presented opportunities for consideration of CEA on a regional scale and along the corridor in a transboundary, trans-national context. CEA in Ghana is also implied in the context of SEA, where developers often take responsibility for addressing issues related to cumulative effects. For example, a full section was dedicated to analysis of ‘secondary, synergistic and cumulative effects’ in the SEA of the Transportation Integration Plan of Ghana (2010) prepared by Mott MacDonald, a Southampton UK-based consulting firm. Ghana In particular, cumulative effects were considered in determining the overall risk levels of environmental sustainability criteria of the Plan with issues such as climate change, air quality, water quality, public health, poverty reduction and cross-sectoral coordination flagged as long term, cumulative effects (Government of Ghana, 2010). The absence of regulatory requirements, and in consequence, good practice guidance, usually means that no rigorous benchmark exists for regulators to evaluate the adequacy of information on cumulative effects provided by proponents. In addition, though discretionary, information on CEA is only found in EIA reports of Schedule 2 (Mandatory List) projects under the EIA Regulation. These projects include large-scale mining, oil and gas exploration, transportation, energy, agricultural and health developments with standardized thresholds; for example, agricultural projects on lands exceeding 40 hectares or affecting more than 20 families or with irrigation components will ideally require that a CEA is carried out. EIA for many of these large-scale projects are often prepared by international consultants or donor agencies who sometimes choose to adopt a good practice approach – which may require doing CEA – to their EIA process. The implication is that CEA for small-scale projects, which constitutes the bulk of assessments, are completely non-existent. This is because the concept of CEA is widely unknown to local practitioners. Other problems include the limited training and proficiency of CEA among regulators, which limits the ability to make sound judgment relating to CEA. Compounding the challenges of the environmental institutions is the general citizen recalcitrance and, to an extent, intransigence toward due process and compliance with environmental regulations. For instance, project proponents tend to relax on the implementation conditions and the prescribed mitigation submitted through their Environmental and Social Management Plans once permits and development licenses are issued. On the other hand, despite provision for follow-up and monitoring in the EIA regulation (EPA Act 490, p. 3), there is no evidence of post-approval communications and interventions by Agency staff once permits have been issued. Thus, CEA process in Ghana lacks the required consistency, cohesion and institutional environment to thrive (Baxter, 2001). The general awareness of the concept of CEA among a section of the Ghanaian EIA community provides opportunity to shape the debate on the legal and policy imperative of CEA practice. Ghana This is a strength in the Ghanaian context as individual practitioners are taking the initiative to consider cumulative effects in the absence of a clear requirement, though at different levels of sophistication in their approach. This discretionary approach suggests the need for a legislative reform that makes CEA an explicit requirement in EIA process, capacity enhancement for regulators and practitioners, and a policy environment that provides guidance for proponents and developers on best practice CEA. 4 Known as Ley General del Equilibrio Ecológico y la Protección al Ambiente in Spanish 5 Known as Reglamento de la LGEEPA en materia de Impacto Ambiental in Spanish 4 Known as Ley General del Equilibrio Ecológico y la Protección al Ambiente in Spanish 5 Known as Reglamento de la LGEEPA en materia de Impacto Ambiental in Spanish 4 Known as Ley General del Equilibrio Ecológico y la Protección al Ambiente in Spanish 5 Known as Reglamento de la LGEEPA en materia de Impacto Ambiental in Spanish 6 Known as Secretaría de Medio Ambiente y Recursos Naturales in Spanish Mexico It is evident that in Mexico the current gaps related to CEA practice are more important than its strengths, but this could be viewed as an opportunity for improvement in the following areas: • Capacity building: there is little to no training for practitioners and authorities in identification and assessment of cumulative impacts. This is evident in the quality of the EIAs submitted by practitioners, in the authorizations granted by authorities, and more critically in the negative effects to the environment manifested after the development of the authorized projects. • CEA understanding: splitting of big projects into smaller ones with the intent of not including cumulative impacts in EIA reports is a recurrent practice allowed by Mexican environmental authorities. For example, a project that involves the construction of a liquid natural gas terminal in Manzanillo, Colima, may include the terminal, a railroad deviation, and a 300 km pipeline from Manzanillo to Guadalajara, Jalisco, in a coastal lagoon planned to be a new port. This project, instead of being submitted as a single EIA or being assessed as a SEA with rigorous consideration of cumulative effects, was split into three single projects with their respective regional EIAs and without considering cumulative impacts. The three projects were independently approved by the authority without weighing the collective impacts of the three projects in addition to other activities in the region. • CEA understanding: splitting of big projects into smaller ones with the intent of not including cumulative impacts in EIA reports is a recurrent practice allowed by Mexican environmental authorities. For example, a project that involves the construction of a liquid natural gas terminal in Manzanillo, Colima, may include the terminal, a railroad deviation, and a 300 km pipeline from Manzanillo to Guadalajara, Jalisco, in a coastal lagoon planned to be a new port. This project, instead of being submitted as a single EIA or being assessed as a SEA with rigorous consideration of cumulative effects, was split into three single projects with their respective regional EIAs and without considering cumulative impacts. The three projects were independently approved by the authority without weighing the collective impacts of the three projects in addition to other activities in the region. • Good practice guidance: the non-existence of proper guidance for the identification and assessment of cumulative impacts within the different types of EIA. Mexico Mexico has specific provisions for CEA in its environmental law and its EIA specific regulation, especially in the General Law for Environmental Protection and Ecological Balance (LGEEPA)4. The LGEEPA, which came into effect from 28 January 1988 (amended on 5 July 2018), in its Chapter IV (Tools for Environmental Policy) Section V (Environmental Impact Assessment), defines the EIA process that certain federal activities and infrastructure are to follow. The specific regulation of this law for EIA (Regulation for Environmental Impact Assessment5), published 30 May 2000 (amended 31 October 2014), defines cumulative environmental impact as ‘the effect in the environment as a result of the increment of the impacts for particular actions caused by the interaction with other past or present impacts’ (Chapter I General Provisions, Article 3). It is important to note that the Mexican legal framework does not include future projects/undertakings in its definition contrary to guidance issued by the Ministry of Environment and Natural Resources6 (SEMARNAT) and other international practice guidelines (Canter & Ross, 2010; European Commission, 2013; Natural England, 2014). At the federal level Mexico has two types of EIA (particular and regional) where cumulative impacts are encouraged to be identified; however, it is in the regional type where these impacts are more likely to be recognized because regional EIA was visualized as a type of SEA for urban development plans or programs, and for ecological zoning of land by Mexican authorities (SEMARNAT, n.d., p. 4), although in practice this type is mostly used in big projects like dams, wind farms, highways, and so on. Currently there is no guidance issued by responsible government department to support the formal and systematic CEA practice in Mexico at any level (federal, state or municipal) (Perevochtchikova & André, 2013). As previously mentioned, a general guidance for regional EIA-type in Mexico exists (SEMARNAT, n.d., p. 61). It draws some recommendations for practitioners, whom according to this guidance, should include in the regional EIA report an analysis of: • The effects of past, present and future activities that have modified the ecosystems of the region. • The foreseeable changes over the environment that could reasonably be expected from the project proposed, in combination with other human activities in the Regional Environmental System. • The assessment of the total sum of similar alterations related with the project proposed, independently of their origin. • The likely interactions between dissimilar sources of an impact. Mexico • SEA use: the lack of formal and systematic use of SEA that must be addressed as SEA remains the most viable tool to address cumulative impacts (Perevochtchikova & André, 2013; Gonzalez et al., 2014) and CEA ‘should be a central feature of these strategic studies’ (Canter & Ross, 2010, p. 262). Despite the above, not all is negative. The research community is leading the narrative for good practice CEA in Mexico. A deliberate attempt at translating research to policy in order to meet international CEA standards is what is missing. Bridging the research- practice gaps in CEA can be fostered in an atmosphere of trust and collaboration, and an institutional environment that is committed to best practice environmental protection and natural resources sustainability. Nigeria At the beginning of the 1990s, provisions regulating environmental impacts in Nigeria emerged from three different fronts: the Environmental Guidelines and Standards for the Petroleum Industry in Nigeria, 1991 (EGAS); the Urban and Regional Planning Decree 88, 1992; and the Environmental Impact Assessment Decree 86, 1992. Among these, the only law with specific provision for CEA was the EIA Decree 86 of 1992, which was established by a decree of the then federal military government, entered into force on 10 December 1992 and amended as an Act in 2004 (CAP. E12 L.F.N. 2004). This Act mirrors the United States National Environmental Protection Act and has been the only official legal provision that mandates project proponents to consider impacts beyond their projects. The Act requires, as part of its provisions for an EIA, that a project proponent conduct ‘an assessment of the likely or potential environmental impacts on the proposed activity and the alternatives, including the direct or indirect cumulative, short-term and tong-term effects’ (s. 4(d)). Information sharing and consultation are also implied in its objectives, particularly to determine if a project is ‘likely to have significant environmental effects on boundary or trans-state or on the environment of bordering towns and villages’ (s. 1(c)). The purpose of the provision was to ensure any cumulative environmental effects that, in the opinion of the responsible Minister (now vested in the National Environmental Standards and Regulations Enforcement Agency), ‘are likely to result from the project in combination with other projects that have been or will be carried out’ (s. 20.4) are included in the review and decision-making process. While the Act provides for two classes of assessment, screening (for all developments not included in the mandatory list) and mandatory study (for all activities listed in Schedule 1 of the Act), no specific trigger exists for cumulative effects. The requirement for CEA applies to all development projects, private or public, including those proposed by institutions of government at the local, state and federal levels. An approval could only be granted by the federal minister (or his designate) if the activity in question, besides being defensible from a project-specific point of view, is deemed environmentally sound from a cumulative effects perspective – at least in theory. In 2017, the Act was amended to include a requirement to conduct a SEA for policies, plans and programs that are deemed to have significant environmental implications (s. 59). Nigeria The Federal Ministry of Environment (the responsible authority) also developed a series of guidelines, including a SEA guideline, a generic EIA procedural guideline and other guidelines specific to oil and gas, urban development, renewable energy and chemical plants. While they are often sketchy with no robust framework to operationalize requirements, a recurrent theme in all the guidelines is the need to address cumulative effects in every major sector and at every scale of development. As of date, no SEA has been developed to test the robustness of CEA consideration within this context. The blanket provision for cumulative effects for all developments represents a move towards best practice in CEA as advocated for in many studies (Hegmann et al., 1999; Olagunju & Gunn, 2013). Hegmann et al. (1999), for instance, consider CEA as EIA ‘done well’. However, translating legal provisions to practice is a huge gap in the Nigerian context. A review of regulatory processes and selected EIA reports shows that neither a strong cumulative effects guideline or framework exists, nor the detailed consideration of cumulative effects implemented in practice. The 1991 Environmental Guidelines and Standards for the Petroleum Industry in Nigeria remains the only document where some form of expectations for CEA is set out for oil and gas development projects, albeit in an anecdotal manner that only captures the impact of multiple stream crossings and the difference between ‘[s]hort term, cumulative and long term impacts’ (s. 5.6.2.iii). The adoption of the US National Environmental Policy Act’s approach to environmental assessment law with its particular attention to cumulative effects may have been the inspiration for similar development in Nigeria (Ogunba, 2004). Thousands of EIA reports are prepared across levels of government and sectors each year, but no indication that sound CEAs are factored in the decision-making process, especially in the absence of a clear guideline that sets out expectations. There is no evidence that case law is playing any role in interpreting EIA requirements and more specifically with CEA in Nigeria. With three independent systems of EIA, the ability to develop a coordinated approach to CEA practice becomes complicated. 7 Nigeria Niger Delta region contains over 300 active gas flare sites with an estimated 15.1 billion cubic metres (bcm) of natural gas flared, with impacts spreading to the entire West Africa sub-region (see Fawole, 2019). A 2018 Regulation (Flare Gas (Prevention of Waste and Pollution) Regulations, 2018) introduced by the Federal government to minimize the environmental and social impact of natural gas flaring in the region is mute on its cumulative effects. geria Niger Delta region contains over 300 active gas flare sites with an estimated 15.1 billion cubic Nigeria There is little harmonization and a lack of consistent standards across sectors regarding treatment of cumulative effects, which thus suggests that vague requirements via a decree without commensurate policy and technical guidance will not be enough for project proponents to identify, characterize and evaluate potential cumulative effects in any substantive manner that fulfills its intentions. A first step to improving CEA practice in Nigeria would be to develop a cumulative effects guideline that explicitly stipulates in legislation or through a similar instrument the issues that are to be taken into account. With a legislated SEA, potential exists for an adoption of an “enlightened” CEA approach that is regional-focused and emphasizes an ecological significance relative to sustainability goals and future scenarios (Gunn & Noble, 2011; Jones, 2016). Similarly, a centralized EIA system where state and local government jurisdictions have no inputs into assessment processes despite being vested with the authority to create local and regional plans (as per Urban and Regional Planning Decree 88, 1992) would not be sufficient. Rather, a switch to a rigorous CEA regime that is focused on an approach that reconciles scientific assessments, plan development and social policy is required (Parkins, 2011; Duinker et al., 2013; Olagunju & Gunn, 2016). This integrated approach to CEA is especially important to reconcile and address the complex mix of environmental pollution (including the notorious issue of gas glaring in the Niger Delta with its local, regional and transboundary effects7), social justice and livelihood sustainability challenges in its oil producing region of Niger Delta (Giwa et al., 2017; Fawole et al., 2019; Matemilola et al., 2019). Some international EIA jurisdictions (for example, the United Kingdom) demonstrate promising ways to overcome such limitations through collaboration between agencies and levels of governments and through engagement with subject matter expertise across sectors (mining, oil and gas, and transportation, for example). As it stands, CEA in Nigeria has the legal basis to succeed but more work is required to ensure practice lives up to minimum acceptable standard for good practice CEA. Regulatory features and variations across jurisdictions Cumulative effects assessment is part of EIA process in many of the countries examined, and is adapted to each individual jurisdiction’s context. The concept has advanced in certain jurisdictions such as Australia and the United Kingdom where policy, legal and research environments, and attention to SEA have shaped practice. However, even in these countries, practice efficiency, political leadership and explicit guidance are still fundamental issues to be addressed. Regulatory and legislative instruments have played an important role in mainstreaming CEA into project EIA processes, although each country differs in the approach and level of sophistication of the CEA requirements. The development in CEA research, along with recognition of interactive effects of many major projects, has enabled some countries to enact and evolve EIA legislation that requires explicit consideration of CEA. However, often such legislative provisions are not accompanied with policy guidance that defines expectations, offers clear methodology, or sets implementation requirements for CEA. In most of the jurisdictions discussed in this chapter, national governments have been responsible for setting the legal framework. Owing to the historical volume of EIA practice in Canada, for example, operational guidelines specific to CEA have been available for more than two decades, with the most recent revision having been provided in 2012. However, this is not the norm. Guidance for CEA practice seems to be scant among the countries profiled. In the United Kingdom, industry-specific guidelines have been developed, for example, that are specific to infrastructure issued by the Planning Inspectorate. Most often, any CEA guidance that does exist seems to have emerged in a piecemeal fashion, driven by sectoral interest such as concern for cumulative effects related to water management, and so on. Unlike in Canada where case law has had at least some (but overall quite minimal) influence in the interpretation of cumulative effects (for example, West Moberly First Nations v. British Columbia (Chief Inspector of Mines), 2011, BCCA 247 (CanLII))8, in New Zealand and other countries, legal challenges involving CEA are reportedly limited, thus legal understandings of expectations for CEA practice in terms of its scope, rigour and implementation seem to be in an early stage of development. All eight countries discussed here are facing some form of capacity challenges in implementing CEA in a way that aligns with best practice principles. 8 The Court of Appeal for the Province of British Columbia (Canada) ruled that project proponents must include the understanding of the nature and extent of historic and cumulative impacts to Aboriginal rights and take a broad, forward-looking approach to cumulative impacts assessment in relation to Aboriginal rights. It is important to note, however, that this ruling is specific to its Aboriginal law context and does not reflect or inform CEA practice as part of the general EIA regime in Canada. Regulatory features and variations across jurisdictions The shortage of experts is a universal challenge, but the problem is especially acute in countries with weak institutions for EIA. Except in Brazil where some form of legal guidance for addressing cumulative effects has been developed for its oil and gas sector, none of the other developing countries has been able to create a CEA-specific policy document to guide practice, in large part due to absence of technical knowhow and adequate institutional capacity to enforce requirements (for example, Ghana). The lack of reconciliation between competing legislative instruments (for example, Nigeria has three EIA laws with overlapping jurisdictions) further complicates the capacity to develop a coherent, consistent approach to CEA. Mexico’s federal law restricts CEA consideration to a retrospective exercise that excludes “the foreseeable future”, and instead directs focus to current changes to project environment. Capacity to effectively implement CEA needs different levels of intervention. In the developed countries, practice in Australia, Canada and the United Kingdom, for example, has been shaped substantially by industry experts, particularly those in the extractive sectors. In part, capacity building has been driven by parliamentary and agency inquiries emphasizing the risks of cumulative effects of the extractive sector on water resources. In Australia and the United Kingdom, external, independent review and evaluation as a complement to proponents’ reports may suggest increased rigour and better quality of assessment. However, these interventions are limited by other institutional challenges, including limited data sharing, lack of consistency and transparency on approach and expectations, and a practice approach that typically excludes small-scale projects which usually constitute a majority of assessments. Project splitting as a way of circumventing the need to do a CEA has been cited in the case of Mexico. This practice, however, may be more universal than is thought, especially in jurisdictions where requirements for CEA is limited to major projects (for example, Canada). Some countries have successfully expanded their legislation to include explicit provisions for SEA (Nigeria, for example) and/or regional EIA (Mexico, for example). These legal developments, as well as voluntary SEAs and REAs in other jurisdictions (Australia, New Zealand and Canada, for example), have the potential of offering meaningful context to CEA practice and are potentially capable of strengthening practice more quickly than the traditional project-based approach. It is important, however, to recognize that SEA approaches in many countries continue to be limited by project proponents’ expertise, subjective experience, and business interests. Regulatory features and variations across jurisdictions Coordination between environmental and planning institutions is evidently a key requirement for strengthening CEA within a SEA framework to realize long-term, strategic outcomes that transcend individual project development. Conclusion Overall, despite being fraught with many challenges, commitment towards improved CEA practice continues to grow, and lessons drawn from the eight countries discussed in this chapter may be helpful in efforts to design and implement reforms that are effective in the long run and contribute to more environmentally responsible decision-making. Acknowledgement Dr. Rebecca Nelson acknowledges the research assistance of Lara Shirley in relation to the Australian component of the chapter and funding from the Australian Research Council (‘Regulating Cumulative Environmental Effects: Designing Global Best Practice’, Project ID #DE180101154). Conclusion This chapter shows that the concept of CEA is evolving from a strictly project-based approach towards a more strategic and regional context that is explicitly being promoted through EIA legislation in many jurisdictions. Despite this apparent progress, realizing best practice in CEA is a continuous work in progress that requires stronger political commitment, improved technical knowledge, and a systems approach that bridges the legal, scientific and development planning imperatives across geographical scales and levels. Developed countries are leading in sector-based innovations, industry-led guidance, and peer review and evaluation, though the need for strengthened government leadership and ownership of the CEA process is common to all the national systems described in this chapter. In developing countries, the foundations for CEA to thrive are being laid through legal provisions and explicit recognition of higher-order assessments such as SEA and REA, but technical know-how is reportedly limited and improved practice will depend also on innovative strategies that address institutional fragmentation and consistency in implementation. Across all countries, increased data sharing is necessary to capture the effects of many small projects on a cumulative basis, and political leadership is required to advance practice. Central to these challenges are questions such as whether developers should be held accountable to cumulative impacts beyond their projects and, with the exclusion of small projects from CEA in many jurisdictions, how close can we get to understanding the ‘full’ cumulative impacts of human developments? These questions remain unanswered. Overall, despite being fraught with many challenges, commitment towards improved CEA practice continues to grow, and lessons drawn from the eight countries discussed in this chapter may be helpful in efforts to design and implement reforms that are effective in the long run and contribute to more environmentally responsible decision-making. REA, but technical know-how is reportedly limited and improved practice will depend also on innovative strategies that address institutional fragmentation and consistency in implementation. Across all countries, increased data sharing is necessary to capture the effects of many small projects on a cumulative basis, and political leadership is required to advance practice. Central to these challenges are questions such as whether developers should be held accountable to cumulative impacts beyond their projects and, with the exclusion of small projects from CEA in many jurisdictions, how close can we get to understanding the ‘full’ cumulative impacts of human developments? These questions remain unanswered. References Abaza, H., Bisset, R. and Sadler, B. (2004). Environmental impact assessment and strategic environmental assessment: Towards an integrated approach. United Nations Environment Programme (1st ed.). ACT Government. (2017). 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https://openalex.org/W3134864044	https://pmr.lf1.cuni.cz/media/pdf/pmr_2021122010025.pdf	English	null	Laparoscopic Pectopexy: An Effective Procedure for Pelvic Organ Prolapse with an Evident Improvement on Quality of Life	Prague Medical Report	2,021	cc-by	4,217	25) 25) Prague Medical Report / Vol. 122 (2021) No. 1, p. 25–33 Introduction Pelvic organ prolapse (POP) is a health problem that affects millions of women worldwide and manifests in 50% of women over 50 years of age (Subak et al., 2001). Although POP is not a life threatening disease, it impairs the quality of life (QOL) significantly and negatively affects the psychosocial status of the patient due to discomfort and urinary and/or intestinal dysfunctions (Ghersel et al., 2019). Therefore, the treatment of this disease is important to precede the isolation from the social life especially in middle and advanced ages. Sacropexy is considered as the gold standard technique and it is the most effective approach for apical prolapse surgery which keeps the physiological axis of the vagina preserved (Nygaard et al., 2009; Maher et al., 2010; Bataller et al., 2019). Unlike open abdominal sacrocolpopexy, laparoscopic and robotic-assisted approaches don’t require a large abdominal incision and minimize bowel manipulation so they provide less postoperative pain and shorter recovery time. Laparoscopic sacrocolpopexy (LSP), which shortens hospital stay compared to open surgery, is an effective method but has some disadvantages in the postoperative period such as defecation disorders and pelvic pain (Akladios et al., 2010). The mesh placed between the sacrum and the vagina creates pelvic discomfort in many patients. Also potential damage to hypogastric nerve can cause dyschezia. The anatomic location of the sigmoid colon may also make it difficult to place the mesh between the vagina and the sacrum. There may be restriction in the movement of the colon after the operation and defecation or pain problems may be seen (Kale et al., 2017). A new technique for POP surgery was defined by Banerjee and Noe in 2011 to prevent the complications of LSP mentioned above.ff We aimed to investigate the effects of this effective and safe technique on quality of life, on psychosexual functions and incontinence. The method becomes widespread in recent years and reduces morbidity rates in patients. Prague Medical Report / Vol. 122 (2021) No. 1, p. 25–33 Prague Medical Report / Vol. 122 (2021) No. 1, p. 25–33 Karslı A.; Karslı O.; Kale A. Laparoscopic Pectopexy: An Effective Procedure for Pelvic Organ Prolapse with an Evident Improvement on Quality of Life Aybike Karslı1, Onur Karslı1, Ahmet Kale2 1Health Science University, Kocaeli Derince Education and Research Hospital, Kocaeli, Turkey; 2Health Science University, Kartal Dr. Lütfi Kırdar Education and Research Hospital, Kartal, Turkey Received March 25, 2020; Accepted January 28, 2021. Received March 25, 2020; Accepted January 28, 2021. Key words: Pelvic organ prolapse – Sacropexy – Pectopexy – Quality of life Abstract: It has been stated that the effectiveness of pectopexy method for the treatment of pelvic organ prolapse (POP) is similar to sacrocolpopexy. We aimed to search the effects of pectopexy method to the quality of life, sexual function and urinary incontinence. Thirty-one patients who were operated for POP with the technique of laparoscopic pectouteropexy/pectocolpopexy between January 2016 and November 2017 were included the study. Exclusion criteria were pelvic inflammatory disease, suspect of malignancy, pregnancy, prior POP or continence surgery. Quality of life inventories were (P-QOL, PISQ-12, UDI-6, IIQ-7) recorded preoperatively and at the postoperative third month. Results were compared statistically. The percentage of patients with menopause was 67.7% (n=21) and with reproductive term was 32.3% (n=10). Mean prolapse related quality of life inventory (P-QOL) score was 83.45 ± 8.7 (64–98) preoperatively and 8.61 ± 6.4 (0–23) postoperatively (p<0.05). The preoperative and postoperative score of quality of life inventories for urinary symptoms were 20 (15–21) and 2 (0–9) for IIQ-7 and 13 (3–18) and 4 (0–11) for UDI-6, respectively (p<0.05). The mean PISQ-12 sexual quality of life inventory score was 29.61 ± 4.8 (14–38) preoperatively and 7.1 ± 3.2 (1–13) postoperatively. According to our results laparoscopic pectopexy offers a feasible, safe and comfortable alternative for apical prolapse surgery. Mailing Address: Onur Karsli, MD., FEBU, Department of Urology, Health Science University, Kocaeli Derince Education and Research Hospital, 41900, Kocaeli, Turkey; Phone: +90 262 317 80 00; Fax: +90 262 233 46 41; e-mail: onurkarsli@yahoo.com Mailing Address: Onur Karsli, MD., FEBU, Department of Urology, Health Science University, Kocaeli Derince Education and Research Hospital, 41900, Kocaeli, Turkey; Phone: +90 262 317 80 00; Fax: +90 262 233 46 41; e-mail: onurkarsli@yahoo.com Laparoscopic Pectopexy for Quality of Life https://doi.org/10.14712/23362936.2021.3 © 2021 The Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0). Laparoscopic Pectopexy for Quality of Life https://doi.org/10.14712/23362936.2021.3 © 2021 The Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0). 26) Material and Methods Thirty-one patients who underwent laparoscopic pectoutero/colpopexy operation in gynecology clinic of Health Science University, Kocaeli Derince Education and Research Hospital with the diagnosis of POP were included to the study. The patients who presented with vaginal fullness, vaginal pressure, sagging sensation, were examined in lithotomy position. Patients with POP-Q stage greater than 2 were included to the study. Transvaginal ultrasonography and pap smear test were routinely applied to all patients. Endometrial sampling was performed in patients with suspected malignancy. The exclusion criteria were previous history of pelvic inflammatory disease, malignancy suspicion, pregnancy, prior POP or continence surgery and patients who did not want to be operated with this technique. In the preoperative evaluation of the patients; data related to the age, body mass index, parity status, history of surgery, gynecological examination were recorded. The validated prolapse quality of life (P-QOL), pelvic organ prolapse/ Karslı A.; Karslı O.; Kale A. Prague Medical Report / Vol. 122 (2021) No. 1, p. 25–33 27) urinary incontinence sexual questionnaire (PISQ-12), urinary distress inventory (UDI-6), incontinence impact questionnaire (IIQ-7) forms that were used in our study were filled preoperatively. P-QOL questionnaire is a simple, reliable and easy-to-understand questionnaire that can assess symptom severity, the impact of these symptoms on quality of life, and treatment outcomes for women with pelvic organ prolapse. UDI-6 is a questionnaire form consisting of six questions and specific to lower urinary tract symptoms. It combines information on irritant, stress, and obstructive/uncomfortable symptoms. IIQ-7 is a quality of life assessment tool specific to urinary incontinence. It consists of seven questions and allows an assessment of physical activity, social life and emotional health. The two query forms are intended to be used in combination. A short form of the (PISQ-12) was used for the assessment of sexual quality of life. The patients were called for control at the first postoperative week and at the third month. Preoperative reproductive quality of life questionnaires were filled in by patients at the postoperative third month and the data were recorded. The preoperative and postoperative quality of life data were compared statistically. Surgical technique There was no need for a special diet or bowel cleansing for the preoperative preparation of the patients. All patients were dressed with embolic compression stockings. Preoperatively 1.5 g Cefazolin was administered intravenously to the all patients for surgical prophylaxis. Laparoscopic Pectopexy for Quality of Life Material and Methods A 30-degree laparoscopic lens was guided into the abdomen with a 10-mm laparoscopic port from a 1 cm incision site on the lower edge of the umbilicus. Abdomen was inflated with carbon dioxide at 12 mm Hg pressure. Two 5-mm ports were placed to 2–4 cm inferomedial area of spina iliaca anterior superior bilaterally. One 15-mm port was placed on the left upper quadrant of abdomen. The round ligament part of 4 cm2 size which contains the lateral part of the ilopectineal ligament was used as the anatomical cue point. The peritoneum adjacent to the round ligament was superficially incised. The soft tissue in the pelvic wall was bluntly dissected until the iliopectineal ligament was seen, and the dissection was extended to the obturator nerve region. The same procedure was applied to the contralateral side. After the ilopectineal ligament was prepared, the peritoneal incision on the 2 sides was bluntly expanded along an imaginary line connecting the vaginal apex and pectineal line. Polypropylene monofilament mesh (3×15 cm) and 2-0 non-absorbable suture 10-mm sent from the port to the surgical area. The proximal end portions of mesh were fixed to the bilateral iliopectineal ligament with 2 sutures and the suture needle was taken out. Cervical bulge or vaginal apex were fixed to the middle of the mesh in the tension to prevent sagging. If the length of the mesh was long, the length of the mesh was shortened before the second iliopectineal ligament was fixed. Laparoscopic tacker was used instead of suture while the mesh was fixed to the Prague Medical Report / Vol. 122 (2021) No. 1, p. 25–33 28) tissues in some patients (Jelovsek et al., 2007). The peritoneum was closed using 2-0 absorbable sutures. After the carbon dioxide was evacuated, the ports were removed. tissues in some patients (Jelovsek et al., 2007). The peritoneum was closed using 2-0 absorbable sutures. After the carbon dioxide was evacuated, the ports were removed. y Statistical analysis was performed with IBM SPSS 20.0 (IBM Corp., Armonk, NY, USA) package program. Normal distribution was assessed by Kolmogorov- Smirnov test. Numerical variables with normal distribution were presented as mean ± standard deviation, numerical variables not showing normal distribution were given as median (25th–75th percentile), and categorical variables as frequency (percentages). Material and Methods The difference between the groups was determined by the student’s t-test for the numerical variables with normal distribution, and the Mann-Whitney U test for the non-normal variables. The t-test in the dependent samples was examined by Wilcoxon t-test when the normal distribution assumption was not provided. For the test of two-way hypotheses, the level of significance p<0.05 was accepted as sufficient. Ethics committee approval was obtained from Kocaeli University Faculty of Medicine and the patients were informed about the clinical and laboratory data to be used for scientific purposes and the confidentiality of the data and the consent forms were signed (registration number KUGOKAEK 2017/132). Results When the demographic data of the patients were examined, the mean age of the 31 female patients was 52.19 ± 11.78 (34–72) years and the mean body mass index was 28.82 ± 3.2 (18.4–33.2) (Table 1). Of the 31 patients who underwent laparoscopic pectopexy; 8 of them were operated due to vaginal vault prolapse, 7 of them were treated for grade 3–4 uterine prolapsus and cystocele, one with cystocele only and 15 for only grade 3–4 uterine prolapsus. Laparoscopic pectouteropexy was performed in 10 patients and laparoscopic pectocolpopexy was performed in 21 patients. Table 1 – Demographic data of patients Demographic data Patient number (n) Reproductive Menopause Age (mean ± SD [min–max]) Number of birth (mean ± SD [min–max]) BMI (mean ± SD [min–max]) 31 10 (32.3%) 21 (67.7%) 52.19 ± 11.78 [34–72] 2.90 ± 1.68 [1–9] 28.82 ± 3.20 [18.4–33.2] SD – standard deviation; BMI – body mass index Table 1 – Demographic data of patients 31 10 (32.3%) 21 (67.7%) 52.19 ± 11.78 [34–72] 2.90 ± 1.68 [1–9] 28.82 ± 3.20 [18.4–33.2] SD – standard deviation; BMI – body mass index Karslı A.; Karslı O.; Kale A. Karslı A.; Karslı O.; Kale A. Prague Medical Report / Vol. 122 (2021) No. 1, p. 25–33 29) Table 2 – Quality of life results of patients Quality of life questionnarie Preoperative score (mean ± SD) (min–max) Postoperative score (mean ± SD) (min–max) P P-QOL UDI-6 IIQ-7 PISQ-12 83.45 ± 8.71 (64–98) 12.52 ± 3.11 (3–18) 19.48 ± 1.56 (15–21) 29.61 ± 4.80 (14–38) 8.61 ± 6.43 (0–23) 4.13 ± 2.36 (0–11) 2.94 ± 2.08 (0–9) 7.10 ± 3.28 (1–13) 0.001 0.001 0.001 0.001 SD – standard deviation Table 2 – Quality of life results of patients When the operative data was analysed, the mean operation time was 33.8 ± 14.6 minutes. There were no perioperative and postoperative complications. Three months postoperatively, one patient had recurrence. The quality of life data of this patient was better than that in the preoperative period, no additional operation was therefore considered. The P-QOL score, which is the quality of life scale of prolapse, was 83.45 ± 8.7 (64–98) in preoperative patients and 8.61 ± 6.4 (0–23) in postoperative patients. There was a statistically significant difference between the quality of life scores of the patients before and after the surgery (p<0.05) (Table 2). UDI-6 quality of life questionnaire used for assessing urinary symptoms, incontinence and pelvic discomfort. The median value of preoperative UDI-6 score was 13 (3–18) and postoperative score was 4 (0–11). The difference was statistically significant (p<0.05). Preoperative and postoperative median IIQ-7 scores were 20 (15–21) and 2 (0–9) respectively and the difference was statistically significant (p<0.05). Laparoscopic pectopexy was performed in all patients. The mesh fixation was made by using suture in 12 patients (38.7%) and with tacker in 19 patients (61.3%). No significant difference in postoperative quality of life was found between these groups. Sexual function score evaluated with PISQ-12 was found as 29.61 ± 4.8 (14–38) in the preoperative patients, and 7.1 ± 3.2 (1–13) in postoperative patients. Postoperative sexual quality of life improved significantly compared to preoperative period and the improvement was statistically significant. Sexual function score evaluated with PISQ-12 was found as 29.61 ± 4.8 (14–38 n the preoperative patients, and 7.1 ± 3.2 (1–13) in postoperative patients. Table 1 – Demographic data of patients i in the preoperative patients, and 7.1 ± 3.2 (1–13) in postoperative patients. Postoperative sexual quality of life improved significantly compared to preoperative period and the improvement was statistically significant. Laparoscopic Pectopexy for Quality of Life Discussion According to the results of this study we consider that laparoscopic pectopexy is an alternative technique to LSP for the functional and anatomic success of POP treatment. Apical prolapse repair was performed laparoscopically for more than 20 years (Whitehead et al., 2007; North et al., 2009), however it depends on the experience of the surgeon. Possible problems may arise during laparoscopic sacrocolpopexy. Laparoscopic Pectopexy for Quality of Life 30) Prague Medical Report / Vol. 122 (2021) No. 1, p. 25–33 Sigmoid colon and sacral promontorium should be identified and care should be taken to avoid damaging the right ureter, presacral veins, hypogastric nerve and sigmoid colon in the sacral area. Another issue is the reason of the difficult surgical field at the ventral side of the sacrum; many surgeons have modified the technique and have fixed the mesh to the top of the promontory. However, this change of mesh localisation results in a positional change in the vaginal axis (Whitehead et al., 2007; Noe et al., 2015). The iliopectineal ligament is an extension of the lacunar ligament that runs on the pectineal line of the pubic bone (Faure et al., 2001). As shown by Cosson et al. (2003), the iliopectineal ligament is a stronger structure than the arcus tendinous of the sacrospinous ligament and pelvic fascia. The structure is strong, and holds suture well. It is also possible to find sufficient material for a suture in the lateral part of the iliopectineal ligament, facilitating reconstruction of the pelvic floor (Noe et al., 2015). This segment of the ligament is situated at the second sacral vertebra (S2) level which is the optimal level for the physiological axis of the vagina. S2 level is the anchor point for the physiological axis of the vagina (Noe et al., 2015). Laparoscopic pectopexy is a new type of endoscopic prolapse surgery using the lateral parts of the iliopectineal ligament for bilateral mesh fixation for the descended structures (Noe et al., 2013). Noe et al. (2013) compared laparoscopic sacrocolpopexy with pectopexy and they found less operation time and bleeding in pectopexy group. On the other hand, there was no difference between the two groups for the mean onset of bowel movements. In another study, Noe et al. (2015) published their comparative results that standard LSP versus laparoscopic pectopexy. Karslı A.; Karslı O.; Kale A. Discussion According to these results there was a statistically significant difference between the incidence of de novo defecation problems following laparoscopic pectopexy (0%) and sacrocolpopexy (19.5%). In our study we did not observe any defecation problems in the patients. This may be explained by the fact that pectopexy neither reduces the space of the pelvis (outlet obstruction) nor carries the risks of trauma to the hypogastric nerves. In classical technique of sacrocolpopexy, due to the narrowness of the minor pelvis, the presence of unexpected vessel variations or the fineness of the anterior longitudinal ligament and the difficulties in mesh fixation may increase the intraoperative complications such as adjacent organ injury or bleeding (Nygaard et al., 2009; Maher et al., 2010; Noe et al., 2013). In our study, 31 patients who underwent laparoscopic pectopexy had no intraoperative complications. The majority of studies that have been published in previous years and evaluated POP treatment usually focus on the anatomical success of the treatment, while other important parts such as vaginal adjustment, bowel problems, QOL or socioeconomic consequences are ignored. The most important benefit of the patient in individual surgery is the regression of symptoms and increased quality of life (Barber et al., 2009). Maher and colleagues (2004) found improvement in disease- specific and overall quality of life after POP surgery in patients with sacrospinous Karslı A.; Karslı O.; Kale A. 31) Prague Medical Report / Vol. 122 (2021) No. 1, p. 25–33 ligament fixation and abdominal sacrocolpopexy. CARE study reported significant improvement in disease-specific quality of life at 3 months and 2 years following sacrocolpopexy (Brubaker et al., 2003, 2008). In our study, postoperative P-QOL showed a significant improvement compared to the preoperative period. ligament fixation and abdominal sacrocolpopexy. CARE study reported significant improvement in disease-specific quality of life at 3 months and 2 years following sacrocolpopexy (Brubaker et al., 2003, 2008). In our study, postoperative P-QOL showed a significant improvement compared to the preoperative period. Improvement of sexual function as well as disease-related quality of life after sacrocolpopexy has also been demonstrated in some studies (Grimminck et al., 2016; Ko et al., 2017). Tahaoglu et al. (2018) reported significant improvement in quality of life and sexual function after laparoscopic pectopexy. In our study, PISQ-12 sexual quality of life score was significantly improved after laparoscopic pectopexy in the postoperative period. Conclusion According to our results, laparoscopic pectopexy offers a feasible, safe, and comfortable alternative for apical prolapse surgery. Pectopexy may increase a surgeon’s technical perspective for apical prolapse surgery. Discussion Urinary complaints are associated with pelvic organ prolapse and with impaired quality of life. Regression of urinary complaints after surgical intervention will contribute to the quality of life of patients. In a multicentre study involving 207 patients operated for apical prolapse, Altman et al. (2018) reported a statistically significant improvement in patients’ UDI-6 score after prolapse surgery. In another study by Coolen et al. (2017) comparing laparoscopic and open sacrocolpopexy, disease-specific quality of life measures, which were evaluated by UDI-6 score, demonstrated the efficacy of sacrocolpopexy as in previous studies, while the results of open and laparoscopic group were similar. In our study, the quality of life was significantly improved in patients who were evaluated with UDI-6 and IIQ-7 quality of life criteria. Since pectopexy technique provides bilateral mesh fixation to the iliopectineal ligament, the pressure is distributed evenly over both sides. However, in the technique of sacrocolpopexy, this pressure is at one point. Noe et al. (2013) suggested that rectosel development or bowel obstruction, which are disadvantages of the anterior fixation method, could be reduced by more physiological lateral fixation. They also reported that the use of monofilament mesh did not cause any erosion in the study in which they defined pectopexy technique. Also, we observed in this study, that the patients who underwent laparoscopic pectopexy had neither rectosel development, nor bowel obstruction and mesh erosion. Akladios, C. Y., Dautun, D., Saussine, C., Baldauf, J. J., Mathelin, C., Wattiez, A. (2010) Laparoscopic sacrocolpopexy for female genital organ prolapse: establishment of a learning curve. Eur. J. Obstet. Gynecol. Reprod. Biol. 149, 218–221. Altman, D., Geale, K., Falconer, C., Morcos, E. (2018) A generic health-related quality of life instrument for assessing pelvic organ prolapse surgery: Correlation with condition-specific outcome measures. Int. Urogynecol. J. 29(8), 1093–1099. References Akladios, C. Y., Dautun, D., Saussine, C., Baldauf, J. J., Mathelin, C., Wattiez, A. (2010) Laparoscopic sacrocolpopexy for female genital organ prolapse: establishment of a learning curve. Eur. J. Obstet. Gynecol. Reprod. Biol. 149, 218–221. Altman, D., Geale, K., Falconer, C., Morcos, E. (2018) A generic health-related quality of life instrument for assessing pelvic organ prolapse surgery: Correlation with condition-specific outcome measures. Int. Urogynecol. J. 29(8), 1093–1099. Laparoscopic Pectopexy for Quality of Life 32) Prague Medical Report / Vol. 122 (2021) No. 1, p. 25–33 Banerjee, C., Noe, K. G. (2011) Laparoscopic pectopexy: A new technique of prolapse surgery for obese patients. Arch. Gynecol. Obstet. 284(3), 631–635. Barber, M. D., Brubaker, L., Nygaard, I. (2009) Defining success after surgery for pelvic organ prolapse. Obstet. Gynecol. 114(3), 600–609. Bataller, E., Ros, C., Anglès, S., Gallego, M., Espuña-Pons, M., Carmona, F. (2019) Anatomical outcomes 1 year after pelvic organ prolapse surgery in patients with and without a uterus at a high risk of recurrence: A randomised controlled trial comparing laparoscopic sacrocolpopexy/cervicopexy and anterior vaginal mesh. Int. Urogynecol. J. 30(4), 545–555. randomised controlled trial comparing laparoscopic sacrocolpopexy/cervicopexy and anterior vaginal h I t U l J 30(4) 545 555 Brubaker, L., Cundiff, G., Fine, P., Nygaard, I., Richter, H., Visco, A., Zyczynski, H., Brown, M. B., Weber, A. (2003) A randomized trial of colpopexy and urinary reduction efforts (CARE): design and methods. Control. Clin. Trials 24(5), 629–642. Brubaker, L., Nygaard, I., Richter, H. E. (2008) Two-year outcomes after sacrocolpopexy with and without burch to prevent stress urinary incontinence. Obstet. Gynecol. 112(1), 49–55. Coolen, A. W. M., van Oudheusden, A. M. J., Mol, B. W. J., van Eijndhoven, H. W. F., Roovers, J. W. R., Bongers, M. Y. 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https://openalex.org/W4205541179	https://europepmc.org/articles/pmc8473451?pdf=render	English	null	Removal of Cyanobacteria and Cyanotoxins in Waters	MDPI eBooks	2,021	cc-by	2,711	Citation: Serrà, A.; Philippe, L.; Gómez, E. Removal of Cyanobacteria and Cyanotoxins in Waters. Toxins 2021, 13, 636. https://doi.org/ 10.3390/toxins13090636 Received: 2 September 2021 Accepted: 8 September 2021 Published: 9 September 2021 In this Special Issue, titled “Removal of Cyanobacteria and Cyanotoxins in Waters,” we have attempted to provide readers with a comprehensive overview of different strategies and alternative treatments currently being studied for the effective removal of cyanobacteria and cyanotoxins from water. The following presents a brief synopsis of the 13 research papers that constitute this Special Issue. Coagulation is a key process employed by conventional water treatment technologies. Worldwide, the most widely applied water treatment technology at water treatment plants involves a combination of coagulation, ﬂocculation, sedimentation, ﬁltration, and disinfec- tion to treat water. Herein, ﬁve papers focus either directly or indirectly on coagulation and its alternatives or on the potential impacts of conventional water treatment technologies on drinking water containing cyanobacteria and/or cyanotoxins [2]. Arruda et al. [2] have demonstrated that the so-called “ﬂoc-and-skin” technique, based on the combination of a ballast (i.e., natural soil or modiﬁed clay) and a coagulant, can effectively remove cyanobac- terial biomass comprised of Dolichospermum circinalis and Microcystis aeruginosa, depending on the ballast’s capacity to adsorb microcystin. At the laboratory scale, the authors studied the effect of the ﬂoc-and-skin technique on the release of microcystins from cyanobacterial biomass in real water from the Funil Reservoir, a eutrophic system in southern Brazil. Their results demonstrate that the technique is more effective than using a sole coagulant (i.e., polyaluminum chloride) to remove cyanobacteria and extracellular microcystins from water [2]. Next, Mucci et al. [3] studied the effect of using chitosan as a coagulant to remove cyanobacteria from the water column. Although chitosan’s coagulation efﬁciency has been extensively studied, little is known about its effect on the viability of cyanobacteria cells and the release of cyanotoxins. The authors’ study conﬁrmed that chitosan was able to damage Microcystis aeruginosa cells by inducing lysis and, consequently, the release of cyanotoxins, Received: 2 September 2021 Accepted: 8 September 2021 Published: 9 September 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Copyright: © 2021 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/). Albert Serrà 1,2,* , Laetitia Philippe 3 and Elvira Gómez 1,2,* 1 Thin Films and Nanostructures Electrodeposition Group (GE-CPN), Department of Materials Science and 1 Thin Films and Nanostructures Electrodeposition Group (GE-CPN), Department of Materials Science and Physical Chemistry, University of Barcelona, Martí i Franquès 1, E-08028 Barcelona, Catalonia, Spain 2 Institute of Nanoscience and Nanotechnology (IN2UB) Universitat de Barcelona 2 Institute of Nanoscience and Nanotechnology (IN2UB), Universitat de Barcelona, E-08028 Barcelona, Catalonia, Spain 3 Manufacture des Montres ROLEX SA, Research & Development, CH-2501 Bienne, Switzerland; laetitia.philippe@gmail.com 3 Manufacture des Montres ROLEX SA, Research & Development, CH-2501 Bienne, Switzerland; laetitia.philippe@gmail.com p pp g * Correspondence: a.serra@ub.edu (A.S.); e.gomez@ub.edu (E.G.) * Correspondence: a.serra@ub.edu (A.S.); e.gomez@ub.edu (E.G.) Harmful cyanobacterial algal blooms and cyanotoxins currently pose a major threat to global society, one that exceeds local and national interests due to their extremely de- structive effects on the environment and human health. In the near future, the formation of harmful cyanobacterial algal blooms and, in turn, cyanotoxins is expected to become widespread, driven by eutrophication and anthropogenic causes such as water pollution and promoted by escalating global temperatures. Such trends, studied since the late 1990s, have attracted increased interest due to (i) the high environmental impact of cyanobacterial blooms and cyanotoxins worldwide, (ii) the ineffective removal of those pollutants by conventional water treatment processes, (iii) the transformational capacity to completely destroy those organic toxins via alternative treatments such as advanced oxidation pro- cesses, and (iv) engineering challenges when transitioning toward the treatment of large volumes of water. The global context of this threat thus urges the innovation of simple, sustainable, low-cost strategies and technologies for water decontamination that can be readily implemented worldwide [1]. Editorial Removal of Cyanobacteria and Cyanotoxins in Waters Albert Serrà 1,2,* , Laetitia Philippe 3 and Elvira Gómez 1,2,* Citation: Serrà, A.; Philippe, L.; Gómez, E. Removal of Cyanobacteria and Cyanotoxins in Waters. Toxins 2021, 13, 636. https://doi.org/ 10.3390/toxins13090636 toxins toxins toxins toxins https://www.mdpi.com/journal/toxins Toxins 2021, 13, 636. https://doi.org/10.3390/toxins13090636 Toxins 2021, 13, 636 2 of 4 which thus inhibit chitosan’s use in lake restoration without directly testing its effects on the natural target biota because, as the authors show, the side effects depend on the strain [3]. Lürling et al. [4] also examined the efﬁciency of deploying the ﬂoc-and-skin technique to control the growth of cyanobacterial blooms using a combination of a coagulant (i.e., polyaluminum chloride) and a ballast (i.e., lanthanum modiﬁed bentonite) from Lake De Kuil and Lake Rauwbraken in the Netherlands. Above all, the authors demonstrate the ne- cessity of testing the technique’s process in the water to be treated, for the technique, when used with similar species of Planktothrix rubescens, yielded different results depending on the lake. After one day, the ﬁlaments of a Planktothrix rubescens biomass from Lake De Kuil resurfaced but remained precipitated in Lake Rauwbraken, possibly due to water matrix effects. By contrast, Pinkanjananavee et al. [5] studied the effectiveness of using conven- tional treatment processes consisting of coagulation, ﬂocculation, sedimentation, ﬁltration, sludge dewatering, and disinfection to remove Microcystis aeruginosa and microcystin-LR prior to disinfection. Noting that several conventional drinking water treatment plants recycle dewatered supernatant from sludge-dewatering operations, the authors aimed to determine the impact of recycling dewatered sludge supernatant on the quality of treated water in laboratory-scale experiments. Ultimately, their study demonstrated that recycling dewatered sludge supernatant can indeed affect the quality of treated water. The ﬁfth paper, by Jalili et al. [6], describes the effects of various cyanobacteria in a treatment plant for drinking water before, during, and after the occurrence of cyanobacterial blooms by monitoring raw water, sludge in the holding tank, and the sludge supernatant. In view of their results, the authors conclude that predicting the cyanobacterial community in the sludge supernatant continues to be a challenge. g p g Three other papers address different advanced oxidation processes used to remove cyanotoxins [7–9]. Advanced oxidation processes involve a set of oxidative water treat- ments, including treatment with UV–O3, UV–H2O2, the Fenton process, the photo-Fenton process, and nonthermal plasmas as well as sonolysis, photocatalysis, and radiolysis, all based on the production of powerful, highly reactive oxidants. Although the authors suggest the potential of using those three advanced oxidation processes to remove various cyanotoxins, unrealistic laboratory settings and conditions that allow the treatment of small volumes of water with highly energy-intensive lamps underscore some of the challenges of applying those technologies in the near future. g pp y g g Because different chemical oxidants can be used to treat water, the effects of some of those oxidants are discussed in three additional papers. In one of those papers, because H2O2 is a highly investigated Fenton reagent used in different water treatment strategies, Lusty et al. [10] investigated the effects of H2O2 on cyanobacteria and microbial commu- nities in order to analyze its use to mitigate potentially toxic freshwater cyanobacterial blooms. The study revealed that H2O2 can effectively reduce but not fully eliminate cyanobacteria from eutrophic bodies of water; thus, the authors conclude that H2O2 is not the best candidate for use in high biomass ecosystems containing harmful cyanobacterial algal blooms. By comparison, Moradinejad et al. [11] investigated variation and shifts in the structure of cyanobacterial communities during chemical oxidation (i.e., with Cl2, KMnO4, O3, and H2O2) using the metagenomic shotgun approach, which allows the considera- tion of the diversity of cyanobacterial communities in pre-oxidant selection in the on-site management of cyanobacterial blooms. According to their results, only pre-oxidation with H2O2 exhibited a clear decrease in the abundance of cyanobacterial biomasses, and the authors add that the selection of the pre-oxidant translates into considerable reductions in cost. Beyond that, the paper by Greenstein et al. [12] provides guidance on how ﬁve oxidants (i.e., Cl2, NH2Cl, ClO2, KMnO4, and O3) affect the delayed release of intracellular microcystins after the partial oxidation of cyanobacteria as a critical parameter to consider, especially for drinking water treatments, because the concertation of cyanotoxins in water can increase after several hours of treatment. From another angle, El Amrani Zerriﬁet al. [13] investigated the potential of em- ploying natural compounds extracted from seaweeds to control harmful algae in aquatic ecosystems. The authors report the results of a series of anti-cyanobacterial assays with different extracts from seaweeds from Morocco, which demonstrate the anti-cyanobacterial properties of Cystoseira tamariscifolia and its potential use in developing environmentally friendly procedures to control the growth of toxic cyanobacteria. Among those oxidants, hydroxyl radicals are the most commonly used and are the strongest known oxidants after ﬂuorine, and, similar to other highly reactive oxidants, can nonselectively destroy the majority of organic matter and can mineralize organic pollutants in water. Against that background, Ferreira et al. [7] investigated the applicability of the Fenton process to miner- alize cylindrosmpermopsin by analyzing how the dosage of Fenton reagents, namely H2O2 and Fe(II), and the H2O2-to-Fe(II) molar ratio affected the removal of cylindrosmpermopsin in ultrapure water (i.e., unrealistic conditions that do not consider water matrix effects or real pH) [7]. Although the Fenton process is a promising advanced oxidation technique that can be easily implemented at full scale worldwide due to its simplicity and highly cost-effective technology, the water matrix effect and large-scale volumes need to be investi- gated to evaluate the process for application in treating cyanotoxins because the presence of scavengers and competing species identiﬁed in real water matrices may affect degradation kinetics. Next, Benamara et al. [8] report that Al-doped ZnO nanoparticles are effective photocatalysts for degrading and mineralizing two typical cyanotoxins, microcystin-LR and anatoxin-A, under visible-light irradiation with light emitting diodes (LEDs). Central to that process is using easily fabricated Al-doped ZnO nanoparticles to act as photocata- lysts for water decontamination. Their paper, describing the visible-light-driven removal of cyanotoxins in the presence of ZnO-based photocatalysts, shows that high removal efﬁciencies can be achieved under simple conditions. The authors also found that the system’s outstanding performance derives from the excellent photocatalytic performance and the high chemical and photochemical stability of Al-doped ZnO nanoparticles under visible-light irradiation with LEDs [8]. In another study, Sorlini et al. [9] investigated the viability of using UV–H2O2 to treat microcystin-LR, particularly by analyzing speciﬁc energy consumption in the waters of Lake Iseo in the Province of Brescia, Lombardy, Italy. Those authors aimed to preliminarily study the effectiveness of UV–H2O2 on real water with respect to water matrix effects in order to clarify how the type of oxidant inﬂuences Toxins 2021, 13, 636 3 of 4 kinetics by analyzing the effects of the initial microcystin-LR concentration and the H2O2 dosage. The authors also examined the total speciﬁc energy consumption of UV–H2O2 compared to UV treatment to determine the optimal operational conditions [9]. Last, the paper by Esterhuizen [14] presents a systematic approach to planning, con- structing, monitoring, and optimizing a phytoremediation system for the treatment of wastewater from aquaculture. In their study, the authors constructed a large-scale Green Liver System requiring minimal maintenance and low construction costs based on the use of macrophytes. Because aquacultural wastewaters are eutrophic, cyanobacterial blooms ﬂourish in them and can often result in the presence of cyanobacterial toxins. However, the authors’ study demonstrated that various microcystins were effectively removed in their bioremediation process. Author Contributions: Writing—original draft preparation, A.S., L.P. and E.G.; writing—review and editing, A.S., L.P. and E.G. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Acknowledgments: The gest editors of this Special Issue, A.S., L.P. and E.G. are grateful to all of the authors for their contributions and particularly to the expert peer reviewers for their rigorous work. Conﬂicts of Interest: The authors declare no conﬂict of interest. 4 of 4 Toxins 2021, 13, 636 References 1. Serrà, A.; Philippe, L.; Perreault, F.; Garcia-Segura, S. Photocatalytic treatment of natural waters. Reality or hype? The case of cyanotoxins remediation. Water Res. 2021, 188, 116543. [CrossRef] [PubMed] y 2. Arruda, R.S.; De Magalh, L.; Coelho, M.; Mesquita, B.; De Almeida, É.C.; Pinto, E.; Lürling, M.; Marinho, M.M. ‘Floc and Sink’ Technique Removes Cyanobacteria and Microcystins from Tropical Reservoir Water. Toxins 2021, 13, 405. [CrossRef] [PubMed] 3. Mucci, M.; Guedes, I.A.; Faassen, E.J.; Lürling, M. Chitosan as a Coagulant to Remove Cyanobacteria Release. Toxins 2020, 12, 711. [CrossRef] [PubMed] 4. Lürling, M.; Mucci, M.; Waajen, G. Removal of Positively Buoyant Planktothrix rubescens in Lake Restoration. Toxins 2020, 12, 700. [CrossRef] [PubMed] [ ] [ ] 5. Pinkanjananavee, K.; Teh, S.J.; Kurobe, T.; Lam, C.H.; Tran, F.; Young, T.M. Potential Impacts on Treated Water Quality of Recycling Dewatered Sludge Supernatant during Harmful Cyanobacterial Blooms. Toxins 2021, 13, 99. [CrossRef] [PubMed] 6. Jalili, F.; Trigui, H.; Guerra Maldonado, J.F.; Dorner, S.; Zamyadi, A.; Shapiro, B.J.; Terrat, Y.; Fortin, N.; Sauvé, S.; Prévost, M. Can Cyanobacterial Diversity in the Source Predict the Diversity in Sludge and the Risk of Toxin Release in a Drinking Water Treatment Plant? Toxins 2021, 13, 25. [CrossRef] [PubMed] 7. Almeida-Ferreira, M.; Silveira, C.C.; Pérez, Y. Oxidation of Cylindrosmpermopsin by Fenton Process: A Bench-Scale Study of the Effects of Dose and Ratio of H2O2 and Fe(II) and Kinetics. Toxins 2021, 13, 604. [CrossRef] 8. Benamara, M.; Gómez, E.; Dhahri, R.; Serrà, A. Enhanced Photocatalytic Removal of Cyanotoxins by Al-Doped ZnO Nanoparticles with Visible-LED Irradiation. Toxins 2021, 13, 66. [CrossRef] [PubMed] 9. Sorlini, S.; Collivignarelli, C.; Carnevale Miino, M.; Caccamo, F.M.; Collivignarelli, M.C. Kinetics of Microcystin-LR Removal in a Real Lake Water by UV/H2O2 Treatment and Analysis of Speciﬁc Energy Consumption. Toxins 2020, 12, 810. [CrossRef] [PubMed] 10. Lusty, M.W.; Gobler, C.J. The Efﬁcacy of Hydrogen Peroxide in Mitigating Cyanobacterial Blooms and Altering Microbial Communities across Four Lakes in NY, USA. Toxins 2020, 12, 428. [CrossRef] [PubMed] dinejad, S.; Trigui, H.; Guerra Maldonado, J.F.; Shapiro, J.; Terrat, Y.; Zamyadi, A.; Dorner, S.; Prévos 11. Moradinejad, S.; Trigui, H.; Guerra Maldonado, J.F.; Shapiro, J.; Terrat, Y.; Zamyadi, A.; Dorner, S Assessment of Toxic Cyanobacterial Blooms during Oxidation Toxins 2020 12 728 [CrossRef] [PubM 11. Moradinejad, S.; Trigui, H.; Guerra Maldonado, J.F.; Shapiro, J.; Terrat, Y.; Zamyadi, A.; Dorner, S.; Prévost, M. Diversity Assessment of Toxic Cyanobacterial Blooms during Oxidation. References Toxins 2020, 12, 728. [CrossRef] [PubMed] j , ; g , ; , J ; p , J ; , ; y , ; , ; , y Assessment of Toxic Cyanobacterial Blooms during Oxidation. Toxins 2020, 12, 728. [CrossRef] [PubMed] 12 Greenstein K E ; Zamyadi A ; Glover C M ; Adams C ; Rosenfeldt E ; Wert E C Delayed Release of Intracellular Microcystin 12. Greenstein, K.E.; Zamyadi, A.; Glover, C.M.; Adams, C.; Rosenfeldt, E.; Wert, E.C. Delayed Release of Intracellular Microcystin following Partial Oxidation of Cultured and Naturally Occurring Cyanobacteria. Toxins 2020, 12, 335. [CrossRef] [PubMed] y y y following Partial Oxidation of Cultured and Naturally Occurring Cyanobacteria. Toxins 2020, 12, 335. [CrossRef] [PubMed] 13 El Amrani ZerriﬁS ; El KhallouﬁF ; Mugani R ; El Mahdi R ; Kasrati A ; Soulaimani B ; Barros L ; Ferreira I C F R ; Amaral 13. El Amrani Zerriﬁ, S.; El Khallouﬁ, F.; Mugani, R.; El Mahdi, R.; Kasrati, A.; Soulaimani, B.; Barros, L.; Ferreira, I.C.F.R.; Amaral, J.S.; Finimundy, T.C.; et al. Seaweed Essential Oils as a New Source of Bioactive Compounds for Cyanobacteria Growth Control: Innovative Ecological Biocontrol Approach. Toxins 2020, 12, 527. [CrossRef] [PubMed] J.S.; Finimundy, T.C.; et al. Seaweed Essential Oils as a New Source of Bioactive Compounds for Cyanobacteria Growth Control: Innovative Ecological Biocontrol Approach. Toxins 2020, 12, 527. [CrossRef] [PubMed] g pp 14. Esterhuizen, M.; Pﬂugmacher, S. 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https://openalex.org/W4392092098	http://www.thieme-connect.de/products/ejournals/pdf/10.1055/s-0044-1779506.pdf	English	null	Higher purpose in life and education were associated with better cognition among older adults	Arquivos de Neuro-Psiquiatria	2,024	cc-by	7,102	© 2024. The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution 4.0 International License, permitting copying and reproduction so long as the original work is given appropriate credit (https://creativecommons.org/licenses/by/4.0/). Thieme Revinter Publicações Ltda., Rua do Matoso 170, Rio de Janeiro, RJ, CEP 20270-135, Brazil Arq. Neuropsiquiatr. 2024;82(3):s00441779506. DOI https://doi.org/ 10.1055/s-0044-1779506. ISSN 0004-282X. 1Universidade de São Paulo, Escola de Artes, Ciências e Humanidades, Departamento de Gerontologia, São Paulo SP, Brazil. Article published online: 2024-02-23 Article published online: 2024-02-23 Article published online: 2024-02-23 Original Article 1Universidade de São Paulo, Escola de Artes, Ciências e Humanidades, Departamento de Gerontologia, São Paulo SP, Brazil. 2Universidade Estadual de Campinas, Faculdade de Ciências Médicas, Departamento de Gerontologia, Campinas SP, Brazil. Address for correspondence Mônica Sanches Yassuda (email: yassuda@usp.br) 1Universidade de São Paulo, Escola de Artes, Ciências e Humanidades, Departamento de Gerontologia, São Paulo SP, Brazil. 2Universidade Estadual de Campinas, Faculdade de Ciências Médicas, Departamento de Gerontologia, Campinas SP, Brazil. p g 2Universidade Estadual de Campinas, Faculdade de Ciências Médicas, Departamento de Gerontologia Campinas SP Brazil Resumo Antecedentes Com o envelhecimento, algumas habilidades cognitivas mudam devido a processos neurobiológicos. A cognição também pode ser inﬂuenciada por aspectos psicossociais. Objetivo Descrever as relações entre uma medida de neuroticismo, sintomas depressivos, propósito de vida e o desempenho cognitivo em pessoas idosas residentes na comunidade. Métodos Trata-se de uma análise transversal com base nos dados da segunda onda do estudo de Fragilidade em Idosos Brasileiros (FIBRA), realizado entre 2016 e 2017. A amostra foi composta por 419 pessoas idosas ( 72 anos) cognitivamente saudáveis e em maior parte com baixa escolaridade. As variáveis de interesse foram as sociodemo- gráﬁcas, domínio Neuroticismo do NEO-PI-R, Escala de Depressão Geriátrica (EDG) e Escala de Propósito de Vida (PV) e um escore cognitivo composto que incluiu o Miniexame de Estado Mental (MEEM) e as pontuações dos subitens do Miniexame Cognitivo de Addenbrooke (M-ACE), a saber, Fluência Verbal (FV) Animal, Teste do Desenho do Relógio (TDR) e Memória Episódica (nome e endereço). Resultados Houve um maior número de mulheres (70%), com idade elevada (mediana ¼ 80 anos, IIQ ¼ 77-82) e baixa escolaridade (mediana ¼ 4 anos, IIQ ¼ 2-5). Nas correlações bivariadas, anos de escolaridade (ρ ¼ 0,415; p < 0,001) e PV (ρ ¼ 0,220; p < 0,001) foram positivamente associadas à cognição. Neuroticismo (ρ ¼ -0,175; p < 0,001) e sintomas depressivos (ρ ¼ -0,185; p < 0,001) foram negativa- mente associados à cognição. Na regressão logística, após a inclusão de variáveis de confusão, as associações entre cognição e PV (OR ¼ 2,04; p ¼ 0,007) e escolaridade (OR ¼ 1,32; p < 0,001) permaneceram signiﬁcativas. Palavras-chave ►Pessoas Idosas ►Neuroticismo ►Depressão ►Propósito de Vida ►Cognição Conclusão Baixo PV e baixa escolaridade foram associados à pior cognição em idosos. Tais resultados podem ser relevantes em programas que visam a melhorar a cognição entre pessoas idosas. Abstract Objetivo Descrever as relações entre uma medida de neuroticismo, sintomas depressivos, propósito de vida e o desempenho cognitivo em pessoas idosas residentes na comunidade. Métodos Trata-se de uma análise transversal com base nos dados da segunda onda do estudo de Fragilidade em Idosos Brasileiros (FIBRA), realizado entre 2016 e 2017. A amostra foi composta por 419 pessoas idosas ( 72 anos) cognitivamente saudáveis e em maior parte com baixa escolaridade. As variáveis de interesse foram as sociodemo- gráﬁcas, domínio Neuroticismo do NEO-PI-R, Escala de Depressão Geriátrica (EDG) e Escala de Propósito de Vida (PV) e um escore cognitivo composto que incluiu o Miniexame de Estado Mental (MEEM) e as pontuações dos subitens do Miniexame Cognitivo de Addenbrooke (M-ACE), a saber, Fluência Verbal (FV) Animal, Teste do Desenho do Relógio (TDR) e Memória Episódica (nome e endereço). Resultados Houve um maior número de mulheres (70%), com idade elevada (mediana ¼ 80 anos, IIQ ¼ 77-82) e baixa escolaridade (mediana ¼ 4 anos, IIQ ¼ 2-5). Nas correlações bivariadas, anos de escolaridade (ρ ¼ 0,415; p < 0,001) e PV (ρ ¼ 0,220; p < 0,001) foram positivamente associadas à cognição. Neuroticismo (ρ ¼ -0,175; p < 0,001) e sintomas depressivos (ρ ¼ -0,185; p < 0,001) foram negativa- mente associados à cognição. Na regressão logística, após a inclusão de variáveis de confusão, as associações entre cognição e PV (OR ¼ 2,04; p ¼ 0,007) e escolaridade (OR ¼ 1,32; p < 0,001) permaneceram signiﬁcativas. Conclusão Baixo PV e baixa escolaridade foram associados à pior cognição em idosos. Tais resultados podem ser relevantes em programas que visam a melhorar a cognição entre pessoas idosas. Palavras-chave ►Pessoas Idosas ►Neuroticismo ►Depressão ►Propósito de Vida ►Cognição Conclusion Low PiL and low education levels were associated with worse cognition among older adults. Such results may be of relevance in programs that aim to improve cognition among older adults. Arquivos de Neuro-Psiquiatria Vol. 82 No. 3/2024 © 2024. The Author(s). Abstract Background With aging, some cognitive abilities change because of neurobiological processes. Cognition may also be inﬂuenced by psychosocial aspects. Background With aging, some cognitive abilities change because of neurobiological processes. Cognition may also be inﬂuenced by psychosocial aspects. Objective To describe the relationship between a measure of neuroticism, depression symptoms, purpose in life, and cognitive performance in community-dwelling older adults. Methods This was a cross-sectional analysis based on the data from the second wave of the Frailty in Brazilian Older Adults (FIBRA) study, carried out between 2016 and 2017. The sample consisted of 419 older people ( 72 years old) cognitively unimpaired and mostly with low education. The variables of interest were sociodemo- graphic, Neuroticism domain from the NEO-PI-R, Geriatric Depression Scale (GDS), Purpose in Life (PiL) scale, and a cognitive composite score which included the Mini- Mental State Examination (MMSE), and the scores for the sub-items of the Mini- Addenbrooke’s Cognitive Examination (M-ACE), namely, Verbal Fluency (VF) – Animal, Clock Drawing Test (CDT), Episodic Memory (name and address). Results There was a greater number of women (70%), with older age (median ¼ 80 years, IQR ¼ 77-82), and low education (median ¼ 4 years, IQR ¼ 2-5). In the bivariate correlations, years of education (ρ ¼ 0.415; p < 0.001) and PiL (ρ ¼ 0.220; p < 0.001) were positively associated with cognition. Neuroticism (ρ ¼ -0.175; p < 0.001) and depression symptoms (ρ ¼ -0.185; p < 0.001) were negatively associated with cogni- tion. In the logistic regression, after including confounding variables, the associations between cognition and PiL (OR ¼ 2.04; p ¼ 0.007) and education (OR ¼ 1.32; p < 0.001) remained signiﬁcant. Keywords ►Older Adults ►Neuroticism ►Depression ►Purpose in Life ►Cognition Keywords ►Older Adults ►Neuroticism ►Depression ►Purpose in Life ►Cognition received July 21, 2023 receive in its ﬁnal form November 29, 2023 accepted December 3, 2023 received July 21, 2023 receive in its ﬁnal form November 29, 2023 accepted December 3, 2023 PiL and education relate to better cognition Oliveira et al. 2 2 Conclusion Low PiL and low education levels were associated with worse cognition among older adults. Such results may be of relevance in programs that aim to improve cognition among older adults. Resumo Antecedentes Com o envelhecimento, algumas habilidades cognitivas mudam devido a processos neurobiológicos. A cognição também pode ser inﬂuenciada por aspectos psicossociais. INTRODUCTION colleagues have documented that higher PiL predicts greater longevity and better health behaviors.11 Recent research has shown that higher PiL can also have a positive impact on cognition. For example, in a longitudinal study, higher PiL was reported as having a protective effect against cognitive decline.5 It was also associated with better coping skills during stressful events and greater longevity.12 In a recent meta- analysis about PiL and cognition, PiL was associatedwith better performance in episodic memory and verbal ﬂuency tasks.13 The inclusion criteria for the follow-up participation were the same as for the baseline assessment: • understanding instructions for tasks, • accepting to participate in the study, and • permanent residence in the census tract. The psychosocial aspects described above represent im- portant mental health parameters that can signiﬁcantly inﬂuence cognition among older people.7,8,12 However, these data originated from high-income countries, where socioeconomic conditions and mental healthcare access tend to be signiﬁcantly better than in middle- and low-income countries. Therefore, we understand that the association between psychosocial aspects and cognition should also be investigated in such adverse contexts. In addition, this asso- ciation has rarely been explored among the oldest old, and no previous studies about the topic were identiﬁed in Brazil. Therefore, the objective of the present study was to investi- gate the associations between sociodemographic, neuroti- cism, DS, PiL variables, and cognitive performance in a sample of community-dwelling older adults, 72þ years, residing in the state of São Paulo, Brazil. We hypothesized that lower neuroticism, lower DS, and higher PiL would be associated with better cognitive performance. The exclusion criteria were as follows: • impaired cognitive and communication skills, suggesting cognitive impairment; • permanent or temporary inability to walk; • aphasia or loss of muscle strength due to stroke sequelae; • severe impairment of speech and motricity related to Parkinson’s disease in an advanced stage; Parkinson’s disease in an advanced stage; • severe hearing and visual disability; and • terminal disease. The informants were the older people themselves or a family member residing in the house- hold. INTRODUCTION worse performance in domains such as memory, function executive, attention, process speed, visuospatial ability, verbal ﬂuency, and numerical reasoning. Another longitudinal study identiﬁed that low neuroticism was associated with less cognitive decline and greater longevity.3 Age-relatedcognitive change isone of the most studied aspects of aging. With age, there may be a reduction in cognitive performance, with implications for daily life and well-being.1 Cognitive changes are related to brain aging, that is, the anatomical and physiological changes that occur throughout the life cycle.2 It has also been documented that cognitive performance in old age may be inﬂuenced by psychosocial aspects, such aspersonality proﬁle, psychiatric conditions such as depression, and certain domains of psychological well-being - purpose in life (PiL), among other factors.3–5 Depression is a mood disorder that signiﬁcantly affects an individual’s ability to function and relate to others. Depression symptoms (DS) have commonly been associated with reduced cognitive performance. DS can inﬂuence cognitive domains, such as learning and memory, attention and concentration, executive functions, and processing speed.4 Also, in a longitu- dinal study, lifetime recurrent depression episodes were pre- dictive of worse cognitive status, and a higher risk of cognitive impairment inearlyold age,8 although thisﬁnding iscontested by other studies (e.g., Burhanullah et al.9). Among psychosocial aspects, studies have suggested that neuroticism, a personality factor related to negative emotional and behavioral reactions to stressful situations, has an associ- ation with cognitive performance.6 This aspect of personality, although present in all individuals, when at elevated levels has often been linked to poor cognitive aging in longitudinal studies. For instance, Sutin et al.7 reported that high scores in neuroticism among respondents were associated with PiL is one of the most important domains of the psychologi- cal well-being construct. It refers to the meaning, purpose, and sense of direction that people assign to their own lives.10 Importantly, higher PiL is considered an important predictor of healthy aging. For instance, in previous work, Ryff and PiL and education relate to better cognition Oliveira et al. 3 (FIBRA) study, carried out between 2016 and 2017. The sample included older Brazilians aged 72þ years living in the city of Campinas and in the Ermelino Matarazzo district in the city of São Paulo, both located in the state of São Paulo. INTRODUCTION For more details regarding the FIBRA study please see Neri et al.14 Among the 1,284 participants at baseline, there were 192 deaths and 543 losses due to various reasons (participants not located [57.9%], refusals [3.5%], withdrawal of permission during data collection [5.5%], exclusion due to study criteria [1.6%], and presenceof risks to interviewers[0.5%]).14A total of 549 older adults underwent assessment of cognitive status, of which 419 scored above the education-adjusted cut-off score for dementia on the Mini-Mental State Examination (MMSE) and completed the follow-up protocol14 which included the measures of interest for the present study (►Figure 1). The Mini-Addenbrooke’s Cognitive Examination (M-ACE) This is a brief cognitive screening instrument for dementia composed of temporal orientation questions, an Episodic Memory test (name and address), Verbal Fluency (VF) - animal category, and the Clock Drawing Test (CDT).16 For the present analyses, the MMSE, and the M-ACE sub- item scores for VF, CDT, and the Episodic Memory delayed recall were used to compose a composite score for cognition. Th M ACE l d id h l f For the present analyses, the MMSE, and the M-ACE sub- item scores for VF, CDT, and the Episodic Memory delayed recall were used to compose a composite score for cognition. The M-ACE total score was not used to avoid the overlap of the temporal orientation sub-items present in the M-ACE and the MMSE. The level of statistical signiﬁcance adopted for all analy- ses was p < 0.05. The statistical software Jamovi (Version 2.3.28, 2022, Australia) and JASP (Version 0.17.2, 2023, The Netherlands) were used. Statistical analyses The interviews were conducted by undergraduate and graduate students who were trained to apply the protocols. Recruitment was carried out in older adults’ homes accord- ing to the list of addresses available in the baseline database. The evaluation was carried out at the residence by a pair of interviewers in one session, with an average duration of 75 minutes. The variables of interest in the study do not have a normal distribution, which is why non-parametric data was used. Therefore, for descriptive analysis of the data, the Mann- Whitey (two categories) and Kruskall-Wallis (three catego- ries or more) tests were used. Mann-Whitney test and the Kruskall-Wallis (with post hoc comparisons, Dwass-Steel- Critchlow-Fligner [DSCF]) to compare continuous variables and the Chi-square test to compare categorical variables. Participants or caregivers completed and signed an in- formed consent form. The project was approved by the ethical committee of the State University of Campinas, under the following protocol numbers - CAAE 49987615.3.0000. 5404 and 92684517.5.1001.5404. The dependent variable was the cognitive composite score including the MMSE and the VF, CDT, and Episodic Memory items from the M-ACE. To create the cognitive composite score, the individual test scores were normalized into z-scores. The z-scores for the tests were added and divided by the number of items, to generate a global cogni- tive score. Considering that the present sample was com- posed only of participants with unimpaired cognition, according to the MMSE, the composite score was trans- formed into a dichotomous variable, z 0 (newZ ¼ 0) to represent lower cognitive performance and z > 0 (newZ ¼ 1) to represent higher cognitive performance. Participants and study design The present study includes cross-sectional analyses of data from the second wave of the Frailty in Brazilian Older Adults Figure 1 Recruitment ﬂowchart and composition of the follow-up sample. FIBRA Study, 2016-2017. A i d N P i i t i V l 82 N 3/2024 © 2024 Th A th ( ) Figure 1 Recruitment ﬂowchart and composition of the follow-up sample. FIBRA Study, 2016-2017. Arquivos de Neuro-Psiquiatria Vol. 82 No. 3/2024 © 2024. The Author(s). PiL and education relate to better cognition Oliveira et al. 4 4 adopted cutoff scores for the MMSE were: 17 points for illiterates, 22 points for 1–4 years of formal study, 24 points for 5–8 years, and 26 points for those with 9 or more.15 with responses on a Likert scale. The higher the score, the higher the purpose in life. Geriatric Depression Scale (GDS) This is a 15-item depression screening instrument for older adults.18 A score 6 is considered to be suggestive of major depression. ►Table 2 characterizes the sample divided by age groups. There were 185 individuals between 72 and 79 years of age (44%) and 234 older adults 80þ years. The younger group showed a lower score on Neuroticism (p ¼ 0.050). Neuroticism The sample was comprised of cognitively unimpaired older adults, mostly with low education. ►Table 1 presents the sociodemographic, psychosocial, and cognitive characteri- zation of the total sample and the sample divided by sex. Among the participants, 70% were women, with a high age (median ¼ 80 years) and low educational level (median ¼ 4 years), with 13% of participants without formal education. In addition, 18% of participants lived alone, with a greater number of women in this living condition. Also, 20% of participants had GDS scores suggestive of major depression and women had a higher level of neuroticism than men. The sample was comprised of cognitively unimpaired older adults, mostly with low education. ►Table 1 presents the sociodemographic, psychosocial, and cognitive characteri- zation of the total sample and the sample divided by sex. Among the participants, 70% were women, with a high age (median ¼ 80 years) and low educational level (median ¼ 4 years), with 13% of participants without formal education. In addition, 18% of participants lived alone, with a greater number of women in this living condition. Also, 20% of participants had GDS scores suggestive of major depression and women had a higher level of neuroticism than men. It represents the domain of the Revised NEO Personality Inventory (NEO-PI-R). It assesses the characteristics of emo- tional instability and individual adjustment. This scale con- sisted of 12 items rated on a Likert scale. The higher the score, the higher the intensity of neuroticism.6,17 The FIBRA study did not collect data on other personality domains besides Neuroticism. Sociodemographic data Age (72-79 and 80þ years old), sex, education (illiterate, 1-4 years of study, 5-8, and 9 þ ), household living arrangement (live alone – yes or no), and family income were considered. MMSE This instrument assesses global cognition, with a maximum score of 30 points.15 Spearman’s correlation tests and logistic regression anal- yses were used to test the associations among the selected variables. For the logistic regression analysis, the cognitive composite score was the dependent variable, and the socio- demographic and psychosocial variables were included as independent variables. The forced entry model was used to include all the independent variables and the cognitive composite score in the regression equation. Arquivos de Neuro-Psiquiatria Vol. 82 No. 3/2024 © 2024. The Author(s). Purpose in Life (PiL) This scale is part of the Psychological Well-Being scale developed by Ryff and Keyes.10,19 It examines the sense of meaning in life, beliefs that give meaning to life, and whether the person has goals for living. It consists of ten questions ►Table 3 demonstrates the differences related to educa- tional level (illiterate, 1-4 years of study, 5-8, and 9 þ ). Among those with higher education (9þ years) income was higher and there was a higher percentage of people Arquivos de Neuro-Psiquiatria Vol. 82 No. 3/2024 © 2024. The Author(s). PiL and education relate to better cognition 5 Oliveira et al. 5 Table 1 Sociodemographic, psychosocial, and cognitive characterization of the total sample and divided by sex (n ¼ 419). FIBRA Study, 2016-2017 Variables Total n ¼ 419 (100%) Sex p-value Women n ¼ 293 (70%) Men n ¼ 126 (30%) Age, n (%) MD (IQR) 80 (77-83) 80 (77-82) 80 (78-83) 0.064þ Education, n (%) Illiterate 54 (13%) 42 (14%) 12 (10%) 0.194þþ Literate 356 (85%) 246 (84%) 110 (87%) Missing 9 (2%) 5 (2%) 4 (3%) MD (IQR) 4 (2-5) 4 (2-5) 4 (3-5.7) 0.221þ Family income, n (%) MD (IQR) 2,400 (1,760-3,600) 2,325 (1,700-3,712) 2,500 (1,874-3,500) 0.876þ Missing 47 33 14 Live alone, n (%) Yes 77 (18%) 61 (21%) 16 (13%) 0.013þþ No 239 (57%) 153 (52%) 86 (68%) Missing 103 (25%) 79 (27%) 24 (19%) MMSE MD (IQR) 25 (23-27) 25 (23-27) 26 (24-27) 0.108þ VF MD (IQR) 11 (9-14) 11 (9-14) 11 (9-14) 0.872þ CDT MD (IQR) 2 (1-4) 3 (1-4) 2 (1-4) 0.688þ Missing 1 0 1 Episodic memory MD (IQR) 3 (0-5) 2 (0-5) 3 (1-5) 0.481þ Missing 2 2 0 Neuroticism MD (IQR) 26 (21-31) 27 (22-32) 24 (19-29) 0.002þ Missing 20 13 7 PiL MD (IQR) 3.6 (3.1-4) 3 (3.1-4) 3.5 (3.1-3.9) 0.467þ Missing 17 11 6 GDS, n (%) 6 82 (20%) 62 (21%) 20 (16%) 0.211þþ 5 337 (80%) 231 (79%) 106 (84%) MD (IQR) 3 (2-5) 3 (2-5) 3 (2-5) 0.407þ Table 1 Sociodemographic, psychosocial, and cognitive characterization of the total sample and divided by sex (n ¼ 419). FIBRA Study, 2016-2017 living alone. The higher the level of education of the partic- ipants, the higher the cognitive scores. Arquivos de Neuro-Psiquiatria Vol. 82 No. 3/2024 © 2024. The Author(s). Abbreviations: CDT, Clock Drawing Test; GDS, Geriatric Depression Scale; IQR, interquartile range; MD, median; MMSE, Mini-Mental State Examination; n, sample size; PiL, Purpose in Life; VF, Verbal Fluence (animal category). Note: þMann Whitney test; þþQui-square test; p-value < 0.05 shows statistical signiﬁcance. DISCUSSION This study explored the relationships between psychosocial and sociodemographic variables with cognitive performance in adults 72þ years. Among the main results, in bivariate correlations, global cognition was signiﬁcantly related to education, Neuroticism, GDS, and PiL. In the logistic regres- sion, with psychosocial and sociodemographic variables included as independent variables, global cognition was signiﬁcantly associated with PiL and number of years of education. ►Table 4 shows the Spearman ρ (rho) bivariate corre- lations between global cognition (composite score) and the psychosocial and sociodemographic variables. There was a signiﬁcant correlation between global cognition and education, Neuroticism, PiL, and GDS scores. PiL was negatively correlated with GDS and Neuroticism. Still, Neuroticism was positively correlated with GDS scores; and global cognition and education were also positively correlated. In the sample, a greater presence of women and older people (80þ years) was observed, similarly to what was found in the study by Freitas et al.,20 among other studies, suggesting greater participation of women in gerontological research. The sample consisted mostly of participants with a low educational level. In Brazil, this has been a common ﬁnding in community-based studies, as it reﬂects the ►Table 5 presents the results of the logistic regression analysis including the variables of interest (sociodemo- graphic, psychosocial, and cognitive variables). This model did not show multilinearity (Tolerance > 0,1; VIF < 10) among the independent variables. The model indicated that education and PiL were signiﬁcantly associated with global cognitive performance. PiL and education relate to better cognition Oliveira et al. 6 6 Table 2 Sociodemographic, psychosocial, and cognitive characterization of the total sample divided by age group (n ¼ 419). DISCUSSION FIBRA Study, 2016-2017 Variables Age group p-value 72-79 n ¼ 185 (44%) 80þ n ¼ 234 (56%) Education, n (%) Illiterate 24 (13%) 30 (13%) 0.931þþ Literate 156 (84%) 200 (85%) Missing 5 (3%) 4 (2%) MD (IQR) 4 (2-6) 4 (2-4) 0.159þ Family income, n (%) MD (IQR) 2,346 (1,800-3,070) 2,490 (1,700-4,000) 0.628þ Missing 17 30 Live alone, n (%) Yes 24 (13%) 53 (23%) 0.659þþ No 81 (44%) 158 (67%) Missing 80 (43%) 23 (10%) MMSE MD (IQR) 25 (23-27) 25 (23-27) 0.772þ VF MD (IQR) 12 (9-14) 11 (8-13) 0.053þ CDT MD (IQR) 2 (1-5) 3 (1-4) 0.242þ Missing 0 1 Episodic memory MD (IQR) 2 (0-5) 3 (1-5) 0.817þ Missing 0 2 Neuroticism MD (IQR) 27 (22-32) 25 (20-30) 0.050þ Missing 7 13 PiL MD (IQR) 3.6 (3.1-4) 3.6 (3.1-4) 0.924þ Missing 8 9 GDS, n (%) 6 34 (18%) 48 (21%) 0.789þþ 5 151 (82%) 186 (79%) MD (IQR) 3 (2-5) 3 (2-5) 0.369þ Table 2 Sociodemographic, psychosocial, and cognitive characterization of the total sample divided by age group (n ¼ 419). FIBRA Study 2016-2017 psychosocial, and cognitive characterization of the total sample divided by age group (n ¼ 419). FIBRA Abbreviations: CDT, Clock Drawing Test; GDS, Geriatric Depression Scale; IQR, interquartile range; MD, median; MMSE, Mini-Mental State Examination; n, sample size; PiL, Purpose in Life; VF, Verbal Fluence (animal category). Notes: þMann Whitney test; þþQui-square test; p-value < 0.05 shows statistical signiﬁcance. Table 3 Sociodemographic, psychosocial, and cognitive characterization of the total sample divided by educational level (n ¼ 419). FIBRA Study, 2016-2017 Table 3 Sociodemographic, psychosocial, and cognitive characterization of the total sample divided by educational level (n ¼ 419). Vol. 82 No. 3/2024 © 2024. The Author(s). Arquivos de Neuro-Psiquiatria Vol. 82 No. 3/2024 © 2024. The Author(s). Arquivos de Neuro-Psiquiatria Vol. 82 No. 3/2024 © 2024. The Author(s). DISCUSSION FIBRA Study, 2016-2017 Variables Educational level p-value Illiterate n ¼ 57 (14%) 1-4 n ¼ 244 (58%) 5-8 n ¼ 68 (16%) 9þ n ¼ 50 (12%) Age, n (%) MD (IQR) 81 (76-84) 80 (77-82) 78 (75-81.3) 79.5 (77-83.8) 0.058 Family income, n (%) MD (IQR) 1,874 (1,264-2,811)c,d 2,000 (1,625-3,000)d 2,500 (1,800-4,000)d,a 4,000 (2,950-7,000)b,c,a <0.001 Missing 12 26 6 3 Live alone, n (%) Yes 8 (14%) 41 (17%) 14 (21%) 14 (28%) <0.001þþ No 36 (63%) 144 (59%) 39 (57%) 20 (40%) Missing 13 (23%) 59 (24%) 15 (22%) 16 (32%) MMSE MD (IQR) 20 (18-23)b,c,d 25 (23-27)c,d,a 26 (25-27.3)b,d,a 27 (26-28)b,c,a <0.001 VF MD (IQR) 10 (8-12)c,d 11 (9-14) 12 (9.7-14.3)a 12 (11-16.8)a 0.005 CDT MD (IQR) 0 (0-1)b,c,d 3 (1-4)d,a 3 (2-4)a 4 (2-5)b,a <0.001 Missing 0 1 0 0 psychosocial, and cognitive characterization of the total sample divided by educational level -2017 Table 3 Sociodemographic, psychosocial, and cognitive characterization of the total sample d (n ¼ 419). FIBRA Study, 2016-2017 PiL and education relate to better cognition Oliveira et al. 7 Table 3 (Continued) Variables Educational level p-value Illiterate n ¼ 57 (14%) 1-4 n ¼ 244 (58%) 5-8 n ¼ 68 (16%) 9þ n ¼ 50 (12%) Episodic memory MD (IQR) 0 (0-3)b,c,d 2 (1-5)d,a 3 (1-5)a 5 (3-6)b,a <0.001 Missing 1 1 0 0 Neuroticism MD (IQR) 27.5 (23.8-33.3) 26.5 (21-31) 24.5 (20-29) 25 (20-30) 0.229 Missing 1 12 6 1 PiL MD (IQR) 3.4 (3-3.9) 3.6 (3.1-4) 3.7 (3.2-4) 3.8 (3.2-4.3) 0.183 Missing 3 10 3 1 GDS, n (%) 6 12 (21%) 49 (20%) 12 (18%) 9 (18%) 0.972þþ 0.211 5 45 (79%) 195 (80%) 56 (82%) 41 (82%) MD (IQR) 3 (2-5) 3 (2-5) 2 (1-4.2) 2 (1-4) Table 4 Spearman’s bivariate correlation between variables of interest in the sample (n ¼ 419). Note. Global cognition (composite score ¼ Mini-Mental State Examination þ Verbal Fluence (animal category) þ Clock Drawing Test þ Episodic Memory (name and address)/4); PiL ¼ Purpose in Life; GDS ¼ Geriatric Depression Scale (total score); ρ ¼ Spearman’s rho; CI ¼ conﬁdence interval; p-value < 0.05 shows statistical signiﬁcance. p p ( g y) Notes: Kruskal-Wallis test (with post hoc comparisons, Dwass-Steel-Critchlow-Fligner [DSCF]); þþQui-square test; p-value < 0.05 shows statistical signiﬁcance. a ¼ different from illiterates (p < 0.05); b ¼ different from 1-4 years (p < 0.05); c ¼ different from 5-8 years (p < 0.05); d ¼ different from 9þ years (p < 0.05). posite score ¼ Mini-Mental State Examination þ Verbal Fluence (animal category) þ Clock Drawing Test þ Episodic Abbreviations: CDT, Clock Drawing Test; GDS, Geriatric Depression Scale; IQR, interquartile range; MD, median; MM Examination; n, sample size; PiL, Purpose in Life; VF, Verbal Fluence (animal category). DISCUSSION FIBRA Study, 2016-2017 Variable 1 Variable 2 ρ 95% CI p-value Lower Upper Age Education -0.060 -0.155 0.036 0.217 Age Family income 0.020 -0.082 0.121 0.704 Age Global cognition -0.068 -0.163 0.028 0.163 Age Neuroticism -0.057 -0.155 0.041 0.253 Age PiL -0.065 -0.162 0.033 0.195 Age GDS 0.003 -0.092 0.099 0.945 Education Family income 0.329 0.235 0.417 <0.001 Education Global cognition 0.415 0.332 0.491 <0.001 Education Neuroticism -0.141 -0.236 -0.043 0.005 Education PiL 0.077 -0.021 0.173 0.125 Education GDS -0.116 -0.210 -0.021 0.017 Family income Global cognition 0.237 0.138 0.330 <0.001 Family income Neuroticism -0.148 -0.248 -0.045 0.005 Family income PiL 0.128 0.025 0.228 0.015 Family income GDS -0.124 -0.223 -0.022 0.017 Global cognition Neuroticism -0.175 -0.268 -0.078 <0.001 Global cognition PiL 0.220 0.125 0.311 <0.001 Global cognition GDS -0.185 -0.276 -0.091 <0.001 Neuroticism PiL -0.332 -0.418 -0.240 <0.001 Neuroticism GDS 0.526 0.451 0.594 <0.001 PiL GDS -0.446 -0.521 -0.364 <0.001 Arquivos de Neuro-Psiquiatria Vol. 82 No. 3/2024 © 2024. The Author(s). PiL and education relate to better cognition Oliveira et al. 8 Table 5 Logistic regression including the study variables of interest with the cognitive composite score as the dependent variable (n ¼ 419). FIBRA Study, 2016-2017 Parameter Estimate OR 95% CI p-value Lower Upper Age -0.054 0.947 -0.115 0.006 0.079 Sex (men) -0.186 0.831 -0.859 0.488 0.589 Education 0.279 1.321 0.168 0.389 <0.001 Family income -0.000 1.000 -0.000 0.000 0.759 Live alone (yes) 0.243 1.275 -0.528 1.014 0.537 Neuroticism -0.032 0.969 -0.075 0.011 0.148 GDS 0.078 1.081 -0.063 0.219 0.277 PiL 0.714 2.043 0.197 1.232 0.007 Note. GDS ¼ Geriatric Depression Scale (total score); PiL ¼ Purpose in Life; OR ¼ odds ratio; CI ¼ conﬁdence interval; p-value < 0.05 shows statistical signiﬁcance. Table 5 Logistic regression including the study variables of interest with the cognitive composite score as the dependent variable (n ¼ 419). DISCUSSION FIBRA Study, 2016-2017 historical reality of lower access to education in older generations.21,22 suggested the negative inﬂuence of depression on cognitive performance.28,29 Studies have shown deﬁcits in memory and executive functions among people with major depres- sion.4,30 Studies have also pointed out that higher neuroti- cism and DS could be associated with faster cognitive decline with age31,32 and a higher incidence of dementia.33 However, Burhanullah et al.9 found no association between depression and dementia. In the present analyses, GDS scores were negatively related to global cognition, yet this association was not signiﬁcant in the multivariable model. It was identiﬁed that 18% of participants lived alone, with a greater percentage of women in this living arrangement. In the National Health Survey in Brazil, carried out in 2013, it was observed that 15.3% of people aged 60 and over lived alone.21 The change from a cohabitation proﬁle towards a greater number of single-person households in recent dec- ades is related to several factors, such as the demographic and epidemiological transition, changes in family arrange- ments, shorter duration of marital unions, and fewer chil- dren or more childless couples.23 Differently from the other psychosocial variables in the present study, PiL was positively associated with global cognition in the bivariate correlation analyses and the asso- ciation remained signiﬁcant in the logistic regression, in the presence of other psychosocial and sociodemographic vari- ables. This ﬁnding suggests that the inﬂuence of PiL on the cognition of older adults may be greater than the inﬂuence of DS and neuroticism. High PiL scores have previously been associated with better cognitive performance, in episodic memory and verbal ﬂuency, and with a lower risk of cogni- tive decline.34,35 It is possible that older people with higher levels of PiL have a more positive view of aging and present more protective behaviors in relation to their health.36 In turn, such behaviors could be associated with greater cogni- tive reserve, such as involvement in intellectually stimulat- ing activities. When the psychosocial variables were compared across sex, it was observed that women had a higher level of Neuroticism. Arquivos de Neuro-Psiquiatria Vol. 82 No. 3/2024 © 2024. The Author(s). PiL and education relate to better cognition 9 Oliveira et al. of healthy behaviors,39 a more positive view of aging and life, greater resilience, optimism, a sense of self-efﬁcacy, and better functioning of biological systems.36 Likewise, higher PiL would be related to low neuroticism, as it could enable emotional self-regulation in the management of adverse events39 and also favor positive affects.37 2 Salthouse TA. Trajectories of normal cognitive aging. Psychol Aging 2019;34(01):17–24. Doi: 10.1037/pag0000288 3 Stephan Y, Sutin AR, Luchetti M, Terracciano A. Facets of consci- entiousness and longevity: Findings from the Health and Retire- ment Study. J Psychosom Res 2019;116:1–5. Doi: 10.1016/j. jpsychores.2018.11.002 4 Pan Z, Park C, Brietzke E, et al. Cognitive impairment in major depressive disorder. CNS Spectr 2019;24(01):22–29. Doi: 10.1017/S1092852918001207 One of the limitations of this study is its cross-sectional design. Longitudinal surveys, with follow-up data over an extended period, could provide informationon thetrajectories of thesevariables andgenerate agreater understandingofsuch relationships. In addition, in the present study, cognition was investigated with the use of a composite score based on screening tests, which precluded the investigation of the association with speciﬁc cognitive domains. It is important to highlight that the validity and reliability of each measure included in the composite score were not assessed. Unfortu- nately, the FIBRA study did not include additional domains of the NEO-PI-R, such as conscientiousness, which would have been important to investigate. On the other hand, the present study ﬁlls an important gap, as the Brazilian gerontological literature lacks studies involving psychosocial variables and cognitive performance in cohorts of older adults. These data are relevant for cross-cultural comparisons. 5 Marone S, Bloore K, Sebastiani P, et al. Purpose in life among centenarian offspring. J Gerontol B Psychol Sci Soc Sci 2020;75 (02):308–315. Doi: 10.1093/geronb/gby023 6 Costa PT, Mccrae RR. NEO-PI-R professional manual: Revised NEO Personality Inventory (NEO-PI-R) and NEO-Five Factor Inventory (NEO-FFI). Odessa, FL:: Psychological Assessment Resources,; 1992 7 Sutin AR, Stephan Y, Luchetti M, Terracciano A. Five-factor model personality traits and cognitive function in ﬁve domains in older adulthood. BMC Geriatr 2019;19(01):343. Doi: 10.1186/s12877- 019-1362-1 8 James SN, Davis D, O’Hare C, et al. Lifetime affective problems and later-life cognitive state: Over 50 years of follow-up in a British birth cohort study. J Affect Disord 2018;241:348–355. Doi: 10.1016/j.jad.2018.07.078 9 Burhanullah MH, Tschanz JT, Peters ME, et al. Neuropsychiatric symptoms as risk factors for cognitive decline in clinically normal older adults: The Cache County Study. Am J Geriatr Psychiatry 2020;28(01):64–71. Acknowledgements The authors thank the staff and participants of the Frailty in Brazilian Older Adults study for their important contributions. 17 Flores-Mendonza CE. Inventário de Personalidade NEO-Revisado. Manual técnico. São PauloVetor Editora,2007 18 Paradela EM, Lourenço RA, Veras RP. [Validation of geriatric de- pression scale in a general outpatient clinic]. Rev Saude Publica 2005;39(06):918–923. Doi: 10.1590/s0034-89102005000600008 PiL and education relate to better cognition Doi: 10.1016/j.jagp.2019.03.023 In conclusion, the present study investigated the associ- ations between psychosocial variables and cognitive perfor- mance among participants from the second wave of FIBRA study. Lower PiL and lower education levels were associated with worse cognition. The association between PiL and global cognitive scores is relevant as it suggests that inter- ventions geared to increase PiL (e.g., Friedman et al.40) might also have a positive effect on cognition. 10 Ryff CD, Keyes CL. The structure of psychological well-being revisited. J Pers Soc Psychol 1995;69(04):719–727. Doi: 10.1037//0022-3514.69.4.719 11 Ryff CD, Kim ES. Extending research linking purpose in life to health: The challenges of inequality, the potential of the arts, and the imperative of virtue. In: Burrow AL, Hill P, eds. The Ecology of Purposeful Living Across the Lifespan [Internet]. Cham:: Springer International Publishing;; 2020:29–58. Doi: 10.1007/978-3-030- 52078-6_3 12 Kim G, Shin SH, Scicolone MA, Parmelee P. Purpose in life protects against cognitive decline among older adults. Am J Geriatr Psychiatry 2019;27(06):593–601. Doi: 10.1016/j.jagp. 2019.01.010 Authors’ Contributions WLO, RCM, MC, DVSF, SSTB, TNO, ALN, MSY: conceptuali- zation, data curation, formal analysis, funding acquisition, investigation, methodology, project administration, resources, software, supervision, validation, visualization, writing – original draft, writing – review & editing; 13 Sutin AR, Luchetti M, Stephan Y, Strickhouser JE, Terracciano A. The association between purpose/meaning in life and verbal ﬂuency and episodic memory: a meta-analysis of >140,000 participants from up to 32 countries. Int Psychogeriatr 2022;34 (03):263–273. Doi: 10.1017/S1041610220004214 Conﬂict of Interest There is no conﬂict of interest to declare. 16 Miranda DDC, Brucki SMD, Yassuda MS. The Mini-Addenbrooke’s Cognitive Examination (M-ACE) as a brief cognitive screening instrument in Mild Cognitive Impairment and mild Alzheimer’s disease. Dement Neuropsychol 2018;12(04):368–373. Doi: 10.1590/1980-57642018dn12-040005 DISCUSSION This ﬁnding has been commonly reported, although, it is poorly understood, especially among older adults.24 In the present study, participants with low education had worse cognitive performance, and global cognition and educational level were positively correlated, replicating the well-known negative impact of low education on cogni- tion.25 High education is understood as an important marker of cognitive reserve, that is, brain characteristics that allow higher cognitive functioning than expected in the face of pathological processes or aging.26 Therefore, this ﬁnding was expected. Elevated neuroticism has been associated with worse cognitive performance. For example, longitudinal analyses of the Health and Retirement Study (HRS) identiﬁed that among individuals with higher neuroticism, there was sig- niﬁcantly lower performance in cognition.7 Another study reported that high neuroticism was associated with lower scores on episodic memory.27 In the present study, there was a signiﬁcant negative correlation between global cognition and neuroticism. However, in the logistic regression, with the inclusion of other psychosocial and demographic varia- bles, the association did not remain signiﬁcant. Logistic regression results revealed that besides higher PiL, higher education was also signiﬁcantly associated with better global cognition. These ﬁndings are in line with the Emory Healthy Aging Study, in which low PiL was identiﬁed as a robust predictor of cognitive decline, especially when associated with risk factors such as low education.35 Additionally, it was possible to observe, in the bivariate correlations, that PiL was negatively correlated with GDS and Neuroticism. These ﬁndings were similar to those found in previous studies. In Ribeiro et al.,37 high PiL was associated with a lower number of SD and in Kim et al.38 to a reduced risk for developing major depression. Other authors postu- lated the idea that higher PiL could lead to greater adoption Similarly, cognition has been negatively associated with elevated DS in previous studies.8 Several studies have PiL and education relate to better cognition Support 14 Neri AL, et al. Follow-up evaluation of the FIBRA Study: socio- demographic, cognitive, and frailty characterization of older adults in Campinas and Ermelino Matarazzo, SP. Rev Bras Geriatr Gerontol 2022;25(05):e210224. Doi: 10.1590/1981-22562022 025.210224.en This work was supported by the São Paulo Research Foun- dation (FAPESP), grant number 16/00084-8, and by the Brazilian National Council for Scientiﬁc and Technological Development (CNPq), grant number 424789/2016-7. 15 Brucki SM, Nitrini R, Caramelli P, Bertolucci PH, Okamoto IH. [Suggestions for utilization of the mini-mental state examination in Brazil]. Arq Neuropsiquiatr 2003;61( 3B):777–781. Doi: 10.1590/s0004-282. Doi: 2003000500014 1 Krivanek TJ, Gale SA, McFeeley BM, Nicastri CM, Daffner KR. Promoting successful cognitive aging: A ten-year update. J Alz- heimers Dis 2021;81(03):871–920. Doi: 10.3233/JAD-201462 References 1 Krivanek TJ, Gale SA, McFeeley BM, Nicastri CM, Daffner KR. Promoting successful cognitive aging: A ten-year update. J Alz- heimers Dis 2021;81(03):871–920. Doi: 10.3233/JAD-201462 19 Ribeiro CC, Neri AL, Yassuda MS. Semantic-cultural validation and internal consistency analysis of the Purpose in Life Scale for Arquivos de Neuro-Psiquiatria Vol. 82 No. 3/2024 © 2024. The Author(s). 10 Oliveira et al. PiL and education relate to better cognition Oliveira et a brazilian older adults. Dement Neuropsychol 2018;12(03): 244–249. Doi: 10.1590/1980-57642018dn12-030004 brazilian older adults. Dement Neuropsychol 2018;12(03): 244–249. Doi: 10.1590/1980-57642018dn12-030004 31 Sadeq NA, Molinari V. Personality and its relationship to depres- sion and cognition in older adults: Implications for practice. Clin Gerontol 2018;41(05):385–398. Doi: 10.1080/07317115.2017. 1407981 20 Freitas JLGDS, Silva JMM, Nóbrega JCL, et al. Health-related quality of life and associated factors: Regional differences among oldest-old in Brazil. Inquiry 2022;59:469580221086922. Doi: 10.1177/00469580221086922 32 Munoz E, Stawski RS, Sliwinski MJ, Smyth JM, MacDonald SWS. 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Doi: 10.1111/acps.13385 40 Friedman EM, Ruini C, Foy CR, Jaros L, Love G, Ryff CD. Lighten UP! A community-based group intervention to promote Eudaimonic Well-Being in older adults: A multi-site replication with 6 month follow-up. Clin Gerontol 2019;42(04):387–397. Doi: 10.1080/ 07317115.2019.1574944 30 James TA, Weiss-Cowie S, Hopton Z, Verhaeghen P, Dotson VM, Duarte A. Depression and episodic memory across the adult lifespan: A meta-analytic review. Psychol Bull 2021;147(11): 1184–1214. Doi: 10.1037/bul0000344
https://openalex.org/W4220652984	https://figshare.com/articles/preprint/The_effect_of_cortical_thickness_and_thread_profile_dimensions_on_stress_and_strain_in_bone-anchored_implants_for_amputation_prostheses/14499759/1/files/27773880.pdf	English	null	The effect of cortical thickness and thread profile dimensions on stress and strain in bone-anchored implants for amputation prostheses	Journal of the mechanical behavior of biomedical materials/Journal of mechanical behavior of biomedical materials	2,022	cc-by	8,734	The effect of cortical thickness and thread profile dimensions on stress and strain in bone-anchored implants for amputation prostheses This paper was downloaded from TechRxiv (https://www.techrxiv.org). SUBMISSION DATE / POSTED DATE SUBMISSION DATE / POSTED DATE 28-04-2021 / 30-04-2021 10.36227/techrxiv.14499759.v1 CITATION Thesleff, Alexander; Ortiz-Catalan, Max; Brånemark, Rickard (2021): The effect of cortical thickness and thread profile dimensions on stress and strain in bone-anchored implants for amputation prostheses. TechRxiv. Preprint. https://doi.org/10.36227/techrxiv.14499759.v1 10.36227/techrxiv.14499759.v1 1 1 Title The effect of cortical thickness and thread profile dimensions on stress and strain in bone-anchored implants for amputation prostheses The effect of cortical thickness and thread profile dimensions on stress and strain in bone-anchored implants for amputation prostheses Authors Alexander Thesleff1, 2, 3, Max Ortiz-Catalán1, 2, 4, 5, Rickard Brånemark5, 6 Abstract Skeletal attachment of limb prostheses ensures load transfer between the prosthetic leg and the skeleton. For individuals with lower limb amputation, these loads may be of substantial magnitude. To optimize the design of such systems, knowledge about the structural interplay between implant design features, dimensional changes, and material properties of the implant and the surrounding bone is needed. Here, we present the results from a parametric finite element investigation on a generic bone-anchored implant system of screw design, exposed to external loads corresponding to average and high ambulatory loading. Of the investigated parameters, cortical thickness had the largest effect on the stress and strain in the bone-anchored implant and in the cortical bone. 36 % – 44 % reductions in maximum longitudinal stress in the bone-anchored implant was observed as a result of increased cortical thickness from 2 mm to 5 mm. Changes in thread depth had larger effect on the maximum stresses in the fixture and the bone than changes in thread root radius within the evaluated parameter space. Stress reductions in the percutaneous abutment were obtained by autologous transplantation of bone tissue distal to the fixture. Results from this investigation may guide structural design optimization for bone-anchored implant systems for attachment of limb prostheses. Authors Alexander Thesleff1, 2, 3, Max Ortiz-Catalán1, 2, 4, 5, Rickard Brånemark5, 6 1. Center for Bionics and Pain Research, Mölndal, Sweden 2. Dept. of Electrical Engineering, Chalmers University of Technology, Gothenburg, Sweden 3. Integrum AB, Mölndal, Sweden 4. Operational Area 3, Sahlgrenska University Hospital, Mölndal, Sweden 5. Department of Orthopaedics, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Sahlgrenska University Hospital, Gothenburg, Sweden 6. Center for Extreme Bionics, Biomechatronics Group, MIT Media Lab, Massachusetts Institute of Technology, Cambridge, MA, USA 1. Center for Bionics and Pain Research, Mölndal, Sweden 2. Dept. of Electrical Engineering, Chalmers University of Technology, Gothenburg, Sweden 3. Integrum AB, Mölndal, Sweden 4. Operational Area 3, Sahlgrenska University Hospital, Mölndal, Sweden 5. Department of Orthopaedics, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Sahlgrenska University Hospital, Gothenburg, Sweden 6. Center for Extreme Bionics, Biomechatronics Group, MIT Media Lab, Massachusetts Institute of Technology, Cambridge, MA, USA Research supported by the Promobilia Foundation, the IngaBritt and Arne Lundbergs Foundation, the Swedish Foundation for Strategic Research (SSF), the Swedish Innovation Agency (VINNOVA), and the Swedish Research Council (Vetenskapsrådet), and Integrum AB. Background For individuals with amputation, the socket-stump interface is the source of several commonly reported problems such as pain, pressure sores, skin problems, poor retention, and perspiration [1]– [6]. To address these issues, a method of bone-anchored attachment of limb prostheses was developed, where a surgically implanted percutaneous skeletal extension allows for direct attachment of the prosthesis [7]. The method builds on the discovery that bone tissue may form an exceptionally intimate and mechanically stable connection with titanium [8]. This phenomenon is now known as osseointegration and is defined as “the firm anchoring of a surgical implant by the growth of bone 2 around it without fibrous tissue formation at the interface” [9]. The first clinical applications of osseointegration were in dental field, but it has since then spread to other applications such as hearing aids, facial reconstructions, and orthopaedic applications including bone-anchored attachment of prosthetic limbs [10]. The method of bone-anchored attachment of prosthetic limbs was developed in Sweden with the first successful surgeries performed in 1990 [11]. A standardized implant system and treatment protocol was introduced in 1999 as the OPRA (Osseointegrated Prostheses for the Rehabilitation of Amputees) implant system (Integrum AB, Mölndal, Sweden). The OPRA implant system is CE-marked for transfemoral, transhumeral, and thumb/finger amputations, and custom- made implants are available for the aforementioned levels, as well as for transtibial and transradial amputations. The treatment has gained increasing clinical acceptance and is currently available in 12 countries. The OPRA implant system was recently granted a pre-market approval (PMA) by the Food and Drug Administration (FDA) in the USA for treatment at the transfemoral level. Following the successful clinical results of the OPRA implant system a few other systems for bone-anchored attachment of prosthetic limbs have been developed, examples are the ILP (Integral Leg Prosthesis, ESKA Orthopaedic Handels GmbH, Germany) (also known as Endo-Exo Prosthesis (EEP)) and the OPL (Osseointegrated Prosthetic Limb, Permedica s.p.a., Italy). With bone-anchored attachment of the prosthesis, a socket is no longer needed and all aforementioned problems related to socket attachment are eliminated. Research on individuals who have undergone treatment with bone- anchored attachment of limb prosthesis shows that it leads to improved quality of life, increase in prosthetic function and use, improved walking ability, walking efficiency, range of motion, sitting comfort, and sensory perception from the prosthesis via a phenomenon known as osseoperception [12]–[21]. Background The OPRA implant system is a modular system, of which the structural components are the fixture and the abutment. The fixture is a cylindrical, externally threaded component, fully implanted into the residual bone. The abutment, which is the percutaneous component has its proximal end press-fitted into the fixture, and its distal end protruding out from the skin and allowing for attachment of the prosthesis. The rigid connection between the prosthesis and the skeleton leads to a more efficient gait compared with the more compliant socket connection [12], [19]. However, it also means that any loads applied to the prosthesis are transferred directly to the bone-anchored implant and the periprosthetic bone which could be at risk of fracture. Several studies involving load cell measurements at the implant prosthesis interface have offered insights in the magnitude of the loading during ambulatory activities of daily living [22]–[24]. This data is valuable for numerical simulations such as finite element (FE) analyses to calculate periprosthetic and implant stress and strain. The objective of this study was to perform a parametric evaluation on the effect of thread profile parameters and cortical thickness, on the peak stress and strains in a generic implant model subjected to external loads corresponding to average and high ambulatory loading. An additional aim was to quantify effects of changes in bone stiffness characteristics and distal bone resorption on the maximum stresses in the implant system. The generic model was inspired by the OPRA implant system; an externally threaded fixture and other components manufactured from medical grade titanium (Ti6Al4V). The intention was to provide knowledge on how the careful selection of design features can minimize the risk of bone or implant fracture during use. This study contrasts earlier FE- based studies which have been either case studies with subject specific anatomy [25]–[32], studies focused exclusively on bone remodelling around the implant [31], [33], or FE-implementations with too low resolution to accurately capture the stress in the threaded region [25], [27]–[30], [32], [34]– [37]. [37]. 3 Method The generic model used in this study is shown in Figure 1. The geometrical parameters that we investigated were thread depth (d), the thread root radius (rroot), and the cortical thickness (t) within the parameter space shown in Figure 1. The thread was a standard V-shaped thread with 30° flank angle, a rounded thread crest with constant radius of 0.1 mm and either a small or a large root radius, of 0.1 mm or 0.5 mm, respectively. Three thread depths were evaluated from 0.75 mm to 1.5 mm. The thread pitch (p) was determined by the thread depth, where a larger thread depth led to a larger pitch. It was kept constant for each thread depth, regardless the value of root radius in order to discern the effect of this parameter. Two cortical thicknesses were evaluated, a small cortical thickness of 2 mm which correspond to the minimum requirement after implantation according to the OPRA surgical protocol [38], and a larger cortical thickness of 5 mm. Figure 1. Generic geometry with boundary conditions and dimensions. Dimensions denoted with variable names were varied within the study, whereas dimensions with numerical values were fixed. Figure 1. Generic geometry with boundary conditions and dimensions. Dimensions denoted with variable names were varied within the study, whereas dimensions with numerical values were fixed. Analytical estimation of load sharing Analytical estimation of load sharing FE-model FE-modelling and simulations were performed using COMSOL Multiphysics version 5.5 (Comsol AB, Sweden). A generic 3D axisymmetric FE-model of the implant system and surrounding bone was created (Figure 1), where the symmetry in geometry allowed for reduction of the model to half of the full geometry. The abutment and fixture were combined into a single component. In accordance with the surgical protocol of the OPRA implant system, the fixture was countersunk 20 mm into the cortical bone with the distal void filled with a less stiff autologous bone transplant [39], [40]. The interfaces between fixture and the cortical bone and between fixture and transplanted bone were modelled with shared nodes, assuming full osseointegration, (which is close to what has been observed from histological assessment of a retrieved fixture [41]), whereas the interface between the abutment and the transplanted bone was modelled assuming no osseointegration and frictionless sliding. The cortical bone extended 20 mm distally and proximally from both ends of the fixture. The proximal end of the cortical bone was constrained in all degrees of freedom. The external load was applied at the distal end of the abutment as a static load case. To meet computational limitations in the hardware while ensuring a sufficiently dense mesh along the full geometry of the threaded implant, a stepwise submodelling scheme was employed (Figure 3). A global model with the full model geometry was created, transversely partitioned at locations indicated in Figure 3. Subsequently, five densely meshed sub-models were created. Global model simulation results (displacements) at the partition surfaces were applied as boundary conditions (prescribed displacement) at the corresponding surfaces in the submodels to generate high resolution simulation results for each of the submodels. Simulation results along longitudinal evaluation profiles in each of the submodels were exported from COMSOL and imported into Matlab (version 2019a, MathWorks Inc., Natick, MA, USA). Custom written scripts were used to integrate the results between the submodels and to visualize the results along the combined evaluation profiles. To avoid non-physical stress singularities at the border of the submodels due to mesh size inconsistencies between the submodels and the global model, a small overlap was included between the neighbouring sub-models (yellow regions in Figure 3). Analytical estimation of load sharing Prior to performing the FE-simulations, an analytical analysis was performed on a simplified geometry, considering the implant and the cortical bone as two uniform cylinders (without threads) with dimensions as indicated in Figure 1. In this analysis both the Ti6Al4V and the cortical bone were assumed to have isotropic properties with Young’s modulus of 110 GPa and 17 GPa, respectively. Assuming a pure bending load case, the analytical load sharing between the implant and the cortical bone was calculated based on respective component’s bending stiffness, defined as the product of Young’s modulus, (E), and the area moment of inertia according, (I), according to Eq. 1, with (do) and (di) being the external and internal diameter respectively. (1) (1) This initial investigation was conducted with the purpose to serve as a point of reference for the FE- simulation results. This initial investigation was conducted with the purpose to serve as a point of reference for the FE- simulation results. 4 FE-model In the data processing stage, the exported simulation results close to the longitudinal end points of the sub-model were discarded so that each submodel only contributed with simulation results starting from the center of the overlapping zone. In addition to eliminating singularities at the submodel borders, this ensured that no duplicate data was used to form the combined simulation results. 5 5 Figure 2. Simulation scheme using submodelling. Top. Global model with submodel partitions. Yellow segments indicate overlapping regions which were included in both of the neighbouring submodels. Middle. Densely meshed submodels. Blue surfaces show where prescribed displacement was assigned from corresponding surfaces in the global model (indicated with blue lines in top image). Bottom. Merging results from each submodel simulation and plotting results along the longitudinal direction at the indicated evaluation profiles. Figure 2. Simulation scheme using submodelling. Top. Global model with submodel partitions. Yellow segments indicate overlapping regions which were included in both of the neighbouring submodels. Middle. Densely meshed submodels. Blue surfaces show where prescribed displacement was assigned from corresponding surfaces in the global model (indicated with blue lines in top image). Bottom. Merging results from each submodel simulation and plotting results along the longitudinal direction at the indicated evaluation profiles. The material models and material properties used in the simulations are presented in Table 1. Transverse isotropy was assumed for the cortical bone, with uniform material properties around the longitudinal axis. Young’s and shear moduli were obtained from the investigation by Reilly and Burstein [42] where the transversely isotropic material properties had been determined based on experimental measurements on human cadaver femora. Single case simulations with the longitudinal Young’s modulus of the cortical bone reduced to 12 GPa were also performed. This reduced stiffness may arise due to increased porosity of the cortical bone tissue, which is natural consequence of aging, osteoporosis, and not loading the skeleton in a physiological way, an example being individuals with a lower limb amputation fitted with a socket prosthesis [43]–[45]. The reduction of the longitudinal Young’s modulus from 17 GPa to 12 GPa was determined based on experimental data from a study by Dong and Guo [46] where the aforementioned values corresponded to cortical porosity of 8.5 % and 19 % respectively. FE-model An additional case of reduced bone support was simulated corresponding to an extreme case of distal resorption, modelled by removing the transplanted bone and the 40 mm most distal portion of the cortical bone. Two versions of transplanted bone were evaluated, immature transplanted bone with a low Young’s modulus representing the state during early rehabilitation, and mature transplanted bone with a higher Young’s modulus representing the steady state after full healing and rehabilitation has taken place. Prior histological observation of a tissue biopsy of the transplanted bone retrieved from a revision surgery of an OPRA implant system showed that the mature transplanted bone obtained a trabecular bone structure with a compact bone layer towards 6 6 the soft tissue and the abutment [39]. For simplicity, the transplanted bone was in this study modelled as a homogeneous isotropic tissue with the same material properties as had been used by Stenlund et al. [39]. The fixture and the abutment are manufactured from Ti6Al4V and were also modelled with isotropic material properties. Table 1. Material models and material properties used in the FE-models Table 1. Material models and material properties used in the FE-models Component Material model Material properties Fixture and abutment Isotropic E = 110 GPa ν = 0.3 Normal cortical bone Transversely isotropic Er = Eϴ = 11.5 GPa, Ez = 17 GPa Grϴ = 3.6 GPa, Grz = Gϴz 3.3 GPa νrϴ = νrz = νϴz = 0.3 Osteoporotic cortical bone Transversely isotropic Er = Eϴ = 11.5 GPa, Ez = 12 GPa Grϴ = 3.6 GPa, Grz = Gϴz 3.3 GPa νrϴ = νrz = νϴz = 0.3 Mature transplanted bone Isotropic E = 4 GPa ν = 0.3 Immature transplanted bone Isotropic E = 0.4 GPa ν = 0.3 From studies obtaining direct load data at distal end of the abutment, it is known that at physiological loading during activities of daily living, the bending moment is the main contributor to the stress state in the implant and the bone, and the longitudinal force is the dominating force component [22]. The studied load cases were therefore reduced to only include only these components. This led to symmetric loading which in combination with the geometric symmetry allowed for the model reduction as described above. The first load case, LC1, refers to high loading ambulation. The highest body weight acceptable for treatment with the OPRA implant system is 100 kg. FE-model During walking this corresponds to a maximum longitudinal force of approximately 1000 N. This value was therefore chosen for the longitudinal force component for LC1. The bending moment was derived from a study conducting load measurements at the distal end of the abutment in 20 individuals with the OPRA implant system at the transfemoral level while performing everyday ambulatory activities [22]. The highest measured bending moment reported during any of the investigated activities within that study was 90 Nm (observed in a single participant during stairs descent). This value was therefore chosen as the value for the bending moment in LC1. The second load case, LC2, refers to average loading during level ground walking. The magnitudes of the longitudinal force and the bending moment were also derived from the aforementioned study. They correspond to the mean peak longitudinal force and mean peak bending moment measured across the study participants during level ground walking. Table 2. Investigated load cases. Load case F M Interpretation LC1 1000 90 High ambulatory loading LC2 625 37 Average ambulatory loading Table 2. Investigated load cases. Load case F M Interpretation LC1 1000 90 High ambulatory loading LC2 625 37 Average ambulatory loading The FE meshes of both the global model and the submodels were created by first creating a structured surface mesh on a longitudinal section of the axisymmetric FE-geometry. The surface mesh was refined in the threaded portion of the geometry The surface mesh was then swept around the The FE meshes of both the global model and the submodels were created by first creating a structured surface mesh on a longitudinal section of the axisymmetric FE-geometry. The surface mesh was refined in the threaded portion of the geometry. The surface mesh was then swept around the longitudinal axis to form a 3D mesh. This meshing strategy was chosen to minimize the number of The FE meshes of both the global model and the submodels were created by first creating a structured surface mesh on a longitudinal section of the axisymmetric FE-geometry. The surface mesh was refined in the threaded portion of the geometry. The surface mesh was then swept around the longitudinal axis to form a 3D mesh. Analytical estimation of load sharing The result from the analysis of the simplified geometry, considering the implant and the cortical bone as two uniform cylinders is shown in Figure 3 along with the estimated changes in the peak stress in the fixture for varying thread depth and cortical thickness. Figure 3. Estimated load sharing between fixture and cortical bone (CB) from analysis of simplified geometry. To the right, the estimated change in peak stress in the fixture for varying thread depth and cortical thickness. Figure 3. Estimated load sharing between fixture and cortical bone (CB) from analysis of simplified geometry. To the right, the estimated change in peak stress in the fixture for varying thread depth and cortical thickness. Results LC1 and LC2 both correspond to repeated loading conditions for which tensile stresses in general are considered more harmful than compressive stresses [47]. For this reason, only the stress and strain state on the tensile side of the implant-bone model is reported in this study. Analytical estimation of load sharing FE-model This meshing strategy was chosen to minimize the number of The FE meshes of both the global model and the submodels were created by first creating a structured surface mesh on a longitudinal section of the axisymmetric FE-geometry. The surface mesh was refined in the threaded portion of the geometry. The surface mesh was then swept around the longitudinal axis to form a 3D mesh. This meshing strategy was chosen to minimize the number of 7 elements in the model while ensuring a high mesh resolution in the threaded region. The resulting mesh consisted of second order hexahedron and prismatic elements. The total number of elements were 90 000-120 000 for the global models and 35 000-80 000 for each sub-model depending on variations in geometry (i.e., variations in t, rroot, d, and p). A mesh convergence analysis along a longitudinal profile along the fixture thread root, the region most influenced by the quality of the mesh, confirmed that further mesh refinement from the chosen mesh parameters led to < 3% change in results anywhere along the profile. Stress in fixture Thread roots constitute stress concentrations with locally elevated stress levels [48]. The thread root of the fixture may therefore be susceptible to high stress levels and could be a potential location of failure if exposed to excessive loading. The longitudinal stress in the thread root of the tensile side of the fixture subjected to LC1 is presented in Figure 4 for three different thread depths and for two different cortical thicknesses. 8 Figure 4. Longitudinal stress in tensile side of the fixture thread root during LC1. Top. Results from simulations of three different values of the thread depth, d. Cortical thickness, t, and fixture thread root radius, rroot are kept constant at 2 mm and 0.5 mm respectively. Middle. Results from simulations of three different values of the thread depth, d. Cortical thickness, t, and fixture thread root radius, rroot are kept constant at 5mm and 0.5 mm, respectively. Bottom. Results from the proximal end of the fixture with cortical thickness t = 2 mm (zoomed in from the dashed regions within the top graphs). The proximal geometry of the fixture is also shown. Figure 4. Longitudinal stress in tensile side of the fixture thread root during LC1. Top. Results from simulations of three different values of the thread depth, d. Cortical thickness, t, and fixture thread root radius, rroot are kept constant at 2 mm and 0.5 mm respectively. Middle. Results from simulations of three different values of the thread depth, d. Cortical thickness, t, and fixture thread root radius, rroot are kept constant at 5mm and 0.5 mm, respectively. Bottom. Results from the proximal end of the fixture with cortical thickness t = 2 mm (zoomed in from the dashed regions within the top graphs). The proximal geometry of the fixture is also shown. At the distal end of the fixture, (z = 0), the peak stresses are reduced in all studied cases. This is also observed at the proximal end of the fixture for the cases with the large cortical thickness, (t = 5 mm). However, for the case with small cortical thickness (t = 2 mm), there is an increase in the peak stress in the fixture towards the proximal end especially for the two more shallow thread depths with a small thread root radius. Stress in fixture Between the z-coordinates, 20 mm and 60 mm along the fixture, the differences between the peak stress profiles are approximately constant, thus the effect of each parameter can be approximated by an average over this region without losing much detail. Such summarized presentation of the parameter evaluation is shown in Figure 5. At the distal end of the fixture, (z = 0), the peak stresses are reduced in all studied cases. This is also observed at the proximal end of the fixture for the cases with the large cortical thickness, (t = 5 mm). However, for the case with small cortical thickness (t = 2 mm), there is an increase in the peak stress in the fixture towards the proximal end especially for the two more shallow thread depths with a small thread root radius. Between the z-coordinates, 20 mm and 60 mm along the fixture, the differences between the peak stress profiles are approximately constant, thus the effect of each parameter can be approximated by an average over this region without losing much detail. Such summarized presentation of the parameter evaluation is shown in Figure 5. 9 Figure 5. Parameter evaluation of the longitudinal stress in the thread root of the fixture during LC1. Circled values denote the average peak longitudinal stress (MPa) in the fixture thread root between z-coordinates 20 mm and 60 mm during LC1. Colored values denote the percentage difference in the results when varying a parameter. Figure 5. Parameter evaluation of the longitudinal stress in the thread root of the fixture during LC1. Circled values denote the average peak longitudinal stress (MPa) in the fixture thread root between z-coordinates 20 mm and 60 mm during LC1. Colored values denote the percentage difference in the results when varying a parameter. Within the investigated parameter space, the difference between 2 mm and 5 mm cortical thickness had the largest effect on the peak stresses in the fixture thread root, with approximately 40 % reduction across different thread depths and root radii. A reduced thread depth from 1.5 mm to 0.75 mm led to 20 % – 22 % reduction with t = 2 mm and 10 % – 18 % reduction with t = 5 mm. Stress in fixture The effect of an increased radius in the thread root from 0.1 mm to 0.5 mm had a less prominent effect, and stress reductions were only noted for the most deep and shallow threads at t = 2 mm (Figure 6) and the deepest thread at t = 5 mm. In the three other simulated cases, no change in the average peak stress was noted. 10 Figure 6. Longitudinal stress in tensile side of the fixture thread root during LC1 for different thread depths and thread root radii Figure 6. Longitudinal stress in tensile side of the fixture thread root during LC1 for different thread depths and thread root radii Figure 6. Longitudinal stress in tensile side of the fixture thread root during LC1 for different thread depths and thre radii The separate effects of reduced cortical stiffness and extreme distal bone resorption on the maximum stress in the thread root of the fixture are shown in Figure 7. On average, a reduction in the longitudinal cortical stiffness from 17 GPa to 12 GPa resulted in 12 % and 30 % increases in maximum longitudinal stress for 2 mm and 5 mm cortical thickness, respectively. In the case of extreme bone resorption, the maximum stress increased by 54 % on average compared with a situation of full uniform cortical bone support (t = 2 mm). 11 Figure 7. Top. The longitudinal stress peaks in the fixture thread root for two different values of longitudinal stiffness in the cortical bone and for two different cortical thicknesses during LC1. Bottom. The effect of extreme distal bone resorption during LC1. Figure 7. Top. The longitudinal stress peaks in the fixture thread root for two different values of longitudinal stiffness in the cortical bone and for two different cortical thicknesses during LC1. Bottom. The effect of extreme distal bone resorption during LC1. Stress in abutment A comparison of the longitudinal stress on the tensile side of the abutment shaft for different configurations of cortical thickness and Young’s modulus for the transplanted bone is presented in Figure 8. With respect to the case without any bone support (extreme distal bone resorption) a maximum stress reduction of 31 % was achieved with 5 mm cortical thickness and a Young’s modulus of 4 GPa in the transplanted bone. Interestingly, even the smallest cortical thickness (t = 2 mm) and a very soft transplanted bone (ETB = 0.4 GPa) resulted in up to 10 % reduction in the stress of the abutment. 12 Figure 8. Longitudinal stress on the tensile side of the abutment shaft during LC1 and LC2. Figure 8. Longitudinal stress on the tensile side of the abutment shaft during LC1 and LC2. Stress and strain in cortical bone The longitudinal stress and strain along the tensile side of the cortical bone during is shown in Figure 9. To show the effect of the cortical thickness and the cortical stiffness, all graphs presented in Figure 10 are from simulations with fixed thread depth d = 1.125 mm and fixed thread root radius rroot = 0.5 mm. There were clear maxima in the longitudinal stress and longitudinal strain at the z-coordinates corresponding to each end of the fixture. Especially large were the maxima at the proximal end for the simulated cases with 2 mm cortical thickness. At the outer cortex at this location the maximum longitudinal stress reached 109 MPa and 43 MPa for LC1 and LC2 respectively for the simulations with ordinary cortical stiffness (EL CB = 17 GPa). For the 5 mm cortical thickness cases the maximum stress was 43 MPa at the outer cortex. At the distal end of the fixture the maximum stress in the cortical bone reached at most 42 MPa and 27 MPa for the simulations with 2 mm and 5 mm cortical thickness, respectively. As a natural consequence of Hooke’s law, a reduction of the cortical stiffness led to reduced stress but increased strains. The strains at the proximal end of the fixture were the largest, locally reaching 7940 µε for the softer cortical bone simulation and 6050 µε for the normal cortical bone simulation with 2 mm bone thickness in LC1. In LC2 the peak strain reached 2370 µε with ordinary cortical bone stiffness. The largest strains were observed for the case with the reduced cortical stiffens (EL CB = 12 GPa) reaching a maximum of 7940 µε locally for LC1. At ordinary cortical stiffness, the corresponding maximum strain was 6050 µε and 2370 µε respectively for LC1 and LC2. 13 Figure 9. Longitudinal stress and strain in the cortical bone during LC1 and LC2. For the results presented in the graphs the parameters d, and rroot, were kept fixed at 1.125 mm and 0.5 mm respectively. Figure 9. Longitudinal stress and strain in the cortical bone during LC1 and LC2. Stress in fixture The initial analytical approximation of the cortical bone and the fixture as two cylindrical components without thread provided preliminary predictions for the results from the FE-simulations. Changes in the cortical thickness and thread depth, each lead to changes in the external diameter of respective component. Changes in the cortical thickness had the largest effect on the longitudinal stresses in the thread root of the fixture. This was expected considering the external diameter’s high influence on the area moment of inertia (Eq. 1). The average reductions of the peak longitudinal stress in the thread root of the fixture, when increasing the cortical thickness from 2 mm to 5 mm, were similar between the FE-simulations (36 % - 44 %) and the predicted values (39 % - 46 %). The effect of the thread depth was influenced by the cortical thickness. With cortical thickness fixed at t = 2 mm, reducing the thread depth from 1.5 mm to 0.75 mm, led to 20 % and 22 % reduction in the average peak stress in the thread root of the fixture with a large and small root radius respectively. With cortical thickness fixed at t = 5 mm, the effect of an identical reduction in thread depth (1.5 mm to 0.75 mm), led to smaller reductions (10 % and 18 % for large and small root radius respectively). The reason for the smaller influence of the thread depth at the larger cortical thickness can be attributed to the same reason as described above, namely the external diameter’s importance for the bending stiffness (Eq. 1). Interestingly, for a cortical thickness of 2 mm and a small root radius, the magnitude of the stress peaks increased towards the proximal end of the fixture for the two shallower threads (Figure 4). At the proximal end of the fixture the internal diameter of the fixture was 11 mm for all models whereas the external diameter of the fixture was dependent on the thread depth (Figure 1). As a result, the external diameter and material thickness of the fixture was larger for models with smaller thread depth. This led to that the proximal stiffness of the fixture was larger in models with small thread depth and that the extent of proximal stiffness reduction of the fixture (due to the chamfered external diameter proximally) was smaller in these FE-models. For the results presented in the graphs the parameters d, and rroot, were kept fixed at 1.125 mm and 0.5 mm respectively. In Figure 10, the effect of the thread profile parameters, d and rroot on the longitudinal stress and longitudinal strain in the cortical bone is shown. The presented graphs are both from LC1 and at cortical thickness t = 2 mm. At the outer cortex along the length of the fixture, higher stress and strains are observed for the simulations with large thread depth, except for at locations corresponding to the ends of the fixture. At the z-coordinate corresponding to the proximal end of the fixture longitudinal stress and longitudinal strain is lower in simulations with large thread depth. The highest values for the longitudinal stress and longitudinal strain in the cortical bone was observed at the proximal end of the fixture for the shallowest thread depth and the smallest fixture thread root radius and smallest cortical thickness. Figure 10. Longitudinal stress and strain during LC1. The parameter t is kept constant at 2 mm in the graphs. Figure 10. Longitudinal stress and strain during LC1. The parameter t is kept constant at 2 mm in the graphs. 14 Stress in fixture This may offer one explanation for the observed increased peak stress at the thread root of the proximal fixture for the shallower threads at 2 mm cortical thickness in Figure 4. The effect of reduced cortical stiffness was larger for the situation with large cortical thickness, since in this case the cortical bone is resisting a larger portion of the total load and thus a reduction in stiffness consequently has a larger effect on the stresses in the Fixture (Eq. 1). The simulation with extreme distal bone resorption led to a 54 % increase in the peak stresses in the fixture thread root compared with the reference case with uniform bone support with 2 mm cortical thickness. Such increase in the stresses might challenge the mechanical integrity of the fixture and thus, the resorption should be addressed before reaching such extremes. Stress in abutment Discussion We conducted an FE-based evaluation of a generic model of a bone-anchored implant system inspired by the OPRA implant system that was subjected to external loads corresponding to average and high ambulatory loading. Within this investigation, a parametric analysis has been carried out to determine how the peak stress and strains in implant system and the periprosthetic bone are affected by the cortical thickness, the thread depth and the thread root radius. Single case investigations have been performed to quantify the effects of reduced cortical stiffness and distal bone resorption. Cortical bone With minimum cortical thickness (2 mm), the maximum longitudinal stress reached above 100 MPa at the outer cortex at the level of the proximal end of the fixture during LC1. For the 5 mm cortical thickness cases, the maximum stress was 43 MPa. At the distal end of the fixture the peak stress in the cortical bone reached at most 42 MPa for the case with 2 mm cortical thickness. For LC2 loading, the maximum stress reached 42 MPa at the level of the proximal end of the fixture. A reduction of the cortical stiffness led to reduced stress but increased strains. The strains at the proximal end of the fixture were the largest, locally reaching up to 8000 µε for the softer cortical bone simulation and around 6000 µε for the normal cortical bone simulation with 2 mm bone thickness in LC1 (Figure 4). These strain levels are in the order of the yield strain reported from previous research [49]. In the FE- simulations, a small thread depth led to higher peak longitudinal stress and longitudinal strain in the cortical bone at to the proximal end of the fixture compared with a larger thread depth (Figure 10). This may be explained by the fact that a smaller thread depth led to reduced proximal cortical bone thickness due to its geometrical definition in the FE-models as t + d (Figure 1). Along the fixture length between the local maxima at each end of the fixture, higher longitudinal stress and longitudinal strains were instead observed for the simulations with large thread depth. This may be explained by the fact that the fixtures with larger thread depth provide less stress shielding of the bone due to their smaller core diameter, thereby leading to increased strains and stresses in the cortical bone. For LC2 the peak strain was 2370 µε for cortical bone with ordinary stiffness and 2 mm cortical thickness. For the case with 5 mm cortical thickness there were only minor local stress and strain peaks at the proximal end of the fixture location. It has been suggested that peak longitudinal strains above 1000 µε leads to endosteal and periosteal bone formation but little change intracortically, whereas peak longitudinal strains below 1000 µε leads to bone loss due to endosteal resorption and increased intracortical porosity [50]. Cortical bone This would suggest that during both LC1 and LC2 new bone formation at the proximal end of the fixture is to be expected, at least locally in the plane which is subjected to the highest bending moment. This would lead to an increased cortical thickness with subsequent, local decrease in stress and strain and a reduced risk of periprosthetic fracture in case of overloading. Stress in abutment The stress in the abutment was higher than the stress in the fixture, an effect of the smaller external diameter and the conscious design decision to make the abutment the weaker component, since it 15 can be more easily replaced if damaged than the fully implanted and osseointegrated fixture. The simulations showed that the transplanted bone can contribute to substantial stress reductions in the abutment, especially proximally corresponding to the region where the abutment exits from the fixture and would otherwise be particularly vulnerable. The transplanted bone bridges the gap between the distal cortical bone and the abutment and thus the cortical bone may contribute to resisting the bending loads. The advantage of the transplanted bone was observed with material properties representing immature transplanted bone, and improved as the stiffness increased. [1] H. E. J. Meulenbelt, J. H. B. Geertzen, M. F. Jonkman, and P. U. Dijkstraa, “Skin problems of the stump in lower limb amputees: 2. influence on functioning in daily life,” Acta Derm. Venereol., 2011. Limitations There are discrepancies between the geometry of FE-model and the clinically used implant system. As mentioned above, the proximal stiffness reduction of the fixture was not uniform between models of different thread depths. This led to that especially the FE-models with shallow thread depths had unrealistically high stiffness proximally. Furthermore, the FE-models lacked longitudinal slots in the proximal fixture, a feature present in the clinically used fixtures. Slots act to reduce proximal stiffness and therefore, it can be assumed that the stress and strain concentrations in the cortical bone at the proximal end of the fixture are more severe in the FE-model compared with the clinically used implant. A detailed investigation of the effect of the proximal fixture stiffness on the stress and strain in the cortical bone at the proximal end of the fixture was outside the scope of this evaluation and would require a separate study. Another difference between the FE-geometry and the clinically used implant 16 system is that the fixture and abutment were modelled as a single component rather than as separate components, thus omitting any effects from the contacting interface. Furthermore, the abutment screw and the preload were disregarded in the FE-model. This led to the omission of the resulting compressive stresses in the abutment shaft and thus, the presented longitudinal stress on the tensile side of the abutment shaft may be exaggerated by approximately 50 MPa compared with the clinical scenario. The geometry of the bone was modelled as a cylinder with uniform external diameter with the implant concentrically placed within the bone. This is a simplification from individual clinical cases which may vary both in the bone geometry and the implant placement. The load cases used in this study were derived from in vivo experimentally measured longitudinal forces and bending moments, but no torsional load components were considered. Furthermore, the variability in loading between users is known to be large [22], thus the magnitudes of the forces and moments may not be applicable to describe any individual user. [2] F. T. Hoaglund, H. E. Jergesen, L. Wilson, L. W. Lamoreux, and R. Roberts, “Evaluation of problems and needs of veteran lower-limb amputees in the San Francisco Bay Area during the period 1977-1980.,” J. Rehabil. R&D, 1983. Conclusion We conducted a numerical simulation (FE) of a bone-anchoring implant system. We found that the cortical thickness had the largest effect on stress and strain in the cortical bone and the fixture. At loads corresponding to high loading ambulation, 5 mm of cortical bone thickness led to approximately 40 % lower maximum longitudinal stresses at the central half of the fixture compared with a cortical thickness of 2 mm. Changes within the investigated range of thread profile parameters had a smaller effect (0 % – 8 % stress reduction for a thread root radius of 0.5 mm as compared with 0.1 mm, and 10 % – 22 % stress reduction for thread depth of 0.75 mm as compared to 1.5 mm). At the location where the abutment exits from the fixture, autologously transplanted bone may provide substantial support for the abutment by activating support from surrounding cortical bone. We observed 10 % and 31 % stress reduction in the abutment for simulations with transplanted bone with Young’s modulus of 0.4 GPa and 4 G Pa, respectively. Reduced cortical bone stiffness led to increased stresses in the fixture. 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https://openalex.org/W3212180673	https://ora.ox.ac.uk/objects/uuid:a859b1a4-7422-493f-9819-bb4adaed2988/files/sqz20st402	English	null	The indirect impact of COVID-19 pandemic on inpatient admissions in 204 Kenyan hospitals: An interrupted time series analysis	PLOS global public health	2,021	cc-by	8,808	PLOS GLOBAL PUBLIC HEALTH PLOS GLOBAL PUBLIC HEALTH The indirect impact of COVID-19 pandemic on inpatient admissions in 204 Kenyan hospitals: An interrupted time series analysis Steven WambuaID1, Lucas Malla2, George MbeviID2, Amen-Patrick NwosuID3, Timothy TutiID2, Chris PatonID3, Samuel Cheburet4, Ayub ManyaID4, Mike EnglishID2,3‡, Emelda A. Okiro1,3‡ Steven WambuaID1, Lucas Malla2, George MbeviID2, Amen-Patrick NwosuID3, Timothy TutiID2, Chris PatonID3, Samuel Cheburet4, Ayub ManyaID4, Mike EnglishID2,3‡, Emelda A. Okiro1,3‡ 1 Population Health Unit, Kenya Medical Research Institute-Wellcome Trust Research Programme, Nairobi, Kenya, 2 Health Services Unit, KEMRI-Wellcome Trust Research Programme, Nairobi, Kenya, 3 Oxford Centre for Global Health Research, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, United Kingdom, 4 Ministry of Health, Nairobi, Kenya a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 ‡ Joint senior authors and contributed equally. * swambua@kemri-wellcome.org OPEN ACCESS The first case of severe acute respiratory coronavirus 2 (SARS-CoV-2) was identified in March 2020 in Kenya resulting in the implementation of public health measures (PHM) to prevent large-scale epidemics. We aimed to quantify the impact of COVID-19 confinement measures on access to inpatient services using data from 204 Kenyan hospitals. Data on monthly admissions and deliveries from the District Health Information Software version 2 (DHIS 2) were extracted for the period January 2018 to March 2021 stratified by hospital ownership (public or private) and adjusting for missing data using multiple imputation (MI). We used the COVID-19 event as a natural experiment to examine the impact of COVID-19 and associated PHM on use of health services by hospital ownership. We estimated the impact of COVID-19 using two approaches; Statistical process control (SPC) charts to visu- alize and detect changes and Interrupted time series (ITS) analysis using negative-binomial segmented regression models to quantify the changes after March 2020. Sensitivity analy- sis was undertaken to test robustness of estimates using Generalised Estimating Equations (GEE) and impact of national health workers strike on observed trends. SPC charts showed reductions in most inpatient services starting April 2020. ITS modelling showed significant drops in April 2020 in monthly volumes of live-births (11%), over-fives admissions for medi- cal (29%) and surgical care (25%) with the greatest declines in the under-five’s admissions (59%) in public hospitals. Similar declines were apparent in private hospitals. Health worker strikes had a significant impact on post-COVID-19 trends for total deliveries, live-births and caesarean section rate in private hospitals. COVID-19 has disrupted utilization of inpatient services in Kenyan hospitals. This might have increased avoidable morbidity and mortality due to non-COVID-19-related illnesses. The declines have been sustained. Recent data suggests a reversal in trends with services appearing to be going back to pre- COVID levels. Citation: Wambua S, Malla L, Mbevi G, Nwosu A- P, Tuti T, Paton C, et al. (2021) The indirect impact of COVID-19 pandemic on inpatient admissions in 204 Kenyan hospitals: An interrupted time series analysis. PLOS Glob Public Health 1(11): e0000029. https://doi.org/10.1371/journal. Introduction Ministry of Health https://hiskenya.org/dhis-web- commons/security/login.action. The datasets used and/or analysed during the current study are available to others from this source on reasonable request. The global impact of the severe acute respiratory coronavirus 2 (SARS-CoV-2) virus has been extensive with over 137.5 million confirmed cases and 2.9 million official deaths globally as of April 13, 2021 [1]. In Kenya, the first case of COVID-19 was confirmed on 13th March 2020. Since then, 147147 cases have been reported with 2394 official deaths confirmed by April 13, 2021 [1] even though the real extent of spread is estimated to be greater [2]. The spread of the pandemic has placed unprecedented challenge on health systems. Funding: This research was funded in whole or in part by the Wellcome Trust Intermediate Fellow [Grant No. 201866]. For the purpose of Open Access, the author has applied a CC-BY public copyright licence to any author accepted manuscript version arising from this submission. Steven Wambua and Emelda A. Okiro are supported through a Wellcome Trust Intermediate Fellow [Grant No. 201866]. Lucas Malla, Amen- Patrick Nwosu, George Mbevi, Timothy Tuti and Chris Paton are supported through Funds from the Wellcome Trust [Grant No. 207522] awarded to Prof. Mike English as a senior Fellowship together with additional funds from a Wellcome Trust core grant awarded to the KEMRI-Wellcome Trust Research Programme [Grant No. 092654]. Steven Wambua, Lucas Malla, George Mbevi, Timothy Tuti, Mike English and Emelda A. Okiro, acknowledge the support of the Wellcome Trust to the Kenya Major Overseas Programme [Grant No. 203077]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. There have been three waves of the pandemic in Kenya. The initial stage of the outbreak fol- lowing the first case triggered implementation of a partial lockdown on April 6,2020, when 158 cases and 6 deaths had been reported nationally. This wave peaked in July/August 2020, and cases started to drop gradually [2]. However, two months later, the country experienced the second tide of the pandemic, which peaked in October/November 2020 [3] and could have been caused by opening of bars and restaurants and phased re-opening of universities. To curb the spread, the government instituted more restrictions which saw a dramatic drop in cases in the following four months. OPEN ACCESS pgph.0000029 Editor: Jake Michael Pry, Centre for Infectious Disease Research Zambia, Lusaka Zambia \| University of California, Davis, ZAMBIA Received: June 10, 2021 Accepted: October 25, 2021 Published: November 17, 2021 Editor: Jake Michael Pry, Centre for Infectious Disease Research Zambia, Lusaka Zambia \| University of California, Davis, ZAMBIA Editor: Jake Michael Pry, Centre for Infectious Disease Research Zambia, Lusaka Zambia \| University of California, Davis, ZAMBIA 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.pgph.0000029 Copyright: © 2021 Wambua 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. Data Availability Statement: Aggregated DHIS2 data is available online with access provided by 1 / 15 PLOS Global Public Health \| https://doi.org/10.1371/journal.pgph.0000029 November 17, 2021 PLOS GLOBAL PUBLIC HEALTH The indirect impact of COVID-19 pandemic on inpatient admissions in 204 Kenyan hospitals Introduction Nonetheless, the third wave was experienced in March 2021, dur- ing which the second partial lockdown was instituted on 27 March 2021. Previous outbreaks, noticeably the Ebola epidemic in West Africa in 2014 have brought to light unintended effects of control measures on utilization of health services during and after an outbreak, where admissions and surgeries reduced significantly [4–8]. This may be more pronounced in countries with weak and fragile healthcare systems [9]. This effect maybe unevenly distributed with women and children more vulnerable [4] and affecting some health- care services more than others [10]. acknowledge the support of the Wellcome Trust to the Kenya Major Overseas Programme [Grant No. 203077]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. To capture the indirect effects of COVID-19, we sought to quantify the influence of COVID-19 confinement measures by the government on admissions and deliveries to assess utilization of inpatient services using data from 204 Kenyan hospitals. Competing interests: The authors declare that they have no competing interests. PLOS Global Public Health \| https://doi.org/10.1371/journal.pgph.0000029 November 17, 2021 Chronology of events COVID-19 preparedness measures. Two months before the first case of COVID-19 was reported in Kenya on 13th March 2020, the government had increased preparedness to the pandemic. These included installation of surveillance systems to detect suspected COVID-19 cases at border points, additional medical staff at international airports and ports, in-country capacity to test and isolate COVID-19 cases, sensitization of healthcare workers on dealing with COVID-19 cases and establishment of a National Emergency Response Committee. COVID-19 intervention measures. The government started introduction of interven- tions to combat COVID-19 spread on 13th March 2020. These included suspension of public gatherings and events, closing of schools, international travel restrictions, fumigation and dis- infection of markets, closure of bars and restaurants, suspension of attendance to places of worship, limit of people attending weddings and funerals and national dust-to-dawn curfew. The month of April 2020 saw cessation of movement in and out of four counties with highest number of COVID-19 cases, restaurants were opened under strict guidelines of social distanc- ing, handwashing and temperature checks. During the month of May 2020, cessation of move- ment into and out of Kenya through Tanzania and Somalia borders was affected while on 10th June 2020 the government launched home-based care for patients with COVID-19 infection. In July 2020, certain measures were relaxed; cessation of movement into the four counties was lifted, phased re-opening of places of worship and resumption of local air travel. On 1st August 2020, international air travel resumed and in September 2020, operation of bars resumed. This 2 / 15 PLOS Global Public Health \| https://doi.org/10.1371/journal.pgph.0000029 November 17, 2021 PLOS GLOBAL PUBLIC HEALTH The indirect impact of COVID-19 pandemic on inpatient admissions in 204 Kenyan hospitals was followed by phased reopening of schools and lifting of suspension on political gatherings in October 2020 and November 2020 respectively. In December 2020, there was a nationwide health care workers strike precipitated by demands for better working conditions such as pro- vision of adequate Personal Protective Equipment (PPE), enhanced risk allowances and a health insurance cover. The strike only affected public facilities. Public and private health facil- ities in Kenya have separate management processes and funding structures affecting service provision hence challenges experienced in the public sector do not mirror the situation in Pri- vate facilities. Data Selection of hospitals. Hospitals were selected based on service availability. Data from varied databases on service availability were consulted. These include; Service Availability and Readiness Assessment (SARAM) [12], Emergency Medicine Foundation of Kenya (EMFK) [13], Harmonised Health Facility Assessment (HHFA) [14], District Health Information Sys- tem (DHIS2) [15] and the Master Facility List (MFL) [16]. A single database of facilities was then created using a combination of the data sources. Five common indicators used across dif- ferent countries to delineate hospitals were used. These were availability of operating theatres, oxygen, blood transfusion, radiology and critical new-born care. Hospitals identified to have the five indicators were then matched with the number of inpatient beds in an iterative process to confirm the provision of inpatient services and to validate the use of inpatient bed numbers in the definition of hospitals. Different iterations were then conducted based on the number of admissions and service availability to identify facilities that were either likely to have been missed by all of the service availability assessment considered or may be classified as lower- level facilities but attract on average 5,408 admissions annually and therefore warrant upgrad- ing to hospital status. A total of 201 hospitals fitted this criterion and were selected. The indica- tors used to select the hospitals is presented in Table 1 in S1 File. Three more hospitals, which are approved internship centres by the Kenya Medical Practitioners and Dentists Council [17] were added to the list totalling to 204 hospitals. The distribution of the 204 hospitals is repre- sented in Fig 1 & Table 2 in S1 File and the definition of the indicators used in this study pre- sented in Table 3 in S1 File. Extraction of data. Monthly data on inpatient admissions and deliveries in five patient groups (maternity, new-born units, paediatric, adult medical and surgical wards) were obtained from the District Health Information System (DHIS2), an open-source software plat- form for data reporting by most health facilities in the country. Data were extracted for the 204 hospitals, which are known to offer Comprehensive Emergency Obstetric and Newborn Care (CEmONC) services for the period January 2018 to March 2021, a total of 39 months. Adult medical data would be expected to include any admissions to general, high-dependency or intensive care wards although few hospitals will have these higher levels of care. Chronology of events These Health worker strikes were as a result of pay disputes, contracts and COVID safety concerns, including availability of personal protective equipment [11]. By early 2021, the test positivity proportion was below 5% and all schools were re-opened on January 4th. On 5th March 2021, the government launched its COVID-19 vaccination campaign and on March 12th, due to cases rising again during the third wave of the pandemic, funerals, wed- dings and other events were capped to including 100 persons and capacity of places worship was restricted. The timeline of events is presented in Fig 1. Fig 1. COVID-19 control measures timeline during the period of study. https://doi.org/10.1371/journal.pgph.0000029.g001 Fig 1. COVID-19 control measures timeline during the period of study. Fig 1. COVID-19 control measures timeline during the period of study. https://doi.org/10.1371/journal.pgph.0000029.g001 https://doi.org/10.1371/journal.pgph.0000029.g001 https://doi.org/10.1371/journal.pgph.0000029.g001 PLOS Global Public Health \| https://doi.org/10.1371/journal.pgph.0000029 November 17, 2021 3 / 15 PLOS GLOBAL PUBLIC HEALTH The indirect impact of COVID-19 pandemic on inpatient admissions in 204 Kenyan hospitals Data We chose 2018 as a starting point for the data extraction because of prolonged health care workers’ strikes in 2017 which affected health services provision [18] and consequently reporting. For each hospital, we extracted data on administrative units, level of hospital (Level 3: comprehen- sive primary health care facility, level 4: primary referral hospitals, Level 5: secondary referral hospitals and Level 6: national teaching and referral hospitals), whether a hospital is privately or publicly owned (ownership) and whether the hospital belongs to a clinical information net- work (CIN). The CIN is a collaboration of selected hospitals, policy makers and health researchers aimed at improving information available on the quality of inpatient paediatric care in Kenya [19–21]. A few hospitals reported data into an alternative parallel database (the hospital administrative database) and these data were merged where appropriate. Interrupted time series analysis Exploratory analyses. Admissions and deliveries were aggregated monthly across all reporting hospitals and stratified by hospital ownership. Temporal trends were plotted sepa- rately for private and public hospitals to identify significant changes in health services and Sta- tistical Process Control (SPC) charts were produced using 2018–2019 monthly averages as baseline to detect significant shifts in monthly admissions in 2020–2021 [27]. We also carried out multiple change point analysis [28, 29] to identify inpatient indicators that were affected by the national healthcare workers strike between December 2020 –February 2021. The assumption was that the strike affected all the public hospitals since we couldn’t obtain a database that tracks strikes in hospitals nationally. Segmented regression. We conducted interrupted-time series analysis using monthly number of admissions by ward and deliveries as outcomes. Analysis was stratified by hospital type. For caesarean sections and admissions in NBU where we had an appropriate denomina- tor (total deliveries and live births respectively), they were used as offset variable to convert them into a rate and adjust for any potential changes in utilization over time [30, 31]. We therefore report caesarean section rates and NBU admission rates. The period running from January 2018 through March 2020 was defined as pre-COVID-19 and April 2020 to March 2021 as post-COVID-19. For each indicator, a segmented regression model was fitted [30, 32]. Statistical analysis Handling missing data. To adjust for incompleteness in reporting during the period of study, multiple imputation (MI) was performed [22–24]. Missing monthly values were imputed using a mixed effects model in a joint modelling framework using the jomo package in R and obtained 100 imputations for each indicator [25, 26]. We assumed the data followed as missing at random (MAR) pattern. The imputed datasets were analysed individually, and the estimates pooled using Rubin’s Rules. Each inpatient indicator was used as an outcome in the imputation model. Ownership (public or private), whether hospital belongs to a clinical 4 / 15 PLOS Global Public Health \| https://doi.org/10.1371/journal.pgph.0000029 November 17, 2021 PLOS GLOBAL PUBLIC HEALTH The indirect impact of COVID-19 pandemic on inpatient admissions in 204 Kenyan hospitals information network (CIN), time (month and year) and COVID-19 binary indicator (0 – months before pandemic and 1 –months post pandemic) were used as covariates with the hos- pital ID as a clustering variable. MI was performed for hospitals with more than 30% of months reported (at least 11/39 months reported) to reduce uncertainty in imputed values and promote generalizability of the estimates. This strategy was supported by results from a simula- tion study we carried out, indicating MI performance and efficiency was best when imputing data for hospitals that reached this threshold of months reported. The imputation model speci- fication is outlined in Handling missing data in S4 File. The number of hospitals included in analyses based on this strategy varied for each ward as summarised in the results with further information presented in Table 4 in S1 File. PLOS Global Public Health \| https://doi.org/10.1371/journal.pgph.0000029 November 17, 2021 Interrupted time series analysis The following equation specifies the model [32]; Yt ¼ b0 þ b1timet þ b2COVID19t þ b3time after COVID19t þ et Yt ¼ b0 þ b1timet þ b2COVID19t þ b3time after COVID19t þ et Yt ¼ b0 þ b1timet þ b2COVID19t þ b3time after COVID19t þ et Where, Yt is the number of admissions in month t; time is a continuous indicator of time in months from January 2018; COVID19 is an indicator of time t occurring before (COVID19 ¼ 0Þ or after (COVID19 ¼ 1Þ the outbreak, which was implemented at April 2021 in the series, where COVID-19 measures were most stringent in Kenya [33]; and time after COVID19 is a continuous variable counting the number of months after COVID-19 at time t: In the model, b0 estimates the baseline level of admissions at time zero; b1 estimates the change in the monthly number of deliveries before the intervention (the pre-existing trend); b2 estimates the level change in monthly number of admissions immediately after COVID-19 outbreak, which is from the end of the preceding segment; b3 estimates the change in the trend of monthly admissions after COVID-19, compared with the pre-existing trend. The random variability at time t, which is not explained in the model is represented by the error term et. A level change (immediate COVID-19 effect) and change in trend after COVID- 19 were hypothesised [30]. A generalized linear model was applied, assuming a Poisson distribution. Since the count data was over dispersed, and after evaluating several models, we fitted the negative binomial model to account for overdispersion [30, 34, 35]. Model checking was conducted for 5 / 15 PLOS Global Public Health \| https://doi.org/10.1371/journal.pgph.0000029 November 17, 2021 PLOS GLOBAL PUBLIC HEALTH The indirect impact of COVID-19 pandemic on inpatient admissions in 204 Kenyan hospitals autocorrelation using the Durbin-Watson statistic and autoregressive moving average (ARMA) models were fitted for indicators with serial autocorrelation [36–38]. For indicators with significant autocorrelation, various combinations of the ARMA parameters (order of the autoregressive and moving average polynomials) were tested and the model resulting to lower Akaike Information Criterion (AIC) selected. Seasonality was adjusted using Fourier terms [39]. Results were pooled across the multiple imputed datasets using Rubin’s rules [40]. The negative binomial segmented ITS model, which accounted for seasonality, autocorrelation and overdispersion was the best model when compared with negative binomial models that were not adjusted. Interrupted time series analysis Sensitivity analyses were performed for indicators affected by the national health care work- ers strike by excluding months affected and comparing the estimates with those from data that included the strike period. In addition, we anticipated differences in reporting across hospitals. It is possible that the impact of the pandemic across hospitals was variable, for instance some hospitals were assigned as isolation centres or closed for some time. Also, the magnitude of the epidemic in some counties was higher. Therefore, in counties with higher case burden, some hospital staff were re-assigned or had to go for quarantine if exposed or isolation if they con- tracted COVID-19, leading to human resources shortages. The intraclass correlation coeffi- cients across hospitals for each indicator is provided in Table 3 in S3 File. Therefore, a Poisson generalized estimating equations (GEE) was fitted for each indicator to test the effect of vary- ing model assumptions, such as accounting for differences in hospitals, on the estimates [41]. Statistical significance was defined as p-values < 0.05. All analyses were performed using R Statistical significance was defined as p-values < 0.05. All analyses were performed using R (version 3.6.3). Ethics approval and consent to participate The manuscript does not contain any individual person’s data. We extracted aggregated monthly data at hospital level and not at patient level. PLOS Global Public Health \| https://doi.org/10.1371/journal.pgph.0000029 November 17, 2021 Results Out of the 204 hospitals evaluated in this study, 109 were public and 95 were private. The num- ber and percentage of hospitals reporting at least 30% of months and were included in the analysis are presented in Table 3 in S1 File. In the sample of 109 public hospitals, all hospitals reporting at least a month were analysed for total deliveries and live births (108/108), caesarean sections (106/107), admissions in medical (102/104), admissions in surgical (48/57), admis- sions in paediatrics (100/106) and admissions in the NBU (61/73). Similarly, all the private hospitals reporting at least a month were included in the analysis for total deliveries and live births (95/95). While caesarean sections (93/94), admissions in medical (89/92), admissions in surgical (67/82), admissions in paediatrics (83/91) and admissions in NBU (32/60) in the sam- ple of private hospitals. SPC charts illustrate that for both public and private hospitals, no reported values fell out- side the 3SD mark in the first three months of 2020 (pre-COVID) (Fig 2). In public hospitals, monthly admissions fell below the 3SD mark in the paediatrics, adult medical and surgical wards starting in April 2020 through June 2020. This substantial drop remained until Novem- ber 2020 for admissions in the medical and paediatrics wards, while admissions in the surgical ward started returning to normalcy during this period. There were similar falls in private hos- pitals, with admissions in medical, paediatric and surgical wards below the 3SD threshold between April 2020 and August 2020, with slight recoveries for admissions in the surgical ward within this period. In this sample of private hospitals, although admissions in the medical and surgical ward started to recover starting September 2020 to the end of the study, 6 / 15 PLOS Global Public Health \| https://doi.org/10.1371/journal.pgph.0000029 November 17, 2021 PLOS GLOBAL PUBLIC HEALTH The indirect impact of COVID-19 pandemic on inpatient admissions in 204 Kenyan hospitals Fig 2. Statistical Process Control chart for both public and private hospitals. Horizontal dashed lines represent the 3-standard deviation mark. https://doi.org/10.1371/journal.pgph.0000029.g002 Fig 2. Statistical Process Control chart for both public and private hospitals. Horizontal dashed lines represent the 3-standard deviation mark. https://doi.org/10.1371/journal.pgph.0000029.g002 https://doi.org/10.1371/journal.pgph.0000029.g002 reductions in admissions in the paediatrics ward remained below the 3SD mark up to Decem- ber 2020, where they started going back to expected levels. Results Numbers of deliveries and live births in public hospitals also fell starting in April 2020 but these were less substantial than for other admissions and the sample of private hospitals utilisation of the maternity ward services began to rise through July 2020 to November 2020. In December 2020, during the nationwide healthcare workers strike, there was a drastic drop for all indicators in public hospitals and the highest increase in admissions for private hospitals (Fig 2). These changes continued to Febru- ary 2021, with services beginning to go back to historical levels in March 2021. Interrupted timeseries results From Table 1, before COVID-19, the trend was generally positive across all the indicators in both public and private hospitals, showing an increase in admissions and deliveries overtime. There was an immediate drop in admissions post-COVID-19 for most inpatient indicators. In public hospitals, there was a 11% (IRR = 0.89 CI: 0.80–0.99), 29% (IRR = 0.71 CI: 0.63– 0.79), 59% (IRR = 0.42 CI: 0.38–0.45) and 25% (IRR = 0.75 CI: 0.64–0.87) reductions in live From Table 1, before COVID-19, the trend was generally positive across all the indicators in both public and private hospitals, showing an increase in admissions and deliveries overtime. p p p g There was an immediate drop in admissions post-COVID-19 for most inpatient indicators. In public hospitals, there was a 11% (IRR = 0.89 CI: 0.80–0.99), 29% (IRR = 0.71 CI: 0.63– 0.79), 59% (IRR = 0.42 CI: 0.38–0.45) and 25% (IRR = 0.75 CI: 0.64–0.87) reductions in live 7 / 15 PLOS Global Public Health \| https://doi.org/10.1371/journal.pgph.0000029 November 17, 2021 PLOS GLOBAL PUBLIC HEALTH The indirect impact of COVID-19 pandemic on inpatient admissions in 204 Kenyan hospitals Table 1. Interrupted time series analysis results showing incidence rate ratios (IRR) and relative risk (RR)for COVID-19 intervention, time and trend alongside 95% confidence intervals. Total deliveries Live births Caesarean sections rate Admissions > 5 Medical Ownership IRR 95%CI P-value IRR 95%CI P-value RR 95%CI P-value IRR 95%CI P-value Public COVID-19 0.89 (0.79–1.02) 0.08 0.89 (0.80–0.99) 0.04 1.10 (1.06–1.14) <0.01 0.71 (0.63–0.79) <0.01 Time 1.00 (0.99–1.01) 0.67 1.00 (0.99–1.01) 0.42 1.01 (1.00–1.01) <0.01 1.00 (0.99–1.00) 0.57 Trend 0.99 (0.97–1.00) 0.11 0.99 (0.97–1.00) 0.07 1.00 0.99–1.00) 0.14 1.01 (0.99–1.03) 0.10 Private COVID-19 0.91 (0.81–1.03) 0.13 0.91 (0.81–1.03) 0.12 1.03 (0.98–1.09) 0.25 0.66 (0.60–0.72) <0.01 Time 1.00 (0.99–1.01) 0.06 1.00 (0.99–1.01) 0.03 1.00 (1.00–1.01) 0.02 1.00 (1.00–1.01) 0.01 Trend 1.03 (1.01–1.04) <0.01 1.03 (1.01–1.04) <0.01 0.99 (0.98–0.99) 0.01 1.03 (1.01–1.04) <0.01 Admissions < 5 Paediatrics Admissions > 5 Surgical NBU Admissions rate Ownership IRR 95%CI P-value IRR 95%CI P-value RR 95%CI P-value Public COVID-19 0.41 (0.38–0.45) <0.01 0.75 (0.64–0.87) <0.01 0.92 (0.83–1.01) 0.09 Time 1.00 (1.00–1.01) <0.01 1.00 (0.99–1.00) 0.46 1.00 (1.00–1.01) 0.02 Trend 1.04 (1.03–1.05) <0.01 1.01 (0.99–1.03) 0.19 1.01 (1.00–1.02) 0.04 Private COVID-19 0.38 (0.33–0.45) <0.01 0.72 (0.64–0.81) <0.01 0.87 (0.72–1.04) 0.13 Time 1.00 (0.99–1.01) 0.38 1.00 (0.99–1.00) 0.82 1.00 (0.99–1.01) 0.21 Trend 1.07 (1.05–1.08) <0.01 1.02 (1.01–1.04) <0.01 0.99 (0.97–1.02) 0.48 Estimates from ARMA models. Interrupted timeseries results The ARMA (p,q) parameters for admissions in medical, surgical and paediatrics are (2,0), (1,0) and (2,0) respectively. errupted time series analysis results showing incidence rate ratios (IRR) and relative risk (RR)for COVID-19 interventio nce intervals Table 1. Interrupted time series analysis results showing incidence rate ratios (IRR) and relative risk (RR)for COVID-19 95% confidence intervals. ults showing incidence rate ratios (IRR) and relative risk (RR)for COVID-19 intervention, time and trend alongside Estimates from ARMA models. The ARMA (p,q) parameters for admissions in medical, surgical and paediatrics are (2,0), (1,0) and (2,0) respectively. births, over-fives admissions in medical, under-fives admissions in paediatrics and over-fives admissions in surgical respectively. Over-fives in medical, under-fives paediatrics and over- fives surgical admissions declined by 34% (IRR = 0.66 CI: 0.60–0.72), 62% (IRR = 0.38 CI: 0.33–0.45) and 28% (IRR = 0.72 CI: 0.64–0.81) respectively in private hospitals. Caesarean sec- tion rate increased by 10% (RR = 1.10 CI: 1.06–1.14) in public hospitals. Although monthly admissions in the postCOVID-19 period (May 2020 onwards) experienced some recovery across all the indicators except for NBU admissions rate in the sample of private hospitals, the recovery was statistically significant for admissions in the paediatrics ward in the sample of public hospitals. There was significant autocorrelation for admissions in medical, surgical and paediatrics wards and estimates presented are produced from the ARMA models. The ITS fit- ted models are presented in Fig 3. Sensitivity analyses Multiple change point analysis showed the national strike between December 2020 –February 2021 coincided with significant reductions in live-births and caesarean sections in the sample of public hospitals. Conversely, during this strike period, total deliveries, live-births and admis- sions in the paediatrics ward increased sharply in private hospitals (Figs 2 and 3 in S2 File). Excluding data for the three months when strike was ongoing showed the estimates for the post-COVID-19 trend were sensitive to changes in services during the strike for total deliver- ies, live-births and caesarean section rates in the sample of private hospitals and for surgical and NBU admissions in public hospitals (Tables 1 & 2 in S3 File). The estimates obtained from the Poisson generalized estimating equations (GEE) were not different from the primary model estimates hence robustness of the estimates (Table 4 in S3 File). PLOS Global Public Health \| https://doi.org/10.1371/journal.pgph.0000029 November 17, 2021 Discussion PLOS Global Public Health \| https://doi.org/10.1371/journal.pgph.0000029 November 17, 2021 9 / 15 PLOS GLOBAL PUBLIC HEALTH The indirect impact of COVID-19 pandemic on inpatient admissions in 204 Kenyan hospitals Interestingly, total deliveries and total live births in the public sector, that we would obvi- ously expect to be closely linked, were less dramatically affected at the start of Kenya’s epi- demic. However, public sector utilisation of maternity services declined slowly over the 12 months’ post-COVID period with a nadir during the December 2020 health workers’ strike. The decline in public sector utilisation of hospital maternity services was associated with a small but significant increase in the proportion of women having a caesarean delivery. Caesar- ean delivery rates fell during the health workers’ strike (Fig 3). Over the same post-COVID period the proportion of live births admitted to newborn units also slowly increased. Taken together these findings may suggest that while public sector delivery volumes were generally declining. The number of women delivering in hospital with complications of pregnancy, and consequently of newborns with problems at birth, did not decline as much. In the private sec- tor, there was no decline in delivery volumes of live births and in fact a slow increase in these volumes over time. The much higher proportion of women in the private sector (35–40%) delivering by caesarean section is also worthy of note while the proportion of live births being admitted to NBU in private hospitals is much lower than in the public sector. The discrepancy between paediatric, adult medical and adult surgical admissions’ indica- tors for which volumes fell sharply and use of maternity services for delivery is worthy of note and somewhat unexpected. The majority of hospitals for which we have data are non-tertiary and at Level 4 and 5 of the Kenyan health system and located in the major towns in counties and in urban and peri-urban settings of Kenya’s cities. It is therefore possible that urban fami- lies prefer to use hospitals for maternity services rather than deliver at home or in smaller clin- ics. Use of public hospitals may also have been sustained by the policy of free maternity services in the public sector policy put in place by the Kenyan government in 2013. Discussion In this study, the unintended impact of COVID-19 control measures on inpatient admissions for a large sample of private and public Kenyan hospitals using monthly data reported to the 8 / 15 PLOS Global Public Health \| https://doi.org/10.1371/journal.pgph.0000029 November 17, 2021 PLOS GLOBAL PUBLIC HEALTH The indirect impact of COVID-19 pandemic on inpatient admissions in 204 Kenyan hospitals S GLOBAL PUBLIC HEALTH The indirect impact of COVID-19 pandemic on inpatient admissions in 204 Kenyan hospitals Fig 3. Fitted lines of segmented regression models for all indicators and by hospital ownership. Vertical lines represent the month (March 2020) COVID-19 was announced in Kenya and as a pandemic by the WHO. https://doi org/10 1371/journal pgph 0000029 g003 on models for all indicators and by hospital ownership. Vertical lines represent the month (March 2020) COVID-19 was y the WHO. Fig 3. Fitted lines of segmented regression models for all indicators and by hospital ownership. Vertical lines represent the month (March 2020) COVID-19 was announced in Kenya and as a pandemic by the WHO. https://doi.org/10.1371/journal.pgph.0000029.g003 https://doi.org/10.1371/journal.pgph.0000029.g003 https://doi.org/10.1371/journal.pgph.0000029.g003 national information system from January 2018 to March 2021 was evaluated. Utilization of inpatient services was rising slowly prior to March 2020 in these non-tertiary hospitals but declined sharply after this co-incidental with the first introduction of national measures to restrict population mixing in the initial wave of Kenya’s epidemic. Paediatric admissions fell especially sharply in both public and private sectors. So too, although to a lesser degree, did adult surgical and medical admissions in these hospitals that would typically not have any sig- nificant high-dependency or intensive care capacity [42]. In the public sector admissions for these three older groups, including adult medical and surgical ward admissions, remained lower than was typical for the following 12 months during what appeared to be Kenya’s first and second wave of SARS-CoV-2 infections. During this period a national health workers’ strike between in December 2020 and February 2021 also likely reduced admissions for inpa- tient care even though our analysis shows that this didn’t significantly affect all the indicators uniformly. In the private sector admission volumes for these three groups slowly rose again during this 12 months’ period but did not exceed pre-COVID volumes. PLOS Global Public Health \| https://doi.org/10.1371/journal.pgph.0000029 November 17, 2021 Strengths of the study This study provides an understanding of the current situation on utilization of inpatient admissions in a large sample of hospitals in Kenya in the face of the COVID-19 pandemic. At a time when most attention is on restrictive measures to combat the pandemic, the unintended effects of these policies might be overlooked and measuring the effects should inform ongoing policy discussions that seek to balance COVID prevention and ensuring people can access care. This study adjusted for potential confounders in assessing changes overtime. Firstly, we adjusted for missing data in DHIS2, which is quite common in routine data but not always done [49, 53]. This may be important as non-reporting may be compounded during COVID- 19 if Health Records and Information Officers work at reduced capacity during the epidemic. Additionally, the WHO in a recent guide on using routine data to monitor the effects of COVID-19 recommended adjusting for low reporting rates [54]. More generally, missing data were often encountered and to gain an accurate and rapid picture of the effects of COVID poli- cies in countries such as Kenya more needs to be done to improve DHIS2 data quality. This will likely require investment in better infrastructure, supervisory support, formal data quality assurance and human resources [55, 56]. A second strength was use of data from a reasonably large number of hospitals from a broad geographic region covering all the counties across the country. Thirdly, compared to previous studies [49, 57], we have used a prolonged pre- COVID-19 period and a full 12 months post-COVID-19 period. This provided enough data points to measure the post-COVID-19 trend with precision and also allow adjustments for sea- sonal trends. Finally, we evaluated the potential effect of a second interruption to services, the health care workers’ strike, and used sensitivity analysis to account for uncertainty in estimates due to hospital level variability. Discussion Despite this there was a suggestion that as deliveries declined in the public sector they increased in the private sector, something likely exacerbated by the public sector health workers’ strike in December 2020. The significant reductions in over-fives admissions in both medical and surgical wards we hypothesize are likely linked to the restrictions in movements, dusk-to-dawn curfews, lack of or high cost of transport that were the result of COVID containment strategies. It is also possi- ble, as public hospital care is not free for these populations, that the loss of income experienced by many in the post-COVID period prevented care seeking. The cases of the particularly large drop in under-fives admissions from April 2020 are not known. We can speculate that, in addition to the factors influencing utilisation of hospitals by adults, parent’s might fear taking their children to hospitals to limit their own and their child’s exposure to contracting COVID- 19 infection [43]. It is also possible, that rates of illness were reduced because families paid greater attention to hygiene (e.g. hand-washing) [44], because of social distancing and because schools were closed limiting transmission of many respiratory illnesses in households [45] as respiratory illness is responsible for a large proportion of paediatric admissions in Kenya [46]. The patterns of decline in admissions post-COVID-19 reflect those observed in previous hospital and population-based studies [41, 47–50]. A study in the US observed dramatic reduc- tions in hospital admissions after onset of COVID-19 [47]. Significant reductions in childcare services were also reported in South Africa following lockdown measures [41]. Although no studies have quantified effects of the pandemic on surgeries in Kenya, early estimates had revealed reductions in surgical case volumes in low- and middle-income countries [51]. Addi- tionally, deficit in the provision of personal protective equipment, surgical masks, gloves, N95 masks, and shoe covers were reported to worry surgeons in a cross-sectional study of neuro- surgeons across African countries, which might affect provision and consequently utilization of these services [52]. 10 / 15 PLOS Global Public Health \| https://doi.org/10.1371/journal.pgph.0000029 November 17, 2021 PLOS GLOBAL PUBLIC HEALTH The indirect impact of COVID-19 pandemic on inpatient admissions in 204 Kenyan hospitals PLOS Global Public Health \| https://doi.org/10.1371/journal.pgph.0000029 November 17, 2021 Conclusion In a large sample of public hospitals significant drops were observed in monthly volumes of live births (11%), over-fives admissions for medical (29%) and surgical care (25%) with espe- cially high declines in admissions in the under 5 age group (59%). Similarly, substantial declines were apparent in private hospitals, where significant reductions in admissions to the medical (34%), surgical (28%) and paediatrics (62%) wards were observed. However, mater- nity ward indicators and NBU admission rates were not affected in this sample of private hos- pitals. The declines have been sustained and recent data suggests a reversal in trends with services appearing to be going back to the historical levels starting March 2021. Supporting information S1 File. Distribution of hospitals and number of health facilities analysed for each indica- tor including health facilities excluded for not reporting any month and those with less than 30% of months reported. The definition of the indicators analysed is outlined. (DOCX) S1 File. Distribution of hospitals and number of health facilities analysed for each indica- tor including health facilities excluded for not reporting any month and those with less than 30% of months reported. The definition of the indicators analysed is outlined. (DOCX) tor including health facilities excluded for not reporting any month and those with less than 30% of months reported. The definition of the indicators analysed is outlined. (DOCX) S2 File. Patterns of missing data across all the health facilities analysed including multiple change point analysis plots. (DOCX) S3 File. Sensitivity analysis estimates. (DOCX) S4 File. Multiple imputation methodology. (DOCX) S2 File. Patterns of missing data across all the health facilities analysed including multiple change point analysis plots. (DOCX) S2 File. Patterns of missing data across all the health facilities analysed including multiple change point analysis plots. (DOCX) S3 File. Sensitivity analysis estimates. (DOCX) S4 File. Multiple imputation methodology. (DOCX) S4 File. Multiple imputation methodology. (DOCX) Limitations We recognize some limitations of the study. There is poor reporting into DHIS2 of private hospitals, especially those offering the equivalent of regional or tertiary level services. This reduces the number or private hospitals in our sample and tends to exclude those with more advanced patient care facilities. We also had to exclude some tertiary and more specialised cen- tres from the public sector because these too reported less than 30% of months data. Our finding that adult medical admissions were reduced may therefore be misleading if these larger hospitals experienced a major rise in admissions if adults with severe COVID-19 sought care at these sites preferentially. Importantly, we sought to analyse data on mortality of the admissions analysed in this report. However, DHIS2 as implemented in Kenya makes this extremely challenging as hospitals are unable to record zero deaths in a month, efforts to enter a zero-value result in a missing value in the actual database. Accounting for this will need more complex imputation procedures which are being explored. Additionally, given the nature of the pandemic there are no potential controls for this study and so while it seems entirely plausible that the effects, we describe are a consequence of the introduction of COVID-19 con- trol measures it remains possible there are other unknown explanations. We also note that the analysis was restricted to events occurring within hospitals and doesn’t account for those occurring in primary health facilities or outside the health system. We evaluated the impact of health worker strike on the analysis assuming a uniform impact across all hospitals due to lack of a database that tracks strikes within hospitals nationally, however the length of the strike might have been shorter for some hospitals. 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Data curation: Steven Wambua, Lucas Malla, George Mbevi, Amen-Patrick Nwosu, Timothy Tuti, Chris Paton, Samuel Cheburet, Ayub Manya, Mike English, Emelda A. Okiro. Formal analysis: Steven Wambua, Lucas Malla, Mike English, Emelda A. Okiro. Funding acquisition: Mike English, Emelda A. Okiro. Investigation: Steven Wambua, Lucas Malla, George Mbevi, Amen-Patrick Nwosu, Timothy Tuti, Chris Paton, Samuel Cheburet, Ayub Manya, Mike English, Emelda A. Okiro. Methodology: Steven Wambua, Lucas Malla, George Mbevi, Amen-Patrick Nwosu, Timothy Tuti, Chris Paton, Samuel Cheburet, Ayub Manya, Mike English, Emelda A. Okiro. Project administration: Steven Wambua, Mike English, Emelda A. Okiro. Resources: Mike English, Emelda A. Okiro. Software: Steven Wambua, Lucas Malla. Software: Steven Wambua, Lucas Malla. Supervision: Mike English, Emelda A. Okiro. 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https://openalex.org/W4390665291	https://www.frontiersin.org/articles/10.3389/fnagi.2023.1240509/pdf?isPublishedV2=False	English	null	Association between intracranial and extracranial atherosclerosis and white matter hyperintensities: a systematic review and meta-analysis	Frontiers in aging neuroscience	2,024	cc-by	7,101	KEYWORDS cerebral small vessel disease, extracranial atherosclerosis, intracranial atherosclerosis, meta-analysis, white matter hyperintensities TYPE Systematic Review PUBLISHED 08 January 2024 DOI 10.3389/fnagi.2023.1240509 TYPE Systematic Review PUBLISHED 08 January 2024 DOI 10.3389/fnagi.2023.1240509 cerebral small vessel disease, extracranial atherosclerosis, intracranial atherosclerosis, meta-analysis, white matter hyperintensities COPYRIGHT © 2024 Zh COPYRIGHT © 2024 Zhang, Fu and Zhan. 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: Twenty-one eligible studies, including 10,841 participants, were identified. Intracranial and extracranial atherosclerotic stenosis was associated with an increased risk of WMHs (OR 1.80, 95% CI 1.25–2.57, I2 = 75%) and increased WMH volumes (SMD 0.40, 95% CI 0.18–0.63, I2 = 63%). Heterogeneity resulted from the WMHs rating method and the location. Extracranial atherosclerotic stenosis (ECAS) was significantly associated with WMHs (OR 2.10, 95% CI 1.22– 3.62, I2 = 71%), but intracranial atherosclerotic stenosis (ICAS) was insignificantly associated with WMHs (OR 1.75, 95% CI 0.97–3.15, I2 = 84%). The association was stable in the subgroup analysis based on WMHs location, which included deep WMHs and periventricular WMHs. Conclusion: Intracranial and extracranial atherosclerotic stenosis is associated with WMHs. This association is significant in ECAS, but attenuated in ICAS. Association between intracranial and extracranial atherosclerosis and white matter hyperintensities: a systematic review and meta-analysis OPEN ACCESS EDITED BY Stefano Tarantini, University of Oklahoma Health Sciences Center, United States REVIEWED BY Ma Yuanyuan, Fudan University, China Alvin S. Das, Harvard Medical School, United States CORRESPONDENCE Wenyuan Zhang zhangwenyuan11@163.com RECEIVED 15 June 2023 ACCEPTED 28 November 2023 PUBLISHED 08 January 2024 CITATION Zhang W, Fu F and Zhan Z (2024) Association between intracranial and extracranial atherosclerosis and white matter hyperintensities: a systematic review and meta-analysis. Front. Aging Neurosci. 15:1240509. OPEN ACCESS EDITED BY Stefano Tarantini, University of Oklahoma Health Sciences Center, United States Wenyuan Zhang 1, Fangwang Fu 2 and Zhenxiang Zhan 3 1 Department of Neurology, Affiliated Yueqing Hospital, Wenzhou Medical University, Yueqing, China, 2 Department of Neurology, The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, China, 3 Department of Neurology, Affiliated Jinhua Hospital, Zhejiang University School of Medicine, Jinhua, China Zhang W, Fu F and Zhan Z (2024) Association between intracranial and extracranial atherosclerosis and white matter hyperintensities: a systematic review and meta-analysis. Background: White matter hyperintensities (WMHs) are key neuroimaging markers of cerebral small vessel diseases. This study aimed to investigate whether intracranial and extracranial atherosclerotic stenosis is associated with WMHs. Front. Aging Neurosci. 15:1240509. doi: 10.3389/fnagi.2023.1240509 Front. Aging Neurosci. 15:1240509. doi: 10.3389/fnagi.2023.1240509 Methods: Following a previously registered protocol (PROSPERO protocol: CRD42023407465), PubMed, Web of Science, and Embase were systematically searched for relevant literature published until March 2023. Cross-sectional studies examining the association between intracranial and extracranial atherosclerotic stenosis and WMHs were included. Random effects models were used to calculate the pooled estimates. Introduction White matter hyperintensities (WMHs) of presumed vascular origin are ischemic manifestations of cerebral small vessel disease that can be detected through neuroimaging (Wardlaw et al., 2013). The prevalence of WMHs increases significantly with age, ranging from approximately 5% in individuals aged 40 years to nearly 100% in those aged 80 years Frontiers in Aging Neuroscience Frontiers in Aging Neuroscience frontiersin.org frontiersin.org 01 Zhang et al. 10.3389/fnagi.2023.1240509 (Zhuang et al., 2018). Severe WMHs have been linked to various negative outcomes, including cognitive impairment (Debette et al., 2019), neuropsychiatric symptoms (Clancy et al., 2021), gait dysfunction (Kim et al., 2016), increased stroke risk (Debette et al., 2019), and poorer stroke recovery (Cheng et al., 2022). However, the underlying mechanisms responsible for the development of WMHs remain incompletely understood, with current research highlighting dysfunctions in cerebral blood flow and the blood–brain barrier as crucial factors (Wardlaw et al., 2019). Previous studies have established an association between intracranial and extracranial atherosclerotic stenosis and white matter hyperintensities (WMHs), although the results of these studies are not consistent (Pu et al., 2009; Lee et al., 2011; Schulz et al., 2013; Park et al., 2015; Duan et al., 2018; Huang et al., 2022). detailed search formula was as follows: (“white matter hyperintensit” OR “white matter lesion” OR “white matter change” OR “white matter disease” OR “white matter damage” OR “leukoaraiosis” OR “leukoencephalopath” OR “Binswanger’s disease”) AND (“intracranial atherosclero” OR “cerebral atherosclero” OR “extracranial atherosclero” OR “arter stenosis” OR “intracranial stenosis” OR “extracranial stenosis” OR “carotid stenosis”). The references of the included articles and relevant reviews were manually searched to identify potential studies missed during the initial literature search. Two independent investigators (ZZ and FF) performed a literature, and differences were resolved by a third investigator (WZ) joining the discussion. , ; , ; , ; g , ) Atherosclerosis is the primary cause of luminal stenosis in both intracranial and extracranial arteries. Approximately 50% of Asian patients with acute ischemic stroke suffer from intracranial and extracranial atherosclerotic stenosis (Wang et al., 2014). Although WMHs are the primary imaging marker representing damage to the brain’s small vessels, some studies have reported that WMHs were more common in patients with intracranial and extracranial atherosclerotic stenosis (Lee et al., 2011; Park et al., 2015; Duan et al., 2018). Methods This systematic review was carried out based on a predefined protocol (PROSPERO registration number: CRD42023407465), adhering to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines (Page et al., 2021). Data extraction Two independent reviewers (ZZ and FF) used a prespecified template to extract information on study characteristics (first author, publication year, country, study design), participant details (sample size, mean age, sex ratio), atherosclerotic stenosis assessment (location, severity, vascular image), WMHs assessment (rating method, Introduction A previous meta-analysis demonstrated that carotid artery stenosis was associated with total WMHs, deep WMHs (DWMHs), and periventricular WMHs (PVWMHs) (Ye et al., 2018). However, it is important to note that this meta-analysis only included a limited number of studies (n = 8). Furthermore, the study conducted by Ye et al. specifically focused on extracranial internal carotid artery stenosis, and there has been no comprehensive summary of research findings regarding intracranial atherosclerotic stenosis (ICAS) and WMHs. The pathogenic mechanisms and hemodynamic changes of ICAS and extracranial atherosclerotic stenosis (ECAS) are not entirely consistent (Kim et al., 2018). Therefore, it is necessary to determine whether ICAS, similar to ECAS, is related to WMHs. To date, a considerable number of recent studies have been published investigating the correlation between intracranial and extracranial atherosclerotic stenosis and white matter hyperintensities (WMHs) (Duan et al., 2018; Ye et al., 2019; Del Brutto et al., 2020; Fang et al., 2020; Benli et al., 2021; Yin et al., 2021; Choi et al., 2022; Ghaznawi et al., 2022; Huang et al., 2022; Wang et al., 2022). In light of this, we conducted an updated systematic review and meta-analysis to examine the potential association between ICAS or ECAS and WMHs. Additionally, the associations between intracranial and extracranial atherosclerotic stenosis and WMHs in different study designs, severity of atherosclerotic stenosis, WMHs rating method, and WMHs location were further analyzed. Study selection This meta-analysis included studies that reported an association between ICAS or ECAS and WMHs in human. The inclusion criteria were as follows: (i) the set criteria for ICAS or ECAS severity was ≥50%; (ii) WMHs were assessed by a quantitative or semiquantitative method based on magnetic resonance imaging (MRI); (iii) the available data for meta-analysis (effect estimates or mean and standard deviation) were reported; and (iv) the article was published in full text in English. The following studies were excluded: (1) studies with unclear criteria for ICAS or ECAS severity; (2) studies with WMHs assessment based on computer tomography (CT); (3) meeting abstracts; and (4) systematic reviews. In the case of duplicated published data, we included the study with the greatest number of participants. ICAS was defined as stenosis in intracranial internal carotid artery, middle cerebral artery, anterior cerebral artery, posterior cerebral artery, intracranial vertebral artery, and basilar artery. ECAS was defined as stenosis in extracranial internal carotid artery, extracranial vertebral artery, external carotid artery, common carotid artery, the proximal portion of subclavian artery, and aortic arch. Imaging examinations to determine the degree of vascular stenosis included Doppler ultrasonography (DUS), CT angiography (CTA), magnetic resonance angiography (MRA), and digital subtraction angiography (DSA). WMHs were defined as hyperintense in the subcortical white matter displayed on T2-weighted sequences (Figure 1) and could be divided into DWMHs and PVWMHs according to their anatomical locations. WMHs within 13 mm from ventricular surface were classified as DWMHs, and WMHs 13 mm or further from the ventricular surface were classified as PVWMHs (Kim et al., 2008). The measurement of WMHs involved quantification methods and semi-quantitative visual rating scales, such as Fazekas Scale (Fazekas et al., 1987), Scheltens Scale (Scheltens et al., 1993), and Age-Related White Matter Changes (ARWMC) Scale (Wahlund et al., 2001). Frontiers in Aging Neuroscience Literature search Figure 2 shows the screening and selection processes of the study. The systematic database search yielded 477, 452, and 578 records from the PubMed, Web of Science, and Embase databases, respectively. After excluding duplicates and reviewing titles and abstracts, 73 articles were considered as potential studies on the association between WMHs and ICAS or ECAS. Following a review of the full texts and a manual search, 21 studies were included in the meta- analyses. Of these, 14 studies were pooled to calculate the OR (Pu et al., 2009; Romero et al., 2009; Muñoz-Cortés et al., 2013; Schulz et al., 2013; Park et al., 2015; Duan et al., 2018; Ye et al., 2019; Del Brutto et al., 2020; Fang et al., 2020; Benli et al., 2021; Yin et al., 2021; Choi et al., 2022; Huang et al., 2022; Wang et al., 2022), and seven studies were pooled to calculate the SMD (Patankar et al., 2006; Chuang et al., 2011; Lee et al., 2011; Cheng et al., 2012; Scherr et al., 2012; Sahin et al., 2015; Ghaznawi et al., 2022). Quality assessment Two independent reviewers (ZZ and FF) assessed the risk of bias in the study design using the Newcastle-Ottawa scale (Stang, 2010). The checklist consists of three domains (selection, comparability, and exposure) with a total quality score ranging from 0 to 9 points. A score equal to or exceeding 7 indicated a high-quality study. Any disagreements were resolved by a third reviewer (WZ). Statistical analysis Summary measures, including OR and standardized mean difference (SMD), were applied to studies reporting WMHs in the form of categorical and continuous variables, respectively. A random- effects meta-analysis model (DerSimonian Laird method) was used to calculate the pooled OR and SMD (Dersimonian and Laird, 1986). When DWMHs and PVWMHs were reported instead of total WMHs in the included studies, DWMH data was selected for the meta-analysis. Search strategy The PubMed, Web of Science, and Embase databases were systematically searched from their inception to February 2023. The Frontiers in Aging Neuroscience 02 frontiersin.org Zhang et al. 10.3389/fnagi.2023.1240509 FIGURE 1 The form of white matter hyperintensities (WMHs), taking Fazekas Scale as an example: (A) mild, (B) moderate, and (C) severe. GURE 1 he form of white matter hyperintensities (WMHs), taking Fazekas Scale as an example: (A) mild, (B) moderate, and (C) severe. FIGURE 1 The form of white matter hyperintensities (WMHs), taking Fazekas Scale as an example: (A) mild, (B) moderate, and (C) severe. statistical tests were two-tailed, and statistical significance was set at p < 0.05. Statistical analyses were performed using the R version 4.2.1 software (R Foundation for Statistical Computing, Vienna, Austria). location), and statistical analyses (effect estimate and corresponding 95% confidence interval [CI], mean and standard deviation). Relevant missing data were requested by emailing the corresponding authors when possible. Otherwise, the study was not included in the subgroup analysis grouped by missing variables. Effect estimates (usually odds ratios [ORs]) were extracted from the most fully adjusted models. When adjusted effect estimates were not directly provided, we chose unadjusted effect estimates or calculated the ORs using 2 × 2 tables. Frontiers in Aging Neuroscience Study characteristics The characteristics of the included studies are summarized in Table 1. Nine studies were conducted in Europe and America, and 12 studies were conducted in East Asia. Five studies compared WMHs in the ipsilateral and contralateral hemispheres of stenotic vessels in patients with ICAS or ECAS, and 16 studies compared WMHs in patients with ICAS or ECAS and controls without ICAS and ECAS. Overall, 21 studies included 10,841 participants (median sample size, n = 212: minimum, n = 29; maximum, n = 2,420). The average age of the participants in the included studies ranged between 51.6 and 72.7 years, and the proportion of females ranged from 8.7 to Heterogeneity was assessed using the Cochran Q statistic and was quantified using the I2 metric. I2 > 50% was considered statistically significant heterogeneity (Higgins et al., 2003). The source of heterogeneity was investigated via meta-regression (if n ≥ 10) and subgroup analyses stratified by multiple different variables. Sensitivity analyses were conducted by excluding one study at a time to examine the robustness of the synthesized results. Publication bias (if n ≥ 10) was assessed using funnel plot and Egger’s test (Egger et al., 1997). All Frontiers in Aging Neuroscience 03 frontiersin.org Zhang et al. 10.3389/fnagi.2023.1240509 FIGURE 2 Flowchart presenting the selection of eligible articles. FIGURE 2 Flowchart presenting the selection of eligible articles. studies), but also patients with intracranial and extracranial atherosclerotic stenosis had increased odds of WMHs in the ipsilateral hemisphere of the stenotic vessel compared to the contralateral side (OR 2.66, 95% CI 1.65–4.31, I2 = 0%, three studies). Intracranial and extracranial atherosclerotic stenosis with a threshold of 50% stenosis was associated with WMHs (OR 1.94, 95% CI 1.31–2.88, I2 = 76%, 11 studies). According to the location of atherosclerotic stenosis, ECAS (OR 2.10, 95% CI 1.22–3.62, I2 = 71%, six studies) but not ICAS (OR 1.75, 95% CI 0.97–3.15, I2 = 84%, seven studies) was associated with WMHs. Both DWMHs (OR 2.97, 95% CI 2.20–4.02, I2 = 0%, five studies), and PVWMHs (OR 1.81, 95% CI 1.01–3.26, I2 = 65%, five studies) were associated with intracranial and extracranial atherosclerotic stenosis. WMHs assessed by ARWMC Scale were not associated with intracranial and extracranial atherosclerotic stenosis. The funnel plot (Supplementary Figure S2) for the studies investigating the association between intracranial and extracranial atherosclerotic stenosis and WMHs showed mild asymmetry. Association of ICAS and ECAS with WMHs based on OR We found intracranial and extracranial atherosclerotic stenosis to be associated with an increased risk of WMHs (OR 1.80, 95% CI 1.25–2.57, I2 = 75%, 14 studies; Figure 3). Sensitivity analyses further revealed a stable association between intracranial and extracranial atherosclerotic stenosis and WMHs (Supplementary Figure S1). Meta- regression analysis revealed that the source of heterogeneity originated from the WMHs rating method and location (both p < 0.05). Subgroup analyses showed that race, age, and sex did not influence this association (Figure 4). Not only patients with intracranial and extracranial atherosclerotic stenosis had increased odds of WMHs compared to controls without intracranial and extracranial atherosclerotic stenosis (OR 1.64, 95% CI 1.07–2.49, I2 = 77%, 11 Study characteristics However, Egger’s tests (p = 0.17) indicated no significant publication bias in this meta-analysis. 81.0%. ICAS were investigated in 11 studies, and ECAS was investigated in 16 studies. The internal carotid artery was the artery of greatest concern (n = 20). The criterion for atherosclerotic stenosis was set as greater than 50% in 13 studies, greater than 60% in one study, and greater than 70% in seven studies. Quantitative assessment was conducted in two studies, semi-quantitative assessment in 18 studies, and qualitative assessment in one study. The commonly used scales were Fazekas Scale (n = seven), Scheltens Scale (n = four), and ARWMC Scale (n = three). Total WMHs were investigated in 14 studies, and DWMHs and PVWMHs were investigated in nine studies. According to the quality assessment, approximately half of the studies were of high quality (Supplementary Table S1). Frontiers in Aging Neuroscience A (mea N 56.2 58.0 64.4 67.0 71.2 70.2 53.5 71.1 67.8 68.2 61.9 67.2 71.0 63.7 72.7 60.2 51.6 0 Association of ICAS and ECAS with WMHs based on SMD Our analyses found that intracranial and extracranial atherosclerotic stenosis to be associated with increased volume of WMHs (SMD 0.40, 95% CI 0.18–0.63, I2 = 63%, seven studies; Figure 5). The result remained stable in sensitivity analyses, excluding one study each time (Supplementary Figure S3). Frontiers in Aging Neuroscience 04 frontiersin.org Age mean, y) Female Location of atherosclerotic stenosis Severity of atherosclerotic stenosis Vascular image WMHs rating method WMHs location NOS NA NA Extracranial ≥70% DUS Scheltens Scale Deep and periventricular 5 6.2 ± 10.4 14.1% Intracranial and extracranial ≥50% MRA ARWMC Scale Total 5 8.0 ± 10.0 53.0% Extracranial ≥50% DUS Volumetric Total 8 4.4 ± 8.1 39.6% Extracranial ≥60% DUS or MRA Fazekas Scale Deep and periventricular 8 7.0 ± 12.4 43.3% Intracranial and extracranial ≥50% MRA Scheltens Scale Total, deep and periventricular 7 1.2 ± 6.6 44.2% Extracranial ≥70% DUS or MRA Scheltens Scale Total 8 0.2 ± 9.1 43.9% Extracranial ≥50% DUS Pantoni Scale Total 7 3.5 ± 7.0 28.0% Extracranial ≥50% DUS Fazekas Scale Total 5 1.1 ± 11.5 44.3% Intracranial and extracranial ≥50% DUS, CTA or MRA ARWMC Scale Total 6 7.8 ± 12.6 41.8% Intracranial and extracranial ≥50% MRA Fazekas Scale Deep and periventricular 6 8.2 ± 9.2 37.9% Extracranial ≥70% CTA, MRA or DSA Scheltens Scale Deep and periventricular 8 1.9 ± 11.2 32.5% Intracranial and extracranial ≥50% MRA Manolio Scale Total 5 7.2 ± 10.2 18.3% Extracranial ≥70% DUS King Scale Deep and periventricular 9 1.0 ± 8.4 57.1% Intracranial ≥50% MRA Pantoni Scale Total 6 3.7 ± 11.6 35.6% Intracranial ≥70% MRA ARWMC Scale Total 5 2.7 ± 9.3 50.7% Extracranial ≥50% DUS Fazekas Scale Deep and periventricular 8 0.2 ± 11.3 27.7% Intracranial ≥50% CTA, MRA or DSA Lesions ≥5 mm in any diameter Total 6 1.6 ± 9.2 13.5% Intracranial ≥50% MRA Fazekas Scale Deep and periventricular 8 7.0 ± 10.0 81.0% Extracranial ≥70% DUS Volumetric Total 6 5.7 ± 8.4 8.7% Intracranial and extracranial ≥50% DSA Fazekas Scale Deep and periventricular 8 9.0 ± 20.0 25.6% Intracranial ≥70% MRA Fazekas Scale Total 7 ultrasonography; DSA, digital subtraction angiography; MRA, magnetic resonance angiography; NA, not applicable; NOS, Newcastle-Ottawa scale; WMHs, white matter Zhang et al. 10.3389/fnagi.2023.1240509 FIGURE 3 Forest plot depicting intracranial and extracranial atherosclerotic stenosis and the risk of WMHs. FIGURE 3 Forest plot depicting intracranial and extracranial atherosclerotic stenosis and the risk of WMHs. Association of ICAS and ECAS with WMHs based on SMD Intracranial and extracranial atherosclerotic stenosis was associated with increased volume of DWMHs (SMD 0.54, 95% CI 0.22–0.86, I2 = 65%, four studies) and PVWMHs (SMD 0.41, 95% CI 0.25–0.57, I2 = 0%, four studies). No significant differences in this association were observed between the different semi-quantitative assessment scales. Subgroup analyses showed that race and sex did not influence this association (Figure 6). The results were similar between comparison the volume of WMHs in patients with intracranial and extracranial atherosclerotic stenosis and controls without intracranial and extracranial atherosclerotic stenosis (SMD 0.42, 95% CI 0.09–0.75, I2 = 75%, five studies) and comparison the volume of WMHs in the ipsilateral hemisphere of the stenotic vessel and contralateral side in patients with intracranial and extracranial atherosclerotic stenosis (SMD 0.40, 95% CI 0.16–0.64, I2 = 0%, two studies). According to the location of atherosclerotic stenosis, both ICAS (SMD 0.52, 95% CI 0.25–0.79, one study) and ECAS (SMD 0.34, 95% CI 0.07–0.62, I2 = 70%, seven studies) were associated with WMHs. Intracranial and extracranial atherosclerotic stenosis was associated with increased volume of DWMHs (SMD 0.54, 95% CI 0.22–0.86, I2 = 65%, four studies) and PVWMHs (SMD 0.41, 95% CI 0.25–0.57, I2 = 0%, four studies). No significant differences in this association were observed between the different semi-quantitative assessment scales. Association of ICAS and ECAS with WMHs based on SMD The correlation between ECAS and WMHs confirmed the findings of a previous meta-analysis by Ye et al. (2018). Consistent with previous meta-analysis, the extracranial artery of interest in the included studies was the carotid artery, particularly the extracranial internal carotid artery, which is the main blood vessel supplying the supratentorial white matter. Compared to the study by Ye et al., which only provided SMD values, our study also provided OR values. Our study was conducted to verify the correlation between ICAS and WMHs; however, the results were borderline. Several studies on ICAS and WMHs were excluded because of the lack of data available for meta-analysis, which reported contradictory results, including positive and null associations (Chutinet et al., 2012; Li et al., 2014; Nam et al., 2017; Zhai et al., 2018; Zhao et al., 2019; Ren et al., 2021; Feng et al., 2023). The inconsistencies in the results might be due to the differences in study subjects, sample size, ICAS assessment method, WMHs rating method and statistical analysis between the studies. Overall, several studies with a large sample size (n > 1,000) reported similar results, indicating that ICAS was associated with WMHs (Nam et al., 2017; Duan et al., 2018; Zhai et al., 2018; Choi et al., 2022). Another strength of our study is to explore the source of heterogeneity using meta-regression and subgroup analyses. The WMHs rating method was an important source of heterogeneity. Fazekas Scale was suitable for the analysis of WMHs in the form of categorical variable, and Scheltens Scale was suitable for the analysis of WMHs in the form of continuous variable. Subgroup analyses showed that race and sex did not influence this association (Figure 6). The results were similar between comparison the volume of WMHs in patients with intracranial and extracranial atherosclerotic stenosis and controls without intracranial and extracranial atherosclerotic stenosis (SMD 0.42, 95% CI 0.09–0.75, I2 = 75%, five studies) and comparison the volume of WMHs in the ipsilateral hemisphere of the stenotic vessel and contralateral side in patients with intracranial and extracranial atherosclerotic stenosis (SMD 0.40, 95% CI 0.16–0.64, I2 = 0%, two studies). According to the location of atherosclerotic stenosis, both ICAS (SMD 0.52, 95% CI 0.25–0.79, one study) and ECAS (SMD 0.34, 95% CI 0.07–0.62, I2 = 70%, seven studies) were associated with WMHs. Discussion This systematic review and meta-analysis showed that intracranial and extracranial atherosclerotic stenosis ≥50% was associated with WMHs in deep and periventricular regions. The association was validated in studies designed for both inter-and intra-individual comparisons. Our study showed that ECAS was significantly associated with WMHs, whereas ICAS was only marginally associated with WMHs. The pathophysiology linking intracranial and extracranial atherosclerotic stenosis to WMHs remains unclear. The shared risk factors (e.g., advanced age, hypertension, and diabetes) of intracranial and extracranial atherosclerotic stenosis and cerebral Frontiers in Aging Neuroscience 06 frontiersin.org Zhang et al. 10.3389/fnagi.2023.1240509 FIGURE 4 The association of intracranial and extracranial atherosclerotic stenosis with the risk of WMHs in subgroup analyses. FIGURE 4 The association of intracranial and extracranial atherosclerotic stenosis with the risk of WMHs in subgroup analyses. 07 07 Frontiers in Aging Neuroscience frontiersin.org Zhang et al. 10.3389/fnagi.2023.1240509 FIGURE 5 Forest plot depicting intracranial and extracranial atherosclerotic stenosis and the volume of WMHs. FIGURE 5 Forest plot depicting intracranial and extracranial atherosclerotic stenosis and the volume of WMHs. association between intracranial and extracranial atherosclerotic stenosis and WMHs at different sites. small vessel disease may play a mediating role. However, the intra-individual comparison design which eliminates the influence of vascular risk factors still showed more severe WMHs in the ipsilateral hemisphere of intracranial and extracranial atherosclerotic stenosis than that in the contralateral hemisphere. This indicates that other important mechanisms link intracranial and extracranial atherosclerotic stenosis with WMHs. Cerebral blood flow and cerebrovascular reactivity are impaired in patients with ICAS (Liu and Li, 2016; Yang et al., 2017) or ECAS (Hartkamp et al., 2018), especially in the ipsilateral hemisphere of the stenotic vessel. Endothelial dysfunction (including cerebral blood flow, cerebrovascular reactivity, intracranial pulsatility) is a pivotal pathogenesis of cerebral small vessel disease (Wardlaw et al., 2019). Three studies reported that WMHs were not directly related to arterial stenosis but were independently associated with the stenosis-induced cerebral hemodynamic changes in patients with ICAS (Fang et al., 2020; Ren et al., 2021; Feng et al., 2023). Intracranial and extracranial atherosclerotic stenosis does not necessarily cause decreased cerebral blood flow (Shakur et al., 2014) as it also depends on collateral blood flow, which may result in a null association between intracranial and extracranial atherosclerotic stenosis and WMHs in some previous studies. Recently, high intracranial vascular pulsatility has been linked to the formation of WMHs in individuals with asymptomatic ICAS (Zhao et al., 2022). Discussion Our meta-analysis was based on cross-sectional studies; therefore, causality between intracranial and extracranial atherosclerotic stenosis and WMHs could not be established. A 1 year follow-up study reported no association between 50–69% carotid artery stenosis and ipsilateral WMHs progression (Kwee et al., 2011). Another 7 years follow-up study also found that carotid artery stenosis ≥50% was not associated with WMHs progression, as assessed using the Fazekas Scale (Ihle-Hansen et al., 2021). The only positive findings were observed in a retrospective longitudinal study that reported that ICAS ≥20% was associated with WMHs progression assessed using the modified Rotterdam Progression scale after a 3 years interval (Zhong et al., 2022). A drawback of these studies is that cerebral perfusion was not considered. Reduced cerebral blood flow has been demonstrated to predict WMHs progression in longitudinal studies (Ten Dam et al., 2007; Bernbaum et al., 2015; Promjunyakul et al., 2018). Because cerebral blood flow can be normal in patients with carotid artery stenosis, it is necessary to conduct follow-up studies on specific patients with carotid artery stenosis who have a decrease in cerebral blood flow. l Our study has some limitations. First, gray literature such as meeting abstracts were not included in our meta-analysis. Second, a significant number of studies were excluded due to insufficient data availability, thereby impacting the comprehensiveness of our analysis on the association between ICAS and WMHs. Consequently, further high-quality studies are necessary to conduct a more robust meta- analysis. Third, substantial heterogeneity was observed in the meta- analyses. To address this, we analyzed the data using a random-effect model and explored the heterogeneity using meta-regression. Fourth, some included studies provided univariate analysis results on ICAS or ECAS and WMHs. Thus, the association may be biased due to confounding factors. Finally, a limitation of this study is that it only provides information that intracranial and extracranial atherosclerotic stenosis is associated with WMHs. Further studies are warranted to Our results showed that the association between intracranial and extracranial atherosclerotic stenosis and WMHs was stronger in the deep white matter than in the periventricular white matter. Neuropathological examination reveals that the main pathological changes are demyelination in DWMHs and the main changes are interstitial edema in PVWMHs (Haller et al., 2013). A cross- sectional study of arterial spin-labeling images showed that ischemia-hypoperfusion is the pathogenesis of DWMHs rather than PVWMHs (Cai et al., 2022). Frontiers in Aging Neuroscience References Dersimonian, R., and Laird, N. (1986). Meta-analysis in clinical trials. 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The Supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fnagi.2023.1240509/ full#supplementary-material Data availability statement 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. The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. Funding determine whether improving intracranial and extracranial atherosclerotic stenosis (e.g., stenting) can prevent WMHs progression. This work was supported by the Medical Science and Technology Project of Zhejiang Province of China (No. 2021KY797). Discussion This difference in underlying mechanisms explains the differences in the strength of the Frontiers in Aging Neuroscience 08 frontiersin.org Zhang et al. 10.3389/fnagi.2023.1240509 with the volume of WMHs in subgroup analyses. e volume of WMHs in subgroup analyses. FIGURE 6 The association of intracranial and extracranial atherosclerotic stenosis with the volume of WMHs in subgroup analyses. RE 6 association of intracranial and extracranial atherosclerotic steno FIGURE 6 The association of intracranial and extracranial atherosclerotic stenosis with the volume of WMHs in subgroup analyses. 09 Frontiers in Aging Neuroscience frontiersin.org Zhang et al. 10.3389/fnagi.2023.1240509 Conflict of interest In summary, despite the considerable heterogeneity and the cross- sectional nature of the included studies, this meta-analysis showed that intracranial and extracranial atherosclerotic stenosis was related to WMHs severity. 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https://openalex.org/W4311495857	https://aura.abdn.ac.uk/bitstream/2164/20012/1/Patel_etal_CEDR_A_Randomized_Clinical_VOR.pdf	English	null	A randomized clinical study to assess the performance of a marketed denture adhesive in a model of food infiltration in healthy, edentulous adults	Clinical and experimental dental research	2,022	cc-by	8,132	\| Revised: 14 November 2022 \| Accepted: 22 November 2022 Received: 11 May 2021 \| Revised: 14 November 2022 \| Accepted: 22 November 2022 DOI: 10.1002/cre2.703 O R I G I N A L A R T I C L E 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. © 2022 The Authors. Clinical and Experimental Dental Research published by John Wiley & Sons Ltd. Nisha Patel1 \| Roshan Varghese2 \| Gary R. Burnett2 \| Mounir Atassi2 \| Kimberly Milleman3 \| Jeffery Milleman3 1GSK, Brentford, Middlesex, UK 2Haleon, Weybridge, Surrey, UK 3Salus Research Inc., Fort Wayne, Indiana, USA A randomized clinical study to assess the performance of a marketed denture adhesive in a model of food infiltration in healthy, edentulous adults Nisha Patel1 \| Roshan Varghese2 \| Gary R. Burnett2 \| Mounir Atassi2 \| Kimberly Milleman3 \| Jeffery Milleman3 Clin Exp Dent Res. 2022;1–10. wileyonlinelibrary.com/journal/cre2 \| 1 1 \| INTRODUCTION results would be found with other foods that fragment into small particles during mastication. Despite the significant improvements dentures offer edentulous patients in terms of esthetics and function, some people remain unsatisfied with their dentures due to issues with prosthesis retention and stability (Alfadda, 2014). Denture adhesives can improve these factors by creating a seal along the inner, gum‐ facing denture borders. This can help improve denture fit, increase retentive hold (Hoke et al., 2017; Papadiochou et al., 2015), and reduce denture movement while chewing, which can, as a result, help with mastication and reduce infiltration of potentially irritating food particles under the denture (Axe et al., 2018; Gosavi et al., 2013; Kumar et al., 2015; Ozcan et al., 2005; Papadiochou et al., 2015; Varghese et al., 2019). In the efficacy study, a marketed denture adhesive (compared to no adhesive) was applied with a built‐in precision nozzle in a defined pattern to maximize the distribution of the adhesive across the denture, to minimize the excess application. Many denture adhesive tubes have wide nozzles with application instructions to apply bands or dabs of adhesive on the denture. The current nozzle is smaller and round, allowing more precise application of a thinner, targeted band of adhesive. In the previous study (Atassi et al., 2019), participants were questioned regarding how their dentures felt during the chewing exercise. Here, in both the pilot and follow‐on efficacy study, secondary objectives included a comparison of the number of participant‐reported denture dislodgements while chewing and evaluation of participant ratings of the amount of peanut particles under their dentures and how bothered they were by them if present. These studies were carried out in participants with fair‐ or well‐fitting dentures only as the denture adhesive under evaluation is not intended as a substitute for poorly fitting dentures. Published methodologies for evaluating the performance of a denture adhesive at reducing food particle infiltration are limited in number. One such study had participants rate the perceived presence of food particles under their dentures with or without a denture adhesive after eating foods including celery, steak, and taffy/toffee apples. A significant difference was found in favor of the adhesive compared to no adhesive (Tarbet et al., 1980). Another study design described a quantitative methodology based on having participants chew a prescribed amount of peanuts while wearing their dentures. 1 \| INTRODUCTION Peanut particles left under the intaglio surfaces were collected and evaluated and a significantly lower mass of peanuts was found when an adhesive was used compared to no adhesive use (Ahmad et al. 2009, 2010). ry.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License K E Y W O R D S K E Y W O R D S adhesives, dentures, denture retention, methods adhesives, dentures, denture retention, methods adhesives, dentures, denture retention, methods Abstract Objectives: An optimized food infiltration methodology was utilized to assess the objective and subjective efficacy of a marketed denture adhesive regarding denture dislodgment and infiltration and perception of food particles under maxillary and mandibular dentures. A pilot study helped optimize methodologies before the efficacy study. Correspondence Roshan Varghese, Haleon, St George's Ave, Weybridge, Surrey KT13 0DE, UK. Email: roshan.x.varghese@haleon.com Funding information Haleon. Correspondence Roshan Varghese, Haleon, St George's Ave, Weybridge, Surrey KT13 0DE, UK. Email: roshan.x.varghese@haleon.com Materials and Methods: Participants were healthy adults (n =48 for both studies) with fair‐ to well‐fitting and well‐made full maxillary and mandibular dentures. In the pilot, groups were a denture adhesive applied in a conventional dabbed‐on pattern, a denture adhesive applied in continuous strips, or no adhesive. In the efficacy study, groups were the Test denture adhesive (continuous strips pattern application) or no adhesive, employed in a crossover design. Food infiltration was investigated through measurement of peanut particle mass retrieved from under each denture (30–32 g chewed). No formal statistical testing was performed in the pilot. Statistical analysis in the efficacy study was performed using analysis of variance. Primary efficacy evaluation was combined peanut particle mass from both dentures. Secondary efficacy evaluations included peanut particle mass under separate dentures, participant‐reported denture dislodgements, and awareness/rates of how bother- some peanut particles under dentures were. Results: In the pilot, the median peanut particle mass was lower with either pattern application compared with no adhesive. In the efficacy study, peanut particle mass under combined dentures was lower with than without adhesive (geometric mean [product of values]: 5.56 vs. 29.13 mg) with a between‐group geometric mean ratio (adhesive over no adhesive) of 0.19 (95% confidence interval: 0.12, 0.30) favoring the Test adhesive (p < .0001). Similar Test adhesive beneficial outcomes in both studies included significantly fewer denture dislodgements and awareness and how bothersome peanut particles under dentures were. Treatments were generally well‐ tolerated. wileyonlinelibrary.com/journal/cre2 \| Clin Exp Dent Res. 2022;1–10. Conclusions: These findings, including reduced peanut particle infiltration, fewer denture dislodgments, and lower ratings of bothersomeness, corroborate those studies investigating the benefits of denture adhesive in preventing food infiltration. K E Y W O R D S adhesives, dentures, denture retention, methods 2 \| PATEL ET AL. 2 \| 2 2 \| PATEL ET AL. Abstract Conclusions: These findings, including reduced peanut particle infiltration, fewer denture dislodgments, and lower ratings of bothersomeness, corroborate those studies investigating the benefits of denture adhesive in preventing food infiltration. 2.1 \| Participants gum, mineral oil; Haleon, Weybridge, UK; USA marketplace) was applied using a preloaded syringe at a dose of 1.0 g to the maxillary denture and 0.6 g to the mandibular denture when participants were randomized to the denture adhesive treatment arm. The treatment groups were as follows: gum, mineral oil; Haleon, Weybridge, UK; USA marketplace) was applied using a preloaded syringe at a dose of 1.0 g to the maxillary denture and 0.6 g to the mandibular denture when participants were randomized to the denture adhesive treatment arm. The treatment groups were as follows: Unless otherwise stated, participant criteria were the same for both studies. Written, informed consent was provided by each participant before any study‐specific procedures. Eligible edentulous participants were aged between 18 and 85 years and in good physical and mental health. Participants had conventional, full, acrylic dentures in maxillary and mandibular arches, which they self‐reported getting food trapped under while eating. Dentures needed to be fair‐ to well‐ fitting, defined as a Kapur Index (Olshan Modification) retention and stability index sum score ≥6 (Supporting Information: 1) for maxillary and mandibular dentures combined, with no individual stability or retention score <1 (Kapur, 1967; Olshan et al., 1992). Dentures were judged by the lead examiner as being well‐made and defined as having adequate vertical dimension, freeway space, horizontal occlusal relationships, and border extension with acceptable porosity, tissue surfaces, polished surfaces, color, and thickness. Additionally, each denture was required to have a peanut particle migration rating at screening >0 (see below). Denture‐bearing tissue scores were assessed (Kapur, 1967) (Supporting Information: 2), but the scores were not used for screening or stratification purposes. • Conventional pattern application: Maxillary denture: One central dab applied to the middle of the palatal region, and one dab each applied to the left and right premolar regions; Mandibular denture: One dab each applied to the left and right premolar regions. • Continuous strips pattern application: Maxillary denture: Two short strips applied in the palatal region, and a single confluent strip applied to the middle of the alveolar ridge region, inside the buccolabial and posterior borders extending the entire arch; Mandibular denture: A confluent strip applied to the middle of the alveolar ridge region, extending the entire arch. • No adhesive. 2.1 \| Participants For the efficacy study, eligible participants were randomized (1:1) to one of two study groups in a crossover manner to first receive either: Participants were ineligible if they were pregnant; breastfeeding; were receiving medication or had a medical condition that might interfere with the study; had any clinically significant or relevant oral abnormality; had an allergy/intolerance to study materials/ingredi- ents or to peanuts/nuts or had received an investigational drug, cosmetic, or medical device within 30 days of screening. Participants could only have nonemergency dental/denture procedures per- formed during the study if allowed by the examiner. • Test adhesive (COREGA Máximo Sellado/Selamento containing sodium–calcium mixed partial salt of polymethylvinyl ether/maleic acid, carboxymethylcellulose, petrolatum, mineral oil; Haleon) applied as detailed above in the continuous strips pattern via the precision nozzle incorporated into the primary packaging for a total dose of 1.0 g (±0.1 g) for the mandibular denture, and 0.6 g (±0.1 g) for the maxillary denture. • No adhesive. 2 \| MATERIALS AND METHODS Both the pilot and efficacy studies were single‐center, controlled, randomized, crossover trials conducted at a US‐based clinical research facility in full compliance with the International Council for Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use, all applicable local good clinical practice regulations and participant privacy requirements, and the ethical principles outlined in the Declaration of Helsinki. The pilot study (ClinicalTrials.gov: NCT03345108) was an open‐label, three‐treatment, three‐period study. The efficacy study (Clinical- Trials.gov: NCT03709810) was a single‐blind (technician weigh- ing migrated peanut particles), two‐treatment, two‐period study. Final study protocols, informed consent forms, and other information that required preapproval were reviewed and approved by an independent institutional review board (US Inves- tigational Review Board, Inc.: U.S.IRB2017SRI/12 and U.S.IRB2018SRl/05). Anonymized individual participant data and study documents can be requested for further research from www.clinicalstudydatarequest.com. A previous study by some of the current authors (Atassi et al., 2019) utilizing the peanut chewing methodology (Ahmad et al., 2009, 2010) did not find a difference in food infiltration when a denture adhesive was compared to no adhesive. This was postulated to be due to noncontrolled amounts of denture adhesive being applied and to a nonoptimized method of peanut particle collection and handling following chewing. Here, a pilot method development study was carried out to ascertain if changes to the previous study methodology (Atassi et al., 2019) could be employed to better evaluate the mass of peanut particles that infiltrated under dentures when chewing, with an assessment of the pattern of denture adhesive application as the primary objective. This was followed by an efficacy study employing the refined methodology developed in the pilot with the primary objective of assessing the performance of a marketed denture adhesive compared to no adhesive. Peanuts were used in both studies as a representative, analyzable food; it is expected that similar PATEL ET AL. 3 2.4 \| Statistical analysis For the pilot study, as the purpose was not to compare treatment arms but to understand if the food occlusion model was applicable to different patterns of adhesive application, the study was not blinded and no formal statistical analysis was performed. Approximately 48 (maximum 50) participants were planned to be enrolled. Recruitment was targeted to achieve 50 ± 10% of participants with low (score of 6–14, clinically fair to good denture retention and stability) or high (score of 15–18, clinically very good denture retention and stability) KO Index sum scores. Maxillary and mandibular dentures, together with their corre- sponding gauze, were placed in separate beakers of warm deionized (DI) water and sonicated for at least 30 min to loosen adhering peanut particles. Any peanut pieces remaining on the prosthesis or gauze were then rinsed out into the beaker with fresh DI water. The gauze pieces were discarded and the dentures were cleaned and returned to the participant. The contents of the beaker (water, adhesive, saliva, and peanut particles) were heated to boiling with frequent stirring to facilitate the dissolution of any remaining adhesive and then strained through a standard #60 testing sieve. The remaining peanut particles were repeatedly washed with hot DI water to remove residual saliva and contaminants, air‐dried over- night, then oven dried for 5 h at 40°C. Once cooled, the mass (mg) of the dry peanut particles that had migrated under each denture was determined. In the efficacy study, it was planned to screen 53 participants and randomize 52 to ensure that 48 evaluable participants would complete the study. It was estimated that this sample size would provide 91% power to detect a multiplicative difference of 2.25 (0.3522 on the logarithmic scale) between the Test adhesive and no adhesive in the mean mass of peanut particles recovered from maxillary and mandibular dentures (based on the pilot study results). These were calculated using power analysis and sample size (PASS) software (NCSS Statistical Software, 2019) for a two‐period cross- over trial and using the Sw option for the variability estimate as 0.5075. An extensive simulation conducted in the PASS study supported the above calculations. 3 \| Efficacy measurements 2.3 with a tick‐box sheet and instructed to record each incident of denture dislodgement (of either denture) that occurred while chewing the peanuts. For both studies, the primary efficacy evaluation was a combined mass of peanuts under both dentures when denture adhesive was applied per the conventional pattern. For the efficacy study, secondary efficacy endpoints included the mass of peanut particles recovered separately under each denture. Additional secondary endpoints were the number of participant‐reported denture dis- lodgements and participant responses to the postpeanut chewing questionnaire. For the pilot study, exploratory efficacy evaluations included these plus a combined mass of peanuts under both dentures when the denture adhesive was applied per continuous strip pattern or when no adhesive was used and the mass of peanuts under combined dentures in participants with a low or high Kapur–Olshan (KO) score with each treatment. After peanut consumption, participants gently rinsed their mouths with water for 5 s in the pilot study. This rinsing step was increased to 10 s for the follow‐on efficacy study as it was discovered that 5 s was not sufficient to remove all residual peanut particles from around the mouth that had not been retained under the dentures. The examiner removed any peanut particles from the vestibular and sublingual areas (maxillary and mandibular), then carefully extracted the mandibular denture. They then removed and collected any peanut particles and residual adhesive, first from the lower edentulous ridge using gauze, then the maxillary denture. Respective gauzes for each denture were retained in separate, marked beakers. Any residual peanut particles present on the denture surface other than those on the intaglio surface were discarded. In each study, with approximately five participants per group, the denture intaglio surface was photographed such that a variety of denture sizes and fits were represented. These images were for visualization purposes only and were not analyzed. 2.2 \| Procedures They were provided For the pilot study, a marketed denture adhesive (Super Poligrip® Free Denture Adhesive Cream containing polymethylvinyl ether/ maleic acid; sodium–calcium mixed partial salt, petrolatum, cellulose PATEL ET AL. 4 \| 2.2 \| Procedures For both studies, the amount of adhesive applied to the denture was controlled by weighing the adhesive to be placed onto the denture. The amounts used (1.0 g to the maxillary and 0.6 g to the mandibular denture) were chosen to be representative of the amounts typically used in practice. At the screening visits for both studies, participants underwent an oral soft tissue (OST) examination and were assessed for denture retention and stability. Participants were also assessed for adequate food migration under the dentures whereby they chewed and consumed a 30–32 g serving of peanuts in individual portions of approximately eight peanut halves at a time. Participants then rinsed their mouths with water for up to 10 s to help remove any peanut particles that had not migrated under the dentures. Visual assess- ment of the amount and degree of peanut particle migration under the dentures was conducted and graded accordingly by the examiner using a 4‐point scale from 0 (no peanut migration) to 3 (extensive peanut migration on the crest of the denture). To ensure only participants who experienced peanut particle infiltration were included in the study, participants with a score of 1–3 were randomized to the treatment sequence in which they would receive each condition according to a predetermined schedule generated by an independent statistical agency. For both studies, during treatment visits, the use of any nicotine or nicotine‐containing products, or any other oral healthcare products other than the study products was prohibited while at the study site. On test days, participants underwent an OST examination, and dentures were cleaned and dried before adhesive application/ denture insertion; participants rinsed their mouth with water and expectorated before denture insertion. Adhesive/no adhesive was applied (per randomization schedule) and dentures were placed in the mouth; then the participant pressed the denture firmly in place and bit down for a few seconds to secure the hold. At 60 (±5) min after denture insertion, each participant was given a standardized portion of peanuts (30–32 g) to consume following a prescribed chewing and swallowing method whereby participants sequentially chewed approximately eight peanut halves for at least 20 s before swallowing. Participants were allowed small sips of water during peanut consumption to aid chewing and swallowing. 3.1 \| Denture‐related characteristics 3.1 Analysis of denture dislodgements (mITT population) was performed by nonparametric testing, applied to obtain inferential statistics, as assumptions underlying the ANOVA model could not be supported. Analysis was performed using the Wilcoxon signed‐rank test and the Hodges–Lehmann method. In the pilot study, current denture history was comparable for maxillary and mandibular dentures, with mean denture ages of 11.4 years (SD 12.31 years; range: 1.0–52.0 years) and 12.0 years (SD 12.25 years; range: 1.0–52.0 years), respectively. Fewer (25%) parti- cipants with high KO scores at screening were recruited than planned, with 75% having a low KO score; however, sufficient participant numbers were recruited in each subgroup (high or low KO scores) to allow for adequate analysis. For participants’ responses to questionnaires, descriptive statis- tics, mean score, and standard error (SE) were calculated for each question by the treatment group and frequency graphs were constructed. Analysis of participants’ responses to questions was performed using the methods described for the number of denture dislodgements. In the efficacy study, participants had an average duration of denture use of 23.0 years (SD 19.14 years; range 0.1–63.0 years) for maxillary dentures, and 22.8 years (SD 18.83 years; range 0.1–63.0 years) for mandibular dentures. Current denture history was comparable for maxillary and mandibular dentures, with mean denture ages of 12.3 years (SD 13.57 years; range: 0.1–54.0 years) and 12.2 years (SD 13.54 years; range: 0.1–54.0 years), respectively. No participants had dentures with “poor retention and stability” (<6 Kapur Index total score); 1 (2.1%) had “fair retention and stability” (6–9 total score); 24 (50.0%) had “good retention and stability” (10–14 total score), and 23 (47.9%) had “very good retention and stability” (>14). The mean combined score of 14.8 for the fit assessment (Kapur Index) was indicative of overall very good denture fit and stability at baseline. Denture adhesive was currently being used at screening on maxillary dentures by 17 participants (35.4%) and on mandibular dentures by 20 participants (41.7%). Denture‐ bearing tissue scores can be found in Supporting Information: Table 1; briefly, the majority of participants had resilient to firm tissue resiliency, and low to medium border tissue attachment. 2.4 \| Statistical analysis wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License In both studies, following food infiltration testing and denture removal on test days, participants completed a questionnaire on their experience during chewing and whether they had been aware of peanut particles under their dentures (Yes/No answer). Participants who answered “yes” completed additional questions relating to their subjective assessment of the amount of peanut particles under the dentures (1 = none to 10 = numerous), and how bothered they were by these (1 = not at all bothered to 10 = extremely bothered). Additionally, in the pilot study, participants rated how irritating the peanut particles were (1 = not at all irritating to 10 = extremely irritating). Primary and secondary efficacy analyses for both studies were based on a modified intent‐to‐treat (mITT) population, including all participants who were randomized, received at least one dose of study treatment, and had at least one assessment of efficacy. For the efficacy study, primary and secondary analysis of food infiltration was performed using a mixed model analysis of variance (ANOVA) with Log10 peanut mass as a response, study treatment and period as fixed explanatory effects, and participant as a random effect. The geometric mean (GM), geometric coefficient of variation (CV), and 95% confidence interval (CI) were presented. All statistical tests of the hypothesis were two‐sided and employed a level of significance of α = .05. The number of denture dislodgements according to the number of participants was presented as a frequency distribution. Safety was assessed by OST examination at screening and before/after each assessment at the treatment visits, through a collection of adverse events (AEs) and medical device incidents. Abnormalities and AEs reported after the participant's first use of treatment were considered treatment‐emergent adverse events (TEAEs). PATEL ET AL. \| 5 5 3.2 \| Peanut particle mass For the efficacy study, the combined GM mass of peanut particles recovered from both maxillary and mandibular dentures was lower when the Test adhesive was used (5.56 mg; CV 1.160 mg) compared to no adhesive (29.13 mg; CV 1.160 mg) (Table 1). The primary analysis of the between‐group GM ratio deduced as Test adhesive over no adhesive, demonstrated a statistically significant difference in favor of the Test adhesive (0.19 [95% CI: 0.12, 0.30]; p < .0001) (Table 1). The GM equates to 5.2 times lower amount of peanut particle infiltration with the GM ratio indicating that the mass of peanut particles under the denture when using theTest adhesive is 19% of that when using no adhesive. Similarly, the GM mass of peanut particles recovered from the maxillary (5.9 times lower) or mandibular (4.8 times lower) dentures separately was statistically significantly lower with theTest adhesive, with, respectively, 17% and 21% of the infiltration detected with no adhesive (Table 1). Figure 2 shows typical photographs of dentures after the chewing challenge with and without adhesive use, demonstrating both residual denture adhesive and peanut particles. It can be deduced from these photos that peanut particles have ingressed under the dentures, especially where no adhesive was applied, with particles particularly present in the areas of the dentures contacting the alveolar ridge in the case of maxillary dentures. Graphs and tables for the pilot study are held in the section of Supporting Information. For the pilot study (Supporting Informa- tion: Table 2 and Figure 1), peanut particle mass data were positively skewed with mean masses being considerably greater than medians; therefore, both median and mean masses are discussed. Without adhesive, a greater median (11.0 mg, range 0.1–700.0 mg) and mean (58.4 mg, SE 20.48 mg) mass of peanut particles migrated under the mandibular dentures than the maxillary ones (median 7.7 mg, range 0.1–213.4 mg; mean 20.1 mg, SE 5.79 mg). This was the same when the adhesive was applied by the conventional pattern (mandibular median 3.4 mg, range 0.1–27.1 mg; mean 5.8 mg, SE 0.94 mg; maxillary median 2.7 mg, range 0.1–89.2 mg; mean 6.0 mg, SE 1.95). 3.2 \| Peanut particle mass However, when applied by the continuous pattern, there was a slightly lower median and mean for the mandibular dentures (median 1.4 mg, range 0.0–20.0 mg; mean 3.8 mg, SE 0.72 mg) compared with the maxillary ones (median 1.8 mg, range 0.0–57.3 mg; mean 5.6 mg, SE 1.41 mg). When assessed by low or high KO scores, there were few differences for the conventional application or no adhesive; however, for the continuous pattern group, the median mass of peanuts under the combined dentures was higher in those with a low KO score (median 5.8, range 0.2–75.6 mg; mean 10.7, SE 2.35) compared with those with a high KO score (median 2.1, range 0.2–36.7 mg; mean 5.7, SE 2.94) (Supporting Information: Table 2) 3 \| RESULTS In the pilot study, the first participant was enrolled in November 2017; the final participant completed the study in December 2017. Of the 49 participants screened, 48 were randomized to a treatment sequence and were included in the mITT and safety populations (Figure 1). Most participants were females (70.8%) and were either of White/European heritage (91.7%%) or African American/African heritage (8.3%). The mean age was 65.0 years (standard deviation [SD] 12.60: range 31–81 years). In the pilot study, the first participant was enrolled in November 2017; the final participant completed the study in December 2017. Of the 49 participants screened, 48 were randomized to a treatment sequence and were included in the mITT and safety populations (Figure 1). Most participants were females (70.8%) and were either of White/European heritage (91.7%%) or African American/African heritage (8.3%). The mean age was 65.0 years (standard deviation [SD] 12.60: range 31–81 years). For the efficacy study, a total of 53 participants were screened for eligibility and 48 were randomized and completed treatment (Figure 3). Most participants were females (n = 33; 68.8%) and were either White/European heritage (n = 43; 89.6%) or African American/ African heritage (n = 4; 8.3%). The mean age was 65.1 years (SD 10.77: range 31–81 years). wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License FIGURE 1 Study flow schematic. mITT, modified intent‐to‐treat. FIGURE 1 Study flow schematic. mITT, modified intent‐to‐treat. 6 \| PATEL ET AL. 6 3.3 \| 7 TABLE 1 Efficacy (follow‐on) study the mass of peanut particles (mg), the geometric mean ratio between treatment (modified intent‐to‐treat cohort) Mass (mg) Adhesive No adhesive Combined Mean (SE) [min–max] 17.16 (7.475) [0.2–346.0] 78.55 (20.695) [1.3–860.0] GM (CV) [95% CI] 5.56 (1.160) [3.72, 8.31] 29.13 (1.160) [19.48, 43.56] GM ratio [95% CI], p value 0.19 [0.12, 0.30], p < .0001 Maxillary Mean (SE) [min–max] 4.85 (2.233) [0.0–106.8] 22.02 (6.835) [0.6–264.7] GM (CV) [95% CI] 1.39 (1.216) [0.92, 2.11] 8.17 (1.216) [5.39, 12.38] GM ratio [95% CI], p value 0.17 [0.11, 0.27], p < .0001 Mandibular Mean (SE) [min–max] 12.31 (7.141) [0.0–345.2] 56.53 (19.129) [0.0–855.5] GM (CV) [95% CI] 2.75 (1.795) [1.63, 4.64] 13.07 (1.795) [7.75, 22.02] GM ratio [95% CI], p value 0.21 [0.10, 0.43], p < .0001 Note: Analysis was performed using the ANOVA model with Log10 peanuts mass as response variable; study product and period as fixed explanatory effects and participant as a random effect; a GM ratio of <1 implies the mass of peanuts under the denture when using the Test adhesive is reduced to that percentage (e.g., 19% for Combined) of no adhesive. Abbreviations: ANOVA, analysis of variance; CI, confidence interval; CV, geometric coefficient of variation; GM, geometric mean, adhesive over no adhesive; SE, standard error. \| Note: Analysis was performed using the ANOVA model with Log10 peanuts mass as response variable; study product and period as fixed explanatory effects and participant as a random effect; a GM ratio of <1 implies the mass of peanuts under the denture when using the Test adhesive is reduced to that percentage (e.g., 19% for Combined) of no adhesive. Abbreviations: ANOVA, analysis of variance; CI, confidence interval; CV, geometric coefficient of variation; GM, geometric mean, adhesive over no adhesive; SE, standard error. 3.3 FIGURE 4 Efficacy (follow‐on) frequency graph of participant ratings of level of awareness of peanut particles under dentures by treatment (modified intent‐to‐treat population) of continuous application, and 70.8% (n = 34) of no adhesive participants reported being aware of peanuts under d FIGURE 3 Efficacy (follow‐on) study frequency graph of participant‐rated number of denture dislodgements by treatment (modified intent‐to‐treat population) FIGURE 4 Efficacy (follow‐on) frequency graph of participant ratings of level of awareness of peanut particles under dentures by treatment (modified intent‐to‐treat population) FIGURE 3 Efficacy (follow‐on) study frequency graph of participant‐rated number of denture dislodgements by treatment (modified intent‐to‐treat population) FIGURE 4 Efficacy (follow‐on) frequency graph of participant ratings of level of awareness of peanut particles under dentures by treatment (modified intent‐to‐treat population) FIGURE 3 Efficacy (follow‐on) study frequency graph of participant‐rated number of denture dislodgements by treatment (modified intent‐to‐treat population) of continuous application, and 70.8% (n = 34) of no adhesive participants reported being aware of peanuts under dentures. For the efficacy study, overall, 12 participants (25.0%) using the Test adhesive and 33 (68.8%) without adhesive reported being aware of peanut particles under their dentures and answered two further questions. When using the Test adhesive, participants reported a mean score of 0.8 (SE 0.29) on the “amount of peanut pieces under denture” rating scale (0 = none to 10 = lots); no adhesive results showed a mean score of 3.8 (SE 0.52). The median between‐group difference was in favor of the Test adhesive versus no adhesive and was statistically significant (−1.50; point estimate −2.50; 95% CI: −4.50, −1.50; p < .0001). (Figure 4) adhesive) following analysis using the Wilcoxon signed‐rank test was −3.00 and was significantly different in favor of the Test adhesive (p < .001). The point estimate (Hodges–Lehmann estimates of median differences) was −3.00 (95% CI −4.50, −2.00). TABLE 1 Efficacy (follow‐on) study the mass of peanut particles (mg), the geometric mean ratio between treatment (modified intent‐to‐treat cohort) 3.3 For the efficacy study, overall, 12 participants (25.0%) using the Test adhesive and 33 (68.8%) without adhesive reported being aware of peanut particles under their dentures and Mass (mg) Adhesive No adhesive Combined Mean (SE) [min–max] 17.16 (7.475) [0.2–346.0] 78.55 (20.695) [1.3–860.0] GM (CV) [95% CI] 5.56 (1.160) [3.72, 8.31] 29.13 (1.160) [19.48, 43.56] GM ratio [95% CI], p value 0.19 [0.12, 0.30], p < .0001 Maxillary Mean (SE) [min–max] 4.85 (2.233) [0.0–106.8] 22.02 (6.835) [0.6–264.7] GM (CV) [95% CI] 1.39 (1.216) [0.92, 2.11] 8.17 (1.216) [5.39, 12.38] GM ratio [95% CI], p value 0.17 [0.11, 0.27], p < .0001 Mandibular Mean (SE) [min–max] 12.31 (7.141) [0.0–345.2] 56.53 (19.129) [0.0–855.5] GM (CV) [95% CI] 2.75 (1.795) [1.63, 4.64] 13.07 (1.795) [7.75, 22.02] GM ratio [95% CI], p value 0.21 [0.10, 0.43], p < .0001 Note: Analysis was performed using the ANOVA model with Log10 peanuts mass as response variable; study product and period as fixed explanatory effects and participant as a random effect; a GM ratio of <1 implies the mass of peanuts under the denture when using the Test adhesive is reduced to that percentage (e.g., 19% for Combined) of no adhesive. Abbreviations: ANOVA, analysis of variance; CI, confidence interval; CV, geometric coefficient of variation; GM, geometric mean, adhesive over no adhesive; SE, standard error. requency graph of dgements by treatment FIGURE 4 Efficacy (follow‐on) frequency graph of participant ratings of level of awareness of peanut particles under dentures by treatment (modified intent‐to‐treat population) \| 7 20574347, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/cre2.703 by University Of Aberdeen, Wiley Online Library on [10/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA article Mass (mg) Adhesive No adhesive Combined Mean (SE) [min–max] 17.16 (7.475) [0.2–346.0] 78.55 (20.695) [1.3–860.0] GM (CV) [95% CI] 5.56 (1.160) [3.72, 8.31] 29.13 (1.160) [19.48, 43.56] GM ratio [95% CI], p value 0.19 [0.12, 0.30], p < .0001 Maxillary Mean (SE) [min–max] 4.85 (2.233) [0.0–106.8] 22.02 (6.835) [0.6–264.7] GM (CV) [95% CI] 1.39 (1.216) [0.92, 2.11] 8.17 (1.216) [5.39, 12.38] GM ratio [95% CI], p value 0.17 [0.11, 0.27], p < .0001 Mandibular Mean (SE) [min–max] 12.31 (7.141) [0.0–345.2] 56.53 (19.129) [0.0–855.5] GM (CV) [95% CI] 2.75 (1.795) [1.63, 4.64] 13.07 (1.795) [7.75, 22.02] GM ratio [95% CI], p value 0.21 [0.10, 0.43], p < .0001 \| 7 PATEL ET AL. 3.3 Pilot study data are presented in Supporting Information: Table 3. Briefly, the mean number of denture dislodgements was 0.35 (SE 0.113) for the conventional application group; 0.25 (SE 0.138) for the continuous application group, and 1.64 (SE 0.255) for the no adhesive group. For the efficacy study, the number of denture dislodgments experienced by participants while chewing is shown in Figure 3. During the chewing procedure, a greater number of participants reported no denture dislodgments when using the Test adhesive (n = 35; 72.9%) compared to without adhesive (n = 11; 22.9%). A mean of 0.6 (SE 0.24) dislodgements were reported when using the Test adhesive compared to a mean of 4.7 (SE 0.80) without adhesive. A lower median number of patient‐reported denture dislodgements was evident with the Test adhesive compared to no adhesive (0.0 vs. 4.0, respectively). The median difference (Test adhesive minus no ary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License FIGURE 2 Representative photographs from the pilot study of dentures before collection of peanut particles and residual denture adhesive (when applied) per (a) conventional application, (b) continuous application, and (c) no adhesive. Photographs adjusted for contrast and brightness. Black arrows, denture adhesive; red arrows, peanut particles. FIGURE 2 Representative photographs from the pilot study of dentures before collection of peanut particles and residual denture adhesive (when applied) per (a) conventional application, (b) continuous application, and (c) no adhesive. Photographs adjusted for contrast and brightness. Black arrows, denture adhesive; red arrows, peanut particles. oxon signed‐rank test was avor of the Test adhesive of continuous application, and 70.8% (n = 34) of no adhesive participants reported being aware of peanuts under dentures. 3.4 Pilot study data are presented in Supporting Information: Table 4. Briefly, 6.3% (n = 3) of conventional application, 6.3% (n = 3) 8 \| 8 \| PATEL ET AL. FIGURE 5 Efficacy (follow‐on) frequency graph of participant ratings of level of how bothersome peanut pieces felt under denture by treatment group (modified intent‐to‐treat population) 8 \| application had only a small effect; however, the continuous strip application resulted in a numerically lower median/mean mass of peanuts recovered from under the dentures compared to the conventional application. It should be noted that in practice, the amount of denture adhesive required to ensure adequate denture hold and to prevent food particle ingress may be more or less than the amount dosed in this study owing to differences in user's denture fit, size of a denture, quality of underlying soft tissue and user preference. Both studies allowed a period of 60 min between the fitting of the denture and the commencement of testing. This was to ensure the adhesive had fully hydrated before testing, allowing the denture hold to stabilize. application had only a small effect; however, the continuous strip application resulted in a numerically lower median/mean mass of peanuts recovered from under the dentures compared to the conventional application. It should be noted that in practice, the amount of denture adhesive required to ensure adequate denture hold and to prevent food particle ingress may be more or less than the amount dosed in this study owing to differences in user's denture fit, size of a denture, quality of underlying soft tissue and user preference. Both studies allowed a period of 60 min between the fitting of the denture and the commencement of testing. This was to ensure the adhesive had fully hydrated before testing, allowing the denture hold to stabilize. FIGURE 5 Efficacy (follow‐on) frequency graph of participant ratings of level of how bothersome peanut pieces felt under denture by treatment group (modified intent‐to‐treat population) In the follow‐on efficacy study, the primary objective was met with a greater than fivefold reduction in peanut infiltration under dentures when using theTest adhesive compared to no adhesive use. The denture adhesive was shown to be effective in reducing the infiltration of peanut particles under both maxillary and mandibular dentures when analyzed separately or together. A higher mass of peanut particles was observed under the mandibular dentures compared to the maxillary ones in both treatment groups. 3.5 \| Safety For the pilot study, there were no TEAEs, serious AEs, medical device incidents, or deaths reported during the study. For the efficacy study, overall, six TEAEs were reported, all classified as oral: hyperkeratosis (n = 1 for Test adhesive; n = 2 for no adhesive); tongue biting (n = 1 for Test adhesive); mouth injury (n = 1 for no adhesive), and a traumatic ulcer (n = 1 for no adhesive). There were no treatment‐related TEAEs, serious TEAEs, deaths during the study, or TEAEs leading to premature study treatment discontinuation. y.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Another finding of the efficacy study was that only 27.1% of those using the Test adhesive reported any denture dislodgments during the chewing procedure used, compared to 77.1% of those when not using adhesive. More participants in the no adhesive groups also reported multiple (>1) dislodgements. This is in agreement with previous studies examining the maximum incisal bite force a person can exert until denture dislodgement, which generally shows that a greater bite force is achievable when denture adhesive is used compared to no adhesive use (Axe et al., 2018; Jose et al., 2018; Varghese et al., 2019). Fewer denture dislodgements with the use of denture adhesive may be related to reduced movement of the denture while chewing (Tarbet et al., 1980) and supports the reduction in peanut particle infiltration observed in the primary analysis (Munoz et al., 2012). The higher infiltration of peanuts under mandibular dentures may be related to a greater number of mandibular denture dislodgements, as has been shown previously (Grasso et al., 2000); however, participants in this study were not asked to report which denture had been dislodged as it was considered too onerous for the participants to be able to accurately report dislodgements separately while chewing peanuts in the prescribed fashion. This could be examined in further studies. 3.4 This was also found in the pilot study and previous studies (Ahmad et al., 2010; Munoz et al., 2012) and is postulated to be due to poorer retention of the mandibular denture based on the activity and movements of the mandible, tongue, and facial musculature during mastication (Bohnenkamp & Garcia, 2007). The pattern of food distribution around the mouth while chewing and the effects of gravity may also result in more peanut particles gathered in the mandibular denture region during chewing. However, while the mass of peanuts under the mandibular dentures was greater, the GM ratio was similar for both dentures as well as the combined dentures, demonstrating that the adhesive works equally well with maxillary and mandibular dentures at preventing food infiltration. When using the Test adhesive, participants reported peanut pieces under their dentures to be less bothersome (mean score 0.7, SE 0.28) than during the no adhesive period (mean score 4.0, SE 0.57) (Figure 5). The median between‐group difference in favor of Test adhesive use versus no adhesive was also statistically significant (−2.00; point estimate −3.00; 95% CI: −4.50, −1.50; p < .0001). REFERENCES Ahmad, M., Ibrahim, D. F. A., Hazmi, N. H., Tarib, N. A., & Kamarudin, K. H. (2009). The effect of denture adhesive on food occlusion. Malaysian Dental Journal, 30, 146–147. Ahmad, M., Ibrahim, D. F. A., Hazmi, N. H., Tarib, N. A., & Kamarudin, K. H. (2010). An investigation into the effect of denture adhesives in limiting the food impaction. Malaysian Dental Journal, 31, 65–70. One potential consideration in these studies was that there was a large range of denture age and denture‐wearing experience. However, as this was a crossover study, there was no difference between the groups regarding this. As dentures were judged on their Kapur Index for retention and stability as well as the visual examination of fit and condition, the age of the denture should not affect the results. Test adhesive use was generally well tolerated with no treatment‐related TEAEs. Alfadda, S. A. (2014). The relationship between various parameters of complete denture quality and patients’ satisfaction. The Journal of the American Dental Association, 145(9), 941–948. https://doi.org/ 10.14219/jada.2013.48 Atassi, M., Milleman, K. R., Burnett, G. R., Sanyal, S., & Milleman, J. L. (2019). A randomized clinical study to evaluate the effect of denture adhesive application technique on food particle accumulation under dentures. Clinical and Experimental Dental Research, 5(4), 316–325. https://doi.org/10.1002/cre2.168 In conclusion, the use of denture adhesive, applied in a specified amount and pattern with a precision application nozzle, was found to result in a lower mass of peanut particles found under dentures and also improve participant perception of such. This could be related to both better denture adherence to the gum as well as to the finding of fewer denture dislodgements while chewing. These findings add further weight to the utility of denture adhesive in those with well‐ fitting and well‐made full maxillary or mandibular dentures. As complaints of food infiltration are common amongst denture wearers, reduction of such could lead to higher enjoyment of food and a greater range of food choices. Axe, A., Jain, R., Varghese, R., Mason, S., DiLauro, T. S., & Cronin, M. (2018). Randomized clinical study comparing three experimental denture adhesives and Fixodent® versus no adhesive. The Journal of Clinical Dentistry, 29, 69–74. Bohnenkamp, D. M., & Garcia, L. T. (2007). Phonetics and tongue position to improve mandibular denture retention: A clinical report. The Journal of Prosthetic Dentistry, 98(5), 344–347. https://doi.org/10. 1016/S0022-3913(07)60117-6 Brunello, D. L., & Mandikos, M. N. (1998). 4 \| DISCUSSION The accumulation of food under dentures can be a considerable issue for wearers, even when dentures have been fitted appropriately (Brunello & Mandikos, 1998; Gosavi et al., 2013). This was highlighted here in individuals with well‐fitting dentures (mostly rated as “good” or “very good” retention and stability scores), where around 70% of participants in both studies reported that they were aware of peanuts under their dentures when denture adhesive was not used. Both studies examined the efficacy of a measured amount of a marketed denture adhesive applied in thin strips with a precision nozzle compared to no adhesive with regard to food infiltration under dentures. This is in contrast to traditional application nozzles, where the denture adhesive is intended to be applied more coarsely as dabs on the denture intaglio surface (the pilot study “conventional application” pattern). In the pilot study, the pattern of adhesive PATEL ET AL. 9 Previous denture adhesive studies have also seen similar improvements in participant ratings for denture confidence, comfort, satisfaction, and movement when denture adhesive was used compared to no adhesive (Atassi et al., 2019; Munoz et al., 2012). In this study, participants were more specifically asked to rate the amount of peanut particles they perceived as being under their dentures. It was shown that significantly fewer participants were aware of peanuts under their dentures while using the Test adhesive (25.0%) compared to no adhesive (68.8%). Scores for the question posed to those who perceived peanut particles under their dentures —“how bothered were you by the peanut pieces under your denture?”—were also significantly lower with the Test adhesive. These results show that participants’ perceived efficacy of the marketed denture adhesive was significantly higher compared to no adhesive and reflects the objective measurements shown when peanut particles were weighed. manuscript, had final responsibility for the decision to submit, and approved the manuscript. manuscript, had final responsibility for the decision to submit, and approved the manuscript. REFERENCES Construction faults, age, gender, and relative medical health: Factors associated with complaints in complete denture patients. The Journal of Prosthetic Dentistry, 79(5), 545–554. https://doi.org/10.1016/s0022-3913(98)70176-3 Gosavi, S. S., Ghanchi, M., Malik, S. A., & Sanyal, P. (2013). A survey of complete denture patients experiencing difficulties with their prostheses. Journal of Contempory Dental Practice, 14(3), 524–527. https://doi.org/10.5005/jp-journals-10024-1355 ORCID Nisha Patel http://orcid.org/0000-0002-9336-342X Mounir Atassi https://orcid.org/0000-0002-4499-1614 Nisha Patel http://orcid.org/0000-0002-9336-342X Mounir Atassi https://orcid.org/0000-0002-4499-1614 Mounir Atassi https://orcid.org/0000-0002-4499-1614 CONFLICTS OF INTEREST R. V. and G. R. B. are employees of the study sponsor GSK Consumer Healthcare, now known as Haleon. M. A. was an employee of GSK Consumer Healthcare at the time of the research. K. M. and J. M. are Directors of Salus Research, which has received funding from Haleon. A previous food infiltration study by a number of the current authors also investigated the use of an adhesive compared to nonadhesive use but did not find a significant difference between them (Atassi et al., 2019). This was postulated to be due to several factors including controlling the amount of adhesive applied to each denture and having a more precise method of collection and processing of the peanut particles. As such, the methodology to apply denture adhesive and collect peanut particles was refined in the pilot study described here. These improvements in the methodology provided results in the efficacy study where statistically significant differences were shown, suggesting that this refined protocol is suitable for examining the effects of treatments on food infiltration under dentures. DATA AVAILABILITY STATEMENT Anonymized individual participant data and study documents can be requested for further research from www.clinicalstudydatarequest.com ACKNOWLEDGMENTS The authors wish to thank David Shaw and Audrey Souverain, previously of GSK Consumer Healthcare, for statistical analysis and advice. Editorial assistance with the preparation of manuscript drafts was provided by Eleanor Roberts of Beeline Science Communica- tions Ltd., funded by GSK Consumer Healthcare, now known as Haleon. AUTHOR CONTRIBUTIONS All authors contributed to the design and reporting of the study, and/ or were involved in its conduct. All authors had access to the final study report, made contributions to the development of the Grasso, J., Gay, T., Rendell, J., Baker, R., Knippenberg, S., Finkeldey, J., Zhou, X., & Winston, J. L. (2000). Effect of denture adhesive on PATEL ET AL. 10 retention of the mandibular and maxillary dentures during function. The Journal of Clinical Dentistry, 11(4), 98–103. 10 \| retention of the mandibular and maxillary dentures during function. The Journal of Clinical Dentistry, 11(4), 98–103. 10 \| Dentistry, 113(5), 391–397. https://doi.org/10.1016/j.prosdent. 2014.11.001 retention of the mandibular and maxillary dentures during function. The Journal of Clinical Dentistry, 11(4), 98–103. Hoke, P., Tiede, M., Grender, J., Klukowska, M., Peters, J., & Carr, G. (2017). Using electromagnetic articulography to measure denture micromove- ment during chewing with and without denture adhesive. Journal of Prosthodontics, 28, e252–e258. https://doi.org/10.1111/jopr.12679 Tarbet, W. J., Boone, M., & Schmidt, N. F. (1980). Effect of a denture adhesive on complete denture dislodgement during mastication. The Journal of Prosthetic Dentistry, 44(4), 374–378. https://doi.org/10. 1016/0022-3913(80)90092-x Jose, A., Varghese, R., Roohpour, N., Mason, S., Jain, R., & Gossweiler, A. (2018). A randomized proof‐of‐principle bite force study of two experimental denture adhesives and a commercially available adhesive. The International Journal of Prosthodontics, 31(4), 351–358. https://doi.org/10.11607/ijp.5628 Varghese, R., Burnett, G. R., Souverain, A., Patil, A., & Gossweiler, A. G. (2019). A randomised bite force study assessing two currently marketed denture adhesive products compared with no‐adhesive control. Clinical and Experimental Dental Research, 5(3), 276–283. https://doi.org/10.1002/cre2.182 Kapur, K. K. (1967). A clinical evaluation of denture adhesives. The Journal of Prosthetic Dentistry, 18(6), 550–558. Kumar, P. R., Shajahan, P. A., Mathew, J., Koruthu, A., Aravind, P., & Ahammed, M. F. (2015). Denture adhesives in prosthodontics: An overview. Journal of International Oral Health, 7(suppl 1), 93–95. SUPPORTING INFORMATION Additional supporting information can be found online in the Supporting Information section at the end of this article. Additional supporting information can be found online in the Munoz, C. A., Gendreau, L., Shanga, G., Magnuszewski, T., Fernandez, P., & Durocher, J. (2012). A clinical study to evaluate denture adhesive use in well‐fitting dentures. Journal of Prosthodontics, 21(2), 123–129. https://doi.org/10.1111/j.1532-849X.2011.00795.x How to cite this article: Patel, N., Varghese, R., Burnett, G. R., Atassi, M., Milleman, K., & Milleman, J. (2022). A randomized clinical study to assess the performance of a marketed denture adhesive in a model of food infiltration in healthy, edentulous adults. Clinical and Experimental Dental Research, 1–10. https://doi.org/10.1002/cre2.703 Olshan, A. M., Ross, N. M., Mankodi, S., & Melita, S. (1992). A modified Kapur scale for evaluating denture retention and stability: Method- ology Study. American Journal of Dentistry, 5(2), 88–90. Ozcan, M., Kulak, Y., de Baat, C., Arikan, A., & Ucankale, M. (2005). The effect of a new denture adhesive on bite force until denture dislodgement. Journal of Prosthodontics, 14(2), 122–126. https://doi. org/10.1111/j.1532-849X.2005.00020.x Papadiochou, S., Emmanouil, I., & Papadiochos, I. (2015). Denture adhesives: A systematic review. The Journal of Prosthetic
https://openalex.org/W2736093205	https://europepmc.org/articles/pmc5504655?pdf=render	English	null	Photoreceptor Mediated Plant Growth Responses: Implications for Photoreceptor Engineering toward Improved Performance in Crops	Frontiers in plant science	2,017	cc-by	13,235	Photoreceptor Mediated Plant Growth Responses: Implications for Photoreceptor Engineering toward Improved Performance in Crops Rising temperatures during growing seasons coupled with altered precipitation rates presents a challenging task of improving crop productivity for overcoming such altered weather patterns and cater to a growing population. Light is a critical environmental factor that exerts a powerful inﬂuence on plant growth and development ranging from seed germination to ﬂowering and fruiting. Higher plants utilize a suite of complex photoreceptor proteins to perceive surrounding red/far-red (phytochromes), blue/UV-A (cryptochromes, phototropins, ZTL/FKF1/LKP2), and UV-B light (UVR8). While genomic studies have also shown that light induces extensive reprogramming of gene expression patterns in plants, molecular genetic studies have shown that manipulation of one or more photoreceptors can result in modiﬁcation of agronomically beneﬁcial traits. Such information can assist researchers to engineer photoreceptors via genome editing technologies to alter expression or even sensitivity thresholds of native photoreceptors for targeting aspects of plant growth that can confer superior agronomic value to the engineered crops. Here we summarize the agronomically important plant growth processes inﬂuenced by photoreceptors in crop species, alongwith the functional interactions between different photoreceptors and phytohormones in regulating these responses. We also discuss the potential utility of synthetic biology approaches in photobiology for improving agronomically beneﬁcial traits of crop plants by engineering designer photoreceptors. Edited by: Gerrit T. S. Beemster, University of Antwerp, Belgium University of Antwerp, Belgium Reviewed by: Roslyn Gleadow, Monash University, Australia Ping Lan, Institute of Soil Science (CAS), China Correspondence: Eros V. Kharshiing eros.kharshiing@gmail.com Reviewed by: Roslyn Gleadow, Monash University, Australia Ping Lan, Institute of Soil Science (CAS) China Correspondence: Eros V. Kharshiing eros.kharshiing@gmail.com *Correspondence: Eros V. Kharshiing eros.kharshiing@gmail.com Specialty section: This article was submitted to Plant Physiology, a section of the journal Frontiers in Plant Science Specialty section: This article was submitted to Plant Physiology, a section of the journal Frontiers in Plant Science Received: 10 May 2017 Accepted: 20 June 2017 Published: 11 July 2017 Keywords: plant growth and development, light signaling, plant photoreceptors, photoreceptor engineering, crop productivity REVIEW published: 11 July 2017 doi: 10.3389/fpls.2017.01181 published: 11 July 2017 doi: 10.3389/fpls.2017.01181 INTRODUCTION Received: 10 May 2017 Accepted: 20 June 2017 Published: 11 July 2017 “Light exerts a powerful inﬂuence on most vegetable tissues, and there can be no doubt that it generally tends to check their growth” – Charles Darwin, 1880 (The Power of Movement in Plants) Edited by: Gerrit T. S. Beemster, University of Antwerp, Belgium Phytochromes Molecular genetics approaches in plant photobiology have led to the isolation and characterization of photoreceptor genes from various species, most of which play critical roles in determining plant survival and development. Phytochromes, which are principal receptors for light in the red/far-red region of the spectrum (600–750 nm), play an essential role in regulating seed germination and seedling establishment in agriculturally important crops such as rice and tomato (Chung and Paek, 2003; Appenroth et al., 2006; Eckstein et al., 2016), while in maize, phytochromes have been implicated to contribute to the transcription of genes essential for photosynthesis (Markelz et al., 2003). Under natural or ﬁeld conditions, establishment of the emergent seedling post germination is a vital developmental process that determines the ability of the plant to survive, grow, and reproduce. In photosynthesising organisms such as green plants, light is a crucial factor in determining the establishment of an emergent seedling. Maximizing photosynthesis during early development and following the formation of gaps during growth under dense canopies could therefore be critical for seedling survival. Under high plant densities, shade provided by neighboring vegetation triggers a series of developmental changes in growing plants involving several of the known photoreceptors (Ballaré and Pierik, 2017). The actions of phytochromes enables a plant to quantify shade around a seedling’s environment by detecting changes in the Red/Far-red ratios (R:FR) and trigger a series of developmental responses that is thought to provide the plant with a competitive advantage over its neighbors (Schmitt et al., 2003, Figure 1). Such responses include stimulation of elongation growth, coupled with reduced leaf development, increased apical dominance, and a reduction in branching (Franklin, 2008). While such developmental plasticity to diminished light, termed as shade avoidance responses (SAR), might increase survival percentage under limiting light conditions, it could result in compromised productivity of crop plants especially in modern intensive cropping methods with high planting densities (Weijschedé et al., 2006; Weinig et al., 2006). Plant growth and development involves complex signaling networks which are tightly regulated by genetic and environmental factors. These factors inﬂuence a plant’s ability to germinate, adapt, survive, and reproduce in natural conditions. In agriculture, the genetic constitution of diﬀerent crop species have been altered via various crop breeding programs for enhancing their survival and productivity under diﬀerent environmental conditions (Osakabe et al., 2011; Slade and Moehs, 2011; Suprasanna and Nakagawa, 2011; Ukai and Nakagawa, 2011). Citation: Light is a critical environmental factor that inﬂuences growth and development in plants. After seed germination in the soil, etiolated growth enables the germinated seedling to grow toward the soil surface in search of light. Upon exposure to light, the seedling undergoes photomorphogenesis characterized by de-etiolation, chlorophyll synthesis and development of chloroplast all of which enable the seedling to establish itself as an independent autotroph. Quality as well as quantity of incident light inﬂuence the developmental and growth of plants (Kami et al., 2010; Li J. et al., 2012). Research in plant photobiology has led to the discovery Mawphlang OIL and Kharshiing EV (2017) Photoreceptor Mediated Plant Growth Responses: Implications for Photoreceptor Engineering toward Improved Performance in Crops. Front. Plant Sci. 8:1181. doi: 10.3389/fpls.2017.01181 July 2017 \| Volume 8 \| Article 1181 1 Frontiers in Plant Science \| www.frontiersin.org Mawphlang and Kharshiing Plant Photoreceptors as Targets for Improved Productivity PHOTORECEPTORS AND PLANT PRODUCTIVITY Photoreceptors are widely distributed among land plants, with most of the commonly cultivated crop species reported to contain members of at least one major photoreceptor family (Kharshiing and Sinha, 2015). While most of our understanding on the roles of photoreceptors in plant development has been derived from research on Arabidopsis, there is an increasing interest in photoreceptor-mediated developmental responses in an agricultural environment (Ballaré et al., 1992; Sawers et al., 2005; Hudson, 2008; Wargent and Jordan, 2013; González et al., 2015) since several plant developmental responses involve action of one or more of these photoreceptors. PHOTORECEPTORS AND PLANT PRODUCTIVITY of several light-absorbing photoreceptor proteins that initiate plant responses to light. These include the red/far-red absorbing phytochromes (Chen and Chory, 2011), blue/UV-A absorbing cryptochromes and phototropins (Christie et al., 2015) and UV-B absorbing UVR8 (Rizzini et al., 2011). Most of these photoreceptors except for UVR8 contain more than one member, with each individual member being encoded by a diﬀerent gene and sharing a high degree of similarity among the individual members of the same family. Higher plants contain multiple phytochromes (phyA to phy E), three cryptochromes (cry1, cry2, and cry3), two phototropins (phot1 and phot2), and one UVR8 photoreceptor. Land plants also contain a family of blue-light absorbing proteins referred to as ZTL/FKF1/LKP2 (ZEITLUPE/FLAVIN-BINDING KELCH REPEAT F-BOX 1/LOV KELCH PROTEIN 2) having a combination of photoreceptor and F-box protein activities within the same protein (Ito et al., 2012). Photoreceptors are widely distributed among land plants, with most of the commonly cultivated crop species reported to contain members of at least one major photoreceptor family (Kharshiing and Sinha, 2015). While most of our understanding on the roles of photoreceptors in plant development has been derived from research on Arabidopsis, there is an increasing interest in photoreceptor-mediated developmental responses in an agricultural environment (Ballaré et al., 1992; Sawers et al., 2005; Hudson, 2008; Wargent and Jordan, 2013; González et al., 2015) since several plant developmental responses involve action of one or more of these photoreceptors. of several light-absorbing photoreceptor proteins that initiate plant responses to light. These include the red/far-red absorbing phytochromes (Chen and Chory, 2011), blue/UV-A absorbing cryptochromes and phototropins (Christie et al., 2015) and UV-B absorbing UVR8 (Rizzini et al., 2011). Most of these photoreceptors except for UVR8 contain more than one member, with each individual member being encoded by a diﬀerent gene and sharing a high degree of similarity among the individual members of the same family. Higher plants contain multiple phytochromes (phyA to phy E), three cryptochromes (cry1, cry2, and cry3), two phototropins (phot1 and phot2), and one UVR8 photoreceptor. Land plants also contain a family of blue-light absorbing proteins referred to as ZTL/FKF1/LKP2 (ZEITLUPE/FLAVIN-BINDING KELCH REPEAT F-BOX 1/LOV KELCH PROTEIN 2) having a combination of photoreceptor and F-box protein activities within the same protein (Ito et al., 2012). Frontiers in Plant Science \| www.frontiersin.org Phytochromes Regulating plant developmental processes such as biomass production, ﬂowering, fruiting and senescence are of particular interest to agricultural scientists for enhancing productivity. Additionally, processes that regulate disease and/or pest resistance (Jeong et al., 2010), starch metabolism (Zeeman et al., 2010), fruit size and quality (Causse et al., 2006), shelf-life of fruits and tubers (Dahmani-Mardas et al., 2010) and plant productivity under high planting index (Warnasooriya and Brutnell, 2014) can also inﬂuence productivity of agricultural crops. Various reports have shown that plant photoreceptor action can inﬂuence plant growth at diﬀerent developmental stages including but not limited to seed germination, plant architecture, ﬂowering, reproduction, biomass accumulation and senescence. Plant photoreceptors and/or their signaling components are therefore attractive targets for altering productivity and yield in crop plants for future food and non-food applications (Kharshiing and Sinha, 2015). Here we discuss various photoreceptor-controlled developmental processes that can aﬀect plant productivity and the practical implications for engineering such photoreceptors toward enhancing productivity of crop plants. In modern agricultural practices, enhanced yield in food crops such as maize have been achieved by use of varieties that perform optimally at high planting densities but require higher inputs of fertilizer (Warnasooriya and Brutnell, 2014). Modulating the responses of crop plants to vegetative shade for increasing harvestable biomass under high-density planting by manipulating light signaling networks that are fundamental to shade response therefore presents itself as an attractive alternative. Mutant lines harboring lesions in PHYA and PHYB, two of the more functionally important phytochrome genes in plants, show extreme SAR (Kendrick et al., 1997; Weller et al., 2001; Franklin, 2008; Casal, 2012). The overexpression of these genes could, however, compensate for reduced available light in densely grown crops resulting in increased yield (Robson et al., 1996; Boccalandro et al., 2003; Garg et al., 2006). July 2017 \| Volume 8 \| Article 1181 Frontiers in Plant Science \| www.frontiersin.org 2 Plant Photoreceptors as Targets for Improved Productivity Mawphlang and Kharshiing FIGURE 1 \| Induction of shade avoidance responses (SAR) in plants growing under shade. (A) Plants growing in full sunlight; (B) Low Red/Far-red ratios perceived by plants growing under shade resulting in SAR. R, red light; Fr, far-red light. (Artwork by Eros Kharshiing). In Arabidopsis, Devlin et al. (2003) have identiﬁed a number of shade responsive genes that are also regulated by phyA and phyB. Phytochromes Recently in Arabidopsis and tomato, phytochromes have been suggested to inﬂuence plant biomass along with carbon Yang et al., 2016). Altering responses of crop plants to light spectral quality by targetting genes such as phytochromes or their signaling components can therefore inﬂuence both resource partitioning and growth patterns in crop plants, FIGURE 1 \| Induction of shade avoidance responses (SAR) in plants growing under shade. (A) Plants growing in full sunlight; (B) Low Red/Far-red ratios perceived by plants growing under shade resulting in SAR. R, red light; Fr, far-red light. (Artwork by Eros Kharshiing). FIGURE 1 \| Induction of shade avoidance responses (SAR) in plants growing under shade. (A) Plants growing in full sunlight; (B) Low Red/Far-red ratios perceived by plants growing under shade resulting in SAR. R, red light; Fr, far-red light. (Artwork by Eros Kharshiing). In Arabidopsis, Devlin et al. (2003) have identiﬁed a number of shade responsive genes that are also regulated by phyA and phyB. Recently in Arabidopsis and tomato, phytochromes have been suggested to inﬂuence plant biomass along with carbon assimilation and starch metabolism (Kharshiing and Sinha, 2016; Yang et al., 2016). Altering responses of crop plants to light spectral quality by targetting genes such as phytochromes or their signaling components can therefore inﬂuence both resource partitioning and growth patterns in crop plants, which would presumably result in higher productivity even July 2017 \| Volume 8 \| Article 1181 Frontiers in Plant Science \| www.frontiersin.org 3 Plant Photoreceptors as Targets for Improved Productivity Mawphlang and Kharshiing under intensive cropping patterns (Warnasooriya and Brutnell, 2014). genetic manipulation could aﬀect agronomically important traits including starch metabolism which would consequently impact productivity in crop plants. p y p p Phototropins are the principal photoreceptors for blue-light phototropism in plants (Briggs and Christie, 2002; Christie et al., 2015). Besides phototropism, phototropins mediate other critical adaptive responses of plants to the surrounding light environment, which serve to enhance the photosynthetic status of the plants. The opening of stomatal pores which allow for exchange of water and carbon dioxide is redundantly controlled by phototropins (Boccalandro et al., 2012; Sharma S. et al., 2014). The movement of chloroplasts in response to diﬀerent light intensities is also regulated by the phototropins. Cryptochromes and Phototropins yp p Apart from phytochromes, two additional classes of photoreceptors have been reported in plants, which absorb blue/UV-A light, viz., cryptochromes and phototropins (Yu et al., 2010; Christie et al., 2015). In plants, the cryptochrome family photoreceptors participate in several plant process, ranging from photomorphogenesis to entrainment of the circadian clock (Lin, 2002; Botto et al., 2003; Millar, 2003; Giliberto et al., 2005). Developmental processes such as plant height and ﬂowering time which are agronomically important traits are also linked to cryptochrome activity (Mockler et al., 2003; Yu and Lin, 2005; Sharma P. et al., 2014). Since the isolation of the ﬁrst CRY gene from Arabidopsis, CRYs have been reported from most major crops investigated (Kharshiing and Sinha, 2015) having roles in seed germination, leaf senescence, stress responses and regulation of transcription (Lopez et al., 2012; Meng et al., 2013; Barrero et al., 2014; Facella et al., 2017). As Arabidopsis is limited as a model plant for examining the molecular networks inﬂuencing agronomically important traits (Kimura and Sinha, 2008) several workers have evaluated the role of photoreceptors in other crop models. In Brassica napus, an oilseed crop, the overexpression of CRY1 produced short-statured plants, which confer resistance to wind and water lodging (Sharma P. et al., 2014), providing such plants with an adaptive advantage in these conditions. Tomato plants overexpressing CRY2 also displayed short hypocotyl and internode length alongwith delayed ﬂowering (Giliberto et al., 2005). Interestingly, in Arabidopsis, cryptochromes have also been reported to promote growth in a shaded environment (Pedmale et al., 2016), suggesting new a molecular target for altering shade responses in plants. Tomato transgenic lines overexpressing CRY2 overproduced anthocyanins and chlorophyll in leaves alongwith enhanced ﬂavonoids and lycopene content in fruits. In soybean, transgenic lines with upregulated CRY2a expression show delayed senescence while senescence is accelerated in lines with downregulated CRY2a expression (Meng et al., 2013). Manipulation of CRY2 also profoundly aﬀects the molecular pathways related to biotic/abiotic stress, photorespiration, photosynthesis, as well as secondary metabolism pathways, such as biosynthesis of phenylpropanoids, phenolic, and ﬂavonoid/anthocyanin in tomato (Lopez et al., 2012). Tomato lines overexpressing CRY2 show major changes in the rhythmic oscillations of several genes involved in the entrainment of the endogenous clock (Facella et al., 2008) suggesting a role for the photoreceptors in the input to the tomato biological clock. Phytochromes When plants are exposed to low/weak light conditions, both phot1 and phot2 induce accumulation of the chloroplasts to the upper surface of the palisade mesophyll cells of leaves to maximize photosynthetic light capture. Under strong light conditions, phot2 mediates the rearrangement of chloroplasts parallel to the direction of the light source so as to minimize photo-damage (Briggs and Christie, 2002; Kasahara et al., 2004). In ﬁeld conditions, Arabidopsis mutants lacking phototropins have been shown to have reduced photosynthesis (Boccalandro et al., 2012), which could be partially due to the inability of the plants to maximally utilize photosynthetically active radiation (PAR) as the lack of phototropin-mediated adjustment of chloroplast position would inhibit optimal capture of PAR. It is also interesting to note that under laboratory conditions, phototropins promote growth in response to blue light under low light environments (Takemiya et al., 2005). While both phot1 and phot2 are involved in growth enhancement, phot1 is more sensitive than phot2 in promoting growth under low blue light, which can aﬀect plant development in natural conditions of low light. On the other hand, phototropin mutants in Chlamydomonas have been shown to display reduced ﬁtness under excessive light indicating a role for phototropins in photosynthetic regulation under high light conditions (Petroutsos et al., 2016). While similar ﬁndings have not yet been reported for higher plants, such reports underline the versatile roles that photoreceptors play in growth and development of plants. Cryptochromes and Phototropins Interestingly, analyses of circadian clock mutants in Arabidopsis, revealed that genes involved in carbohydrate metabolism including starch synthesis and degradation were aﬀected in all mutant lines tested (Graf et al., 2017). This suggests that inputs to the endogenous clock is vital to the proper regulation of critical physiological and developmental responses in plants. Since cryptochromes play critical roles in the entrainment of the endogenous clock in response to light (Millar, 2003), its Frontiers in Plant Science \| www.frontiersin.org UVR8 and ZTL/FKF1/LKP2 Conversely, promotion of photosynthetic eﬃciency of Arabidopsis seedlings exposed to elevated levels of UV-B occurs via UVR8 (Davey et al., 2012). Furthermore, lettuce seedlings exposed to UV- B radiation at early stages of development also resulted in increase in productivity and biomass (Tsormpatsidis et al., 2010; Wargent et al., 2011), which could be due to the photoprotective eﬀects of early UV-B exposure. UV-B radiation has also been reported to induce accumulation of secondary metabolites such as ﬂavonoids (Treutter, 2005), which is reportedly controlled by UVR8 (Jenkins, 2014). Such simultaneous activation could thus result in signaling networks where action of one photoreceptor can be aﬀected by the activity of other photoreceptors. While some developmental responses in plants are speciﬁcally triggered by activation of a single photoreceptor, there are several instances where signaling networks downstream of light perception of more than one photoreceptor of the same or diﬀerent family are co-ordinately activated to ensure proper growth and development. Such developmental processes include seedling germination (Bertram et al., 2004; Dechaine et al., 2009), photomorphogenesis (Neﬀ and Chory, 1998; Weller et al., 2001; Kami et al., 2010), plant and leaf architecture (Takemiya et al., 2005; Kozuka et al., 2013), ﬂowering (Más et al., 2000; Weller et al., 2001; Mockler et al., 2003; Endo et al., 2016), and fruit quality (Gupta et al., 2014; González et al., 2015) all of which eventually aﬀect plant productivity. The availability of photomorphogenic mutant and transgenic lines in Arabidopsis, rice, tomato, and other model species have provided valuable information into the functional redundancy and interactions of the signaling pathways of diﬀerent photoreceptors during plant development (Table 2). These interactions become especially relevant in the ﬁeld where plants are subject to uncontrolled environmental conditions of light, temperature, moisture, etc. A detailed understanding of such functional interactions between photoreceptors would further enable researchers to utilize the information for enhancing productivity of crop plants for food and non-food applications. Land plants also contain a family of LOV (Light Oxygen or Voltage) photoreceptors referred to as ZEITLUPE/FLAVIN- BINDING KELCH REPEAT, F-BOX1/LOV KELCH PROTEIN2 (ZTL/FKF1/LKP2) having both photoreceptor and F-box protein activities within the same protein (Ito et al., 2012). Interestingly genes coding for this family of photoreceptors are phyllogenetically divided into two groups in dicots and monocots (Boxall et al., 2005; Taylor et al., 2010), suggesting diﬀerent functions for these genes. UVR8 and ZTL/FKF1/LKP2 However, the high levels of structural conservedness of ZTL and FKF1 homologs among diﬀerent monocots and dicots may be suggestive of a certain level of functional conservedness of these genes across species (Taylor et al., 2010). Indeed, mutant analyses in Arabidopsis and other species have revealed that ZTL/FKF1/LPK2 regulate similar developmental pathways across diﬀerent species (Somers et al., 2004; Yon et al., 2016). In Arabidopsis ZTL mutants, presence of FKF1 and LKP2 compensates for the absence of ZTL in as far as in determining circadian rhythm (Baudry et al., 2010) suggesting some level of functional redundancy between these photoreceptors even though they are reported to have distinct roles in photoperiodic ﬂowering (Song et al., 2014). In crop plants, such as soybean GmZTL3 a homolog of Arabidopsis ZTL has also been suggested to function as a photoreceptor controlling timing of ﬂowering (Xue et al., 2012), a critical developmental response for crop plants. For a comprehensive review on structure and functions of ZTL/FKF1/LKP2 proteins, readers can refer the works of Suetsugu and Wada (2013) and Zoltowski and Imaizumi (2014). Table 1 provides a summary of the eﬀects of photoreceptors on agronomic traits in crops. Seed Germination Light is an important signal that functions as a developmental switch in germination and photomorphogenesis. Seedling emergence from soil and subsequent photomorphogenic development involve a vast array of photoreceptors which enable the establishment of emergent seedlings. Activation of these photoreceptors by light results in a range of signaling events which co-ordinate plant growth and development that direct seedling emergence from soil and establish them as autotrophs. There is growing evidence that most of these signaling cascades involve interaction of light and phytohormones signaling pathways (Wang et al., 2013). Since the initial proposal of the Cholodny–Went theory regarding the asymmetric distribution of auxin during shoot phototropism (Koepﬂi et al., 1938), much information has emerged on the links between light perception and hormonal regulation in plant growth responses. In plants seed germination is inhibited by abscisic acid (ABA) while gibberellic acid (GA) induces germination (Jacobsen et al., 2002; Seo et al., 2006). It is now known that light induces seed germination via the interaction of phytochromes and its partner PIF1 (PHYTOCHROME INTERACTING FACTOR 1) also known as PIF3-LIKE 5 Light is an important signal that functions as a developmental switch in germination and photomorphogenesis. Seedling emergence from soil and subsequent photomorphogenic development involve a vast array of photoreceptors which enable the establishment of emergent seedlings. Activation of these photoreceptors by light results in a range of signaling events which co-ordinate plant growth and development that direct seedling emergence from soil and establish them as autotrophs. UVR8 and ZTL/FKF1/LKP2 Ultraviolet-B radiation (UV-B) is a key component of the radiation environment that is utilized by plants as a signal for UV acclimation and survival in sunlight (Jansen, 2002; Jenkins, 2014). The discovery of the UV-B responsive UVR8 locus in plants (Kliebenstein et al., 2002; Rizzini et al., 2011) and resultant works (reviewed in Tilbrook et al., 2013; Jenkins, 2014) has tremendously progressed our understanding of how this photoreceptor responds to UV-B at the molecular and biochemical levels. While our knowledge of UVR8 function in vivo is still at a very nascent stage, its manipulation might have potential applications in crop improvement. The vegetative phase of plant growth is characterized by key developmental processes such as cell division and elongation, directional growth and branching. Low ﬂuence UV-B regulates several of these developmental responses in plants right from seedling to adult stages (Suesslin and Frohnmeyer, 2003; Shinkle et al., 2004; July 2017 \| Volume 8 \| Article 1181 4 Plant Photoreceptors as Targets for Improved Productivity Mawphlang and Kharshiing Wargent et al., 2009). In adult plants, morphogenic responses to UV-B include alterations in leaf characteristics such as leaf area, leaf thickness, leaf mass, and stomatal index (Wargent et al., 2009; Robson et al., 2015), Plants exposed to UV-B radiation also show decrease in chlorophyll content and in chla/b ratio (Lidon and Ramalho, 2011) alongwith decrease in photosynthetic eﬃciency (Lidon et al., 2012). Conversely, promotion of photosynthetic eﬃciency of Arabidopsis seedlings exposed to elevated levels of UV-B occurs via UVR8 (Davey et al., 2012). Furthermore, lettuce seedlings exposed to UV- B radiation at early stages of development also resulted in increase in productivity and biomass (Tsormpatsidis et al., 2010; Wargent et al., 2011), which could be due to the photoprotective eﬀects of early UV-B exposure. UV-B radiation has also been reported to induce accumulation of secondary metabolites such as ﬂavonoids (Treutter, 2005), which is reportedly controlled by UVR8 (Jenkins, 2014). Wargent et al., 2009). In adult plants, morphogenic responses to UV-B include alterations in leaf characteristics such as leaf area, leaf thickness, leaf mass, and stomatal index (Wargent et al., 2009; Robson et al., 2015), Plants exposed to UV-B radiation also show decrease in chlorophyll content and in chla/b ratio (Lidon and Ramalho, 2011) alongwith decrease in photosynthetic eﬃciency (Lidon et al., 2012). INTER AND INTRACLASS INTERACTIONS OF PHOTORECEPTORS AFFECTING PLANT DEVELOPMENT Since natural light is composed of diﬀerent wavelengths, higher plant growing under natural conditions will invariably have activation of more than one photoreceptor at the same time. July 2017 \| Volume 8 \| Article 1181 Frontiers in Plant Science \| www.frontiersin.org 5 Plant Photoreceptors as Targets for Improved Productivity Mawphlang and Kharshiing TABLE 1 \| Agronomic traits in few crops affected by mutations in photoreceptor genes or by altered expression of photoreceptor genes. Crop Locus affected Traits Reference Oryza sativa PHYB Single base insertion Increased drought tolerance, alleviation of chilling induced photoinhibition Liu et al., 2012; Yang et al., 2013 PHYA PHYB PHYC Insertion of retrotransposon Tos17 Single base insertion Insertion of retrotransposon Tos17 Age related resistance to blast fungus Xie et al., 2011 PHYA Overexpression of At PHYA Reduced plant height and increased grain yield Garg et al., 2006 CRY2 Silencing of CRY2 Delayed ﬂowering under long-day and short-day Hirose et al., 2006 Zea mays PHYB2 Deletion Acceleration of ﬂowering under long-day and short-day Sheehan et al., 2007 Triticum sp. PHYC Single base substitution; nonsense mutation Acceleration of ﬂowering under long-day Chen et al., 2014 Hordeum vulgare CRY1a/b Downregulated gene expression Increased germination percentages Barrero et al., 2014 Solanum tuberosum PHYA Increased gene expression Increased tuber formation Yanovsky et al., 2003 PHYB Enhanced gene expression Increased tuber yield at high planting densities Boccalandro et al., 2003 Solanum lycopersicum CRY2 Increased gene expression Enhanced pigmentation and lycopene content of fruits Giliberto et al., 2005 PHYA PHYB1 PHYB2 Single base transition Nonsense mutation Three base substitutions, nonsense mutation Accelerated transition of fruit ripening stages Gupta et al., 2014 Glycine max PHYA3 40 bp deletion Early ﬂowering and pod maturation Watanabe et al., 2009 CRY1a Enhanced gene expression Early ﬂowering Zhang et al., 2008 Brassica sp. CRY1 Increased gene expression Reduced plant stature Sharma P. et al., 2014 Pisum sativum PHYA Single base substitution Early photoperiod-independent ﬂowering Weller et al., 2004 TABLE 2 \| Examples of plant developmental processes involving functional interaction of two or more photoreceptors. INTER AND INTRACLASS INTERACTIONS OF PHOTORECEPTORS AFFECTING PLANT DEVELOPMENT Plant function/response Interacting photoreceptors Reference Seed germination phyA, phyB2 phyA, phyB Bertram et al., 2004; Lee and Lopez-Molina, 2012 Photomorphogenesis phyB, cry2 phyA, phyB phyA,phyB,cry1 Más et al., 2000; Weller et al., 2001; Neff and Chory, 1998 Plant architecture phot1, phot2 Takemiya et al., 2005 Leaf architecture phyB, phot1, phot2 Kozuka et al., 2013 Stomatal development phyA, phyB, cry1, cry2 Kang et al., 2009 Flowering/Timing of ﬂowering phyB, cry2 phyA, phyB phyA, cry1, cry2 Más et al., 2000; Weller et al., 2001; Mockler et al., 2003 Fruiting/Fruit quality phyA, phyB1, phyB2 phys, crys Gupta et al., 2014; González et al., 2015 (PIL5), which in turn regulates both ABA and GA signaling through the same downstream regulators (Bae and Choi, 2008; Seo et al., 2008; Oh et al., 2009; de Wit et al., 2016). It seems that phytochrome-mediated degradation of PIF1 is therefore a central mechanism by which light induces seed germination by altering ABA and GA metabolism in seeds. PIFs are also implicated to play a role during seedling transition from a skotomorphogenic to a photomorphogenic mode of development in light by regulating GA levels (Oh et al., 2007). In Arabidopsis, many regulators of hormone signaling including auxin and cytokinin (Cluis et al., 2004), ABA (Chen et al., 2008), ethylene (Yu et al., 2013), jasmonic acid (JA) (Prasad et al., 2012), GA (Alabadí et al., 2008), and brassinosteroid (Li and He, 2016) are reported to share a common signaling node involving the basic leucine zipper (bZIP) transcription factor ELONGATED HYPOCOTYL 5 (HY5), whose cellular accumulation involves light signaling transduction through phytochromes and cryptochromes (Osterlund et al., 2000; Wang et al., 2001). TABLE 2 \| Examples of plant developmental processes involving functional interaction of two or more photoreceptors. plants are categorized into those that ﬂower when length of daylight exceeds a particular length (called critical length) or long-day plants, those that ﬂower when length of daylight is below the critical length or short-day plants and those that ﬂower independent of the critical length or day-neutral plants. phyA, phyB, and cry2 were the ﬁrst photoreceptors Flowering Time Similarly in Arabidopsis anther dehiscence is regulated by auxin synthesis (Cecchetti et al., 2008) and also involves the auxin transporters ABCB1 and ABCB19 (Cecchetti et al., 2015). The involvement of ABCB19 as a substrate target for phot1 during shoot phototropism in Arabidopsis (Christie et al., 2011) is further illustration of the overlap of photoreceptor and hormone signaling events in plants. Furthermore, while phytochromes are reported to regulate fruit development and ripening in tomato (Gupta et al., 2014), the overproduction of nitric oxide, a secondary messenger in signaling pathways for several plant hormones, in the shr mutant of tomato suppresses fruit growth and ripening (Negi et al., 2010; Bodanapu et al., 2016) implying a possible cross-talk of the two signaling pathways. Targeted Mutagenesis for Improving Plant Traits The myriad developmental processes regulated by light suggest that the manipulation of genes involved in light signaling pathways could be a viable tool for crop improvement (Kharshiing and Sinha, 2015). Rapid advancement in genomics is generating new tools for editing genomes which can be utilized for modiﬁcation of molecular components regulating development in plants (Belhaj et al., 2013; Song et al., 2016). Functional genomics studies, large-scale sequencing and eco-tilling strategies are rapidly identifying polymorphisms between cultivars and landraces resulting in large datasets of molecular diversity among crop plants. Such data is likely to result in the identiﬁcation of natural alleles of photomorphogenic genes, as well as those that have arisen as a consequence of crop breeding. Genome projects of crops such as rice, maize, tomato, soybean, and others are increasingly generating information which can be used to eﬀect targeted dwarﬁng, alter SAR, enhance yield and regulate fruiting and ripening in fruit crops by modifying photoreceptors and/or their signaling pathways. Unlike genetically modiﬁed transgenics, the upregulation or downregulation of genes in an organism does not necessarily require the introduction of foreign genes into the organism. Several reports on mutation studies also provide evidence that traits in an organism can be modiﬁed without the introduction of foreign genes into the system. Many agronomically valuable phenotypes and naturally occurring variants of crop plants have been caused by point, or only a few, mutations. During the last 70 years, more than 3200 crop varieties derived directly as mutant or their progenies have been released worldwide (Pathirana, 2011; Manova and Gruszka, 2015). Many of these mutant-derived varieties have signiﬁcant economic value such as shorter growth cycle (Ahloowalia et al., 2004), semi-dwarf habit, high harvest index and drought tolerance (D’Souza et al., 2009) and disease resistance (D’Souza et al., 2009; Pathirana, 2011). Flowering Time Timing of ﬂowering is an essential developmental process in ﬂowering plants that is important for reproduction. In plants, the timing of ﬂowering is determined by the length of the daylight period during a day (or photoperiod). Broadly, ﬂowering July 2017 \| Volume 8 \| Article 1181 Frontiers in Plant Science \| www.frontiersin.org 6 Plant Photoreceptors as Targets for Improved Productivity Mawphlang and Kharshiing reported to be involved in regulating the timing of ﬂowering in Arabidopsis as well as in crop plants. In rice, mutations in PHYB or PHYC cause moderate alteration in ﬂowering while lines carrying mutations in PHYA coupled with mutations in either PHYB or PHYC show very early ﬂowering (Takano et al., 2005). Under long-day conditions, both PHYC and PHYB-null mutants of wheat also exhibit severe delay in ﬂowering (Chen et al., 2014; Pearce et al., 2016). A study of the transcriptomes of PHYB-null and PHYC-null mutants indicate that phyB also plays a prominent role in regulating GA, BR, auxin, ABA, and ethylene biosynthesis (Pearce et al., 2016), all of which have been implicated in the photoperiodic control of ﬂowering (Galvão and Schmid, 2014). Of these, the regulation of endogenous GA levels may be important for light- induced ﬂowering. Downregulation of the GA biosynthesis gene GA20OX showed delayed ﬂowering in FR enriched environment suggesting a role for this gene in phytochrome-dependent ﬂowering (Hisamatsu and King, 2008). While the association of light and auxin signaling is well established, the tomato pct1-2 mutant having enhanced polar transport of auxin shows delayed phototropism (Kharshiing et al., 2010a,b) as well as increased number of ﬂowers which bloom at the same time as compared to wild type which have lesser ﬂowers and which bloom sequentially (Al-Hammadi et al., 2003). However, the ﬂowers of the pct1-2 mutant are male sterile because the anthers lack dehiscence. Similarly in Arabidopsis anther dehiscence is regulated by auxin synthesis (Cecchetti et al., 2008) and also involves the auxin transporters ABCB1 and ABCB19 (Cecchetti et al., 2015). The involvement of ABCB19 as a substrate target for phot1 during shoot phototropism in Arabidopsis (Christie et al., 2011) is further illustration of the overlap of photoreceptor and hormone signaling events in plants. Flowering Time Furthermore, while phytochromes are reported to regulate fruit development and ripening in tomato (Gupta et al., 2014), the overproduction of nitric oxide, a secondary messenger in signaling pathways for several plant hormones, in the shr mutant of tomato suppresses fruit growth and ripening (Negi et al., 2010; Bodanapu et al., 2016) implying a possible cross-talk of the two signaling pathways. to Ballaré (2014), Lucas and Prat (2014); de Wit et al. (2016) and references mentioned therein. The reports mentioned here are few examples of how light and hormone metabolism seem to aﬀect related aspects of plant development that could eventually impact productivity (Figure 2). As the mechanisms of signal integration of light and hormone signaling pathways are starting to become clearer, we will be in a better position to understand how light signaling interacts with hormone signaling to regulate traits of agronomic interest in plants. reported to be involved in regulating the timing of ﬂowering in Arabidopsis as well as in crop plants. In rice, mutations in PHYB or PHYC cause moderate alteration in ﬂowering while lines carrying mutations in PHYA coupled with mutations in either PHYB or PHYC show very early ﬂowering (Takano et al., 2005). Under long-day conditions, both PHYC and PHYB-null mutants of wheat also exhibit severe delay in ﬂowering (Chen et al., 2014; Pearce et al., 2016). A study of the transcriptomes of PHYB-null and PHYC-null mutants indicate that phyB also plays a prominent role in regulating GA, BR, auxin, ABA, and ethylene biosynthesis (Pearce et al., 2016), all of which have been implicated in the photoperiodic control of ﬂowering (Galvão and Schmid, 2014). Of these, the regulation of endogenous GA levels may be important for light- induced ﬂowering. Downregulation of the GA biosynthesis gene GA20OX showed delayed ﬂowering in FR enriched environment suggesting a role for this gene in phytochrome-dependent ﬂowering (Hisamatsu and King, 2008). While the association of light and auxin signaling is well established, the tomato pct1-2 mutant having enhanced polar transport of auxin shows delayed phototropism (Kharshiing et al., 2010a,b) as well as increased number of ﬂowers which bloom at the same time as compared to wild type which have lesser ﬂowers and which bloom sequentially (Al-Hammadi et al., 2003). However, the ﬂowers of the pct1-2 mutant are male sterile because the anthers lack dehiscence. Frontiers in Plant Science \| www.frontiersin.org Plant Defense Response In plants, while the eﬀect of light on photomorphogenesis and photosynthesis has been known for a long time, it is now becoming increasingly evident that light signaling is also an integral component in determining the outcome of plant-pathogen interactions (Griebel and Zeier, 2008; Ballaré et al., 2012; Demkura and Ballaré, 2012; Erb et al., 2012). Several studies have shown that phytochromes and UVR8 inﬂuence plant defense responses (reviewed in Ballaré, 2014; Mazza and Ballaré, 2015; Gommers et al., 2017) while cryptochrome 2 and phototropin 2 have been reported to mediate resistance protein-mediated plant defense against viral but not bacterial pathogens (Jeong et al., 2010). These photoreceptors have been shown to inﬂuence plant defense responses by regulating hormone signaling pathways such as that of salicylic acid (SA) and JA (Xie et al., 2011; Moreno and Ballaré, 2014). For a more detailed review on light and hormone signaling, readers can refer Gene targeting via site-directed mutagenesis is a technique that is commonly used in molecular biology to introduce mutations in deﬁned site(s) on the genome. Apart from its utility in studying gene function, the versatility of these techniques also enables researchers to produce gene knockouts or point mutations in plants which can be used for molecular breeding purposes. For a more detailed summary of the various gene Frontiers in Plant Science \| www.frontiersin.org July 2017 \| Volume 8 \| Article 1181 7 Plant Photoreceptors as Targets for Improved Productivity Mawphlang and Kharshiing FIGURE 2 \| Schematic representation of few plant growth responses involving photoreceptor and hormone signaling. SA, salicylic acid; JA, jasmonic acid; GA, gibberellic acid; ABA, abscisic acid; CN, cytokinins; ETH, ethylene; NO, nitric oxide; BR, brassinosteroids; AU, auxin. (Artwork by Rimeia C. Lyngdoh and Eros Kharshiing). FIGURE 2 \| Schematic representation of few plant growth responses involving photoreceptor and hormone signaling. SA, salicylic acid; JA, jasmonic acid; GA, gibberellic acid; ABA, abscisic acid; CN, cytokinins; ETH, ethylene; NO, nitric oxide; BR, brassinosteroids; AU, auxin. (Artwork by Rimeia C. Lyngdoh and Eros Kharshiing). FIGURE 2 \| Schematic representation of few plant growth responses involving photoreceptor and hormone signaling. SA, salicylic acid; JA, jasmonic acid; GA, gibberellic acid; ABA, abscisic acid; CN, cytokinins; ETH, ethylene; NO, nitric oxide; BR, brassinosteroids; AU, auxin. (Artwork by Rimeia C. Lyngdoh and Eros Kh hii ) targeting strategies in higher plants via site-directed mutagenesis, the reader can refer to Osakabe and Osakabe (2014). Plant Defense Response While gene targeting in plant photobiology has been commonly utilized for elucidating photoreceptor function and/or signaling, such strategies have been commonly employed for improving the eﬃciency of reporter genes such as GFP (Cinelli et al., 2000) or iLOV (Christie et al., 2012) which is a derivative of the Light Oxygen Voltage (LOV) domains of phototropin blue-light receptors (Chapman et al., 2008). Similarly, the availability of sequence information of various plant photoreceptors coupled with information from functional genomic studies presents an ideal situation for researchers to engineer photoreceptors or even their downstream signaling partners for improving agronomically valuable traits in crop plants. These could include July 2017 \| Volume 8 \| Article 1181 Frontiers in Plant Science \| www.frontiersin.org 8 Plant Photoreceptors as Targets for Improved Productivity Mawphlang and Kharshiing altering the sensitivity thresholds, photocycles or kinase activity of the diﬀerent photoreceptor proteins. The emergence of large-scale screening technologies such as TILLING (McCallum et al., 2000; Henikoﬀet al., 2004; Kurowska et al., 2011) would also allow researchers to couple random mutagenesis with targeted-selected mutagenesis for selecting mutations in photoreceptor genes for subsequent evaluation for desirable traits without the involvement of transgenic modiﬁcations. rapid progress in recent years. Within the last few years, synthetic biology approaches for regulating plant responses has been successfully demonstrated in several crop species (Table 3). As gene editing techniques become more precise, the modulation of photoreceptor activity by engineering designer photoreceptors provides novel opportunities for improving productivity of crop plants. While diﬀerent engineered photoreceptor systems may require diﬀerent designs for optimal function (Schmidt and Cho, 2015), such photoreceptor systems can be utilized to determine how the light input aﬀects agronomically beneﬁcial phenotypes. Synthetic Biology Approaches to Crop Improvement p For many centuries, humans have been constantly modifying plants that are beneﬁcial to them. With advancements in crop improvement practices, modiﬁcation of plant characteristics that were beneﬁcial especially in terms of yield, were favorably targeted. For many decades, traditional breeding practices along with mutation breeding programs have been instrumental in modifying agricultural traits in crop plants. However, a growing global population coupled with altered weather patterns, present multifaceted challenges to future agricultural production both for food and non-food applications. Synthetic biology has emerged as a viable technology for rapid, precise, and robust engineering of organisms for useful societal purposes (Shih et al., 2016). Synthetic biology attempts to create user-designed biological systems including plants which can display various characteristics such as responses to nutrition status, infections or to changes in environment. One deﬁning emphasis of synthetic biology is the designed control of gene expression. Through various approaches, gene expression can be controlled at the DNA, RNA, or protein level, depending on the strategy or application. The increasing availability of resources to eﬃciently inactivate or replace genes in complex organisms such as plants, has led to a revolution in genome editing in the plant science community. Gene editing tools allow targeting of speciﬁc DNA sequences within the plant genome thereby enabling researchers to engineer genes and genomes with higher precision than would have been possible earlier. The various tools and technologies utilized in plant synthetic biology have been discussed in previous reviews (Bortesi and Fischer, 2015; Schaeﬀer and Nakata, 2015; Puchta, 2016; Weeks et al., 2016). While most of the focus of synthetic biology has been on microbes, plant synthetic biology has made July 2017 \| Volume 8 \| Article 1181 Frontiers in Plant Science \| www.frontiersin.org REFERENCES R., and Christie, J. M. (2002). Phototropins 1 and 2: versatile plant blue-light receptors. Trends Plant Sci. 7, 204–210. doi: 10.1016/S1360-1385(02) 02245-8 Bae, G., and Choi, G. (2008). Decoding of light signals by plant phytochromes and their interacting proteins. Annu. Rev. Plant Biol. 59, 281–311. doi: 10.1146/ annurev.arplant.59.032607.092859 Cardi, T. (2016). 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Conservation and divergence of circadian clock operation in a stress-inducible crassulacean acid metabolism species reveals clock compensation against Stress. Plant Physiol. 137, 969–982. doi: 10.1104/ pp.104.054577 Araki, M., and Ishii, T. (2015). Towards social acceptance of plant breeding by genome editing. Trends Plant Sci. 20, 145–149. doi: 10.1016/j.tplants.2015. 01.010 Briggs, W. FUNDING by diﬀerent photoreceptors either alone or in concert with other photoreceptors and signaling pathways favorably places these genes as most suitable candidates for molecular breeding approaches for enhancing the agronomic value of domesticated lines. EK is grateful to the Science and Engineering Research Board (Department of Science and Technology), New Delhi, India for supporting research in his laboratory vide grants no. SB/EMEQ- 152/2014 and SR/FT/LS-82/2011. EK is grateful to the Science and Engineering Research Board (Department of Science and Technology), New Delhi, India for supporting research in his laboratory vide grants no. SB/EMEQ- 152/2014 and SR/FT/LS-82/2011. ACKNOWLEDGMENT EK designed and directed the study, OM and EK carried out the study, EK wrote the manuscript. All authors read and approved the manuscript. The authors thank Rimeia C. Lyngdoh, Department of Botany, St. Edmund’s College for assisting with the artwork. CONCLUSION Genomic studies have shown that light induces extensive reprogramming of gene expression patterns in plants (Li L. et al., 2012; Petrillo et al., 2014; Perrella and Kaiserli, 2016). The emergence of more precise and robust gene modiﬁcation technologies provides researchers with exciting options to engineer photoreceptors and/or their signaling components for modulating the response of plants to light inputs. Such targeted gene editing technologies would allow for subsequent engineering of light responses for development of useful agronomic traits in both food and non-food crops. As seen in Table 1, alterations in gene function arising due to mutations or altered expression levels of photoreceptors can produce agronomically desirable traits in crops. While research on crops is gaining momentum, the rapid pace of research in Arabidopsis and other established model species is continually contributing large amounts of information on photoreceptor signaling which is yet to be translated to research in crops. However, the information gained from such studies can assist researchers to engineer photoreceptors via genome editing technologies to alter expression or even sensitivity thresholds of native photoreceptors for targeting aspects of plant development that can confer superior agronomic value to the engineered crops. While genome editing in crop plants may have perceived safety concerns (Cardi, 2016) which would inﬂuence the future integration of these crops into society (Araki and Ishii, 2015), the myriad of agronomically desirable traits that are developmentally regulated TABLE 3 \| Examples of agronomically beneﬁcial traits in some crop plants through application of genome editing technologies. Synthetic tools Crop Trait affected Reference Meganuclease Cotton Herbicide tolerance D’Halluin et al., 2013 Maize Production of male sterile plants Djukanovic et al., 2013 TALENs Potato Improvement of cold storage and accumulation of reducing sugars in tubers Clasen et al., 2016 Soybean Improved oil quality Haun et al., 2014 Rice Disease resistance Li T. et al., 2012 Wheat Heritable resistance to powdery mildew Wang et al., 2014 CRISPR/CAS9 Tomato Early ﬂowering and early ripening Soyk et al., 2017 Tomato Broad-spectrum disease resistance de Toledo Thomazella et al., 2016 Zinc ﬁnger nucleases (ZFN) Zea mays Tolerance to multiple herbicides Shukla et al., 2009 Zea mays Resistance to multiple herbicides Ainley et al., 2013 TABLE 3 \| Examples of agronomically beneﬁcial traits in some crop plants through application of genome editing technologies. 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https://openalex.org/W3133650060	https://www.research.ed.ac.uk/files/199046510/PIIS2213671121000837.pdf	English	null	Modulation of APLNR Signaling Is Required during the Development and Maintenance of the Hematopoietic System	Stem cell reports	2,021	cc-by	13,504	General rights C i h f h General rights Copyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. Edinburgh Research Explorer Modulation of Aplnr signaling is required during the development and maintenance of the hematopoietic system Edinburgh Research Explorer Citation for published version: Jackson, M, Fidanza, A, Taylor, H, Rybtsov, S, Axton, R, Kydonaki, M, Meek, S, Burdon, T, Medvinsky, A & Forrester, LM 2021, 'Modulation of Aplnr signaling is required during the development and maintenance of the hematopoietic system', Stem Cell Reports. https://doi.org/10.1016/j.stemcr.2021.02.003 Citation for published version: Jackson, M, Fidanza, A, Taylor, H, Rybtsov, S, Axton, R, Kydonaki, M, Meek, S, Burdon, T, Medvinsky, A & Forrester, LM 2021, 'Modulation of Aplnr signaling is required during the development and maintenance of the hematopoietic system', Stem Cell Reports. https://doi.org/10.1016/j.stemcr.2021.02.003 Document Version: Publisher's PDF, also known as Version of record INTRODUCTION and Herbomel, 2010; Taoudi et al., 2005, 2008). Research on the molecular mechanisms associated with the develop- ment of HSPCs in the embryo has instructed the design of culture protocols to model hematopoiesis in vitro from pluripotent stem cells (PSCs) (Ivanovs et al., 2017). However, it has proven challenging to generate bone ﬁde HSCs that are functionally capable of long-term reconstitution. The small number of reports that claim to have succeeded, albeit with very low efﬁciency, have done so by employing trans- genic strategies and/or providing an in vivo environment for their maturation (Ditadi et al., 2017; Sugimura et al., 2017; Suzuki et al., 2013). It is unclear whether the failure to detect transplantable HSCs from differentiating iPSCs re- ﬂects deﬁciencies in their generation or whether they are produced but fail to be maintained. Our recent ﬁnding that HS-like cells are generated transiently during human iPSC differentiation indicates that their maintenance is a sig- niﬁcant problem (Fidanza et al., 2020). Further insight into the cellular and molecular mechanisms associated with both the production and maintenance of HSCs in vivo and in vitro will aid in designing improved culture conditions for the efﬁcient in vitro production of functional HSCs. Deﬁnitive hematopoietic stem and progenitor cells (HSPCs) generate all cells of the blood and immune system, with the most potent of these, the hematopoietic stem cell (HSC), be- ing capable of repopulating the entire blood system upon transplantation. HSPCs arise through a complex process, precisely coordinated at a number of anatomical sites throughout embryonic development. In the mouse embryo the ﬁrst wave of hematopoietic development originates in the yolk sac around embryonic day 7.25 (E7.25) and gives rise to primitive embryonic erythrocytes, megakaryocytes, and macrophages (Palis, 2014). The second wave also origi- nates from the yolk sac from E8.25 and gives rise to erythro- myeloid progenitors that are deﬁned by their ability to also generate granulocytes and natural killer cells (McGrath et al., 2015; Dege et al., 2020). The third wave originates in the ventral region of the developing dorsal aorta within the aorta-gonad mesonephros (AGM) at E10.5–E11.5, where HSCs, which can repopulate the entire hematopoietic sys- tem upon transplantation, emerge (Medvinsky and Dzier- zak, 1998; Medvinsky et al., 2008). Take down policy Take down policy The University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorer content complies with UK legislation. If you believe that the public display of this file breaches copyright please contact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately and investigate your claim. Download date: 24. Oct. 2024 Please cite this article in press as: Jackson et al., Modulation of APLNR Signaling Is Required during the Development and Maintenance of the Hematopoietic System, Stem Cell Reports (2021), https://doi.org/10.1016/j.stemcr.2021.02.003 Stem Cell Reports j Vol. 16 j 1–14 j April 13, 2021 j ª 2021 The Author(s). 1 This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). SUMMARY Apelin receptor (APLNR/AGTRLl1/APJ) marks a transient cell population during the differentiation of hematopoietic stem and progenitor cells (HSPCs) from pluripotent stem cells, but its function during the production and maintenance of hematopoietic stem cells is not clear. We generated an Aplnr-tdTomato reporter mouse embryonic stem cell (mESC) line and showed that HSPCs are generated exclusively from mesodermal cells that express Aplnr-tdTomato. HSPC production from mESCs was impaired when Aplnr was deleted, implying that this pathway is required for their production. To address the role of APLNR signaling in HSPC maintenance, we added APELIN ligands to ex vivo AGM cultures. Activation of the APLNR pathway in this system impaired the generation of long-term reconstituting HSPCs and appeared to drive myeloid differentiation. Our data suggest that the APLNR signaling is required for the generation of cells that give rise to HSCs, but that its subsequent downregulation is required for their maintenance. Modulation of APLNR Signaling Is Required during the Development and Maintenance of the Hematopoietic System Melany Jackson,1,4,5 Antonella Fidanza,1,5 A. Helen Taylor,1 Stanislav Rybtsov,1,2 Richard Axton,1 Maria Kydonaki,1 Stephen Meek,3 Tom Burdon,3 Alexander Medvinsky,1,2 and Lesley M. Forrester1,* 1MRC Centre for Regenerative Medicine, University of Edinburgh, 5 Little France Drive, Edinburgh EH16 4UU, UK 2Institute for Stem Cell Research, Centre for Regenerative Medicine, 5 Little France Drive, Edinburgh EH16 4UU, UK 3Roslin Institute, University of Edinburgh, Easter Bush, Midlothian EH25 9RG, UK 4Present address: Roslin Technologies Limited, Roslin Innovation Centre, University of Edinburgh, Easter Bush, Midlothian EH25 9RG, UK 5These authors contributed equally Roslin Technologies Limited, Roslin Innovation Centre, University of Edinburgh, Easter Bush, Midlothian EH25 9RG, UK ntributed equally Correspondence: l.forrester@ed.ac.uk https://doi.org/10.1016/j.stemcr.2021.02.003 Correspondence: l.forrester@ed.ac.uk https://doi.org/10.1016/j.stemcr.2021.02.003 INTRODUCTION The expression proﬁle of the genes encoding APELIN and its ligands (APELIN and APELA) during early mouse embryonic development indicates that this signaling pathway is also active in the mesoderm and its derivatives (D’Aniello et al., 2009; Devic et al., 1999). In differentiating human PSCs APLNR is expressed in Mixl1-ex- pressing mesodermal progenitors, and the addition of APE- LIN increased the production of blast colonies, which are considered to be derived from a common precursor to endo- thelial and hematopoietic cells (Yu et al., 2012; Vodyanik et al., 2010). Our previous research has also implicated the APLNR pathway during hematopoietic development in vitro. We showed that the expression of genes encoding APLNR and one of its ligands, APELIN, correlated with the increased production of HSPCs when the transcription fac- tor, HOXB4 was activated in differentiating mouse and hu- man PSCs (Jackson et al., 2012, 2016). Although all of these studies implicate a role for APLNR signaling during hemato- poietic development, the speciﬁc function of this pathway has not been addressed directly. To this end, we generated an Aplnr-tdTomato reporter mouse ESC (mESC) line and have shown that the Aplnr-tdTomato reporter marks a pop- ulation of differentiating mesoderm cells that has the poten- tial to form hematopoietic and endothelial lineages. We then generated an Aplnr-null mESC line and demonstrated that HSPCs production was signiﬁcantly impaired, implying that this signaling pathway is indeed required for their gen- eration. To assess the role of APLNR signaling in HSC matu- ration and maintenance, we added APELIN ligands to AGM explant cultures and observed a marked decrease in the number of long-term reconstituting HSCs and an increase in the differentiation of myeloid cells. p g The timing of expression of the Aplnr-tdTomato reporter during mESC differentiation was compared with the cell sur- face expression of E-cadherin (ECAD), an epithelial marker that is expressed at a high level in undifferentiated PSCs but downregulated upon differentiation into mesoderm lin- eages (Malaguti et al., 2013). Because the APLNR is ﬁrst ex- pressed in the developing mesoderm of the mouse embryo, we predict a mutually exclusive expression pattern with ECAD. As expected, we observed ECAD expression in undif- ferentiated ESCs (day 0) and expression was gradually reducedasdifferentiationprogressed(Figure1C).Incontrast, the Aplnr-tdTomato reporter was not expressed in undiffer- entiated cells, and expression increased during differentia- tion. INTRODUCTION HSCs arise from hemo- genic endothelium through the progressive loss of endothelial markers, such as VE-cadherin and gain of he- matopoietic markers, such as CD41 and KIT (Dzierzak and Medvinsky, 2008; Ivanovs et al., 2011; Medvinsky and Dzierzak, 1996; Rybtsov et al., 2011; Taoudi and Medvinsky, 2007). HSCs have been visualized budding from intra-aortic hematopoietic clusters before they are released into the cir- culation (Bertrand et al., 2010; Boisset et al., 2010; Kissa The Apelin receptor gene (Aplnr/Agtrl1/Apj) encodes a member of the G protein-coupled receptor family and has been implicated in cardiac, endothelial, and hematopoietic development in a number of model systems (D’Aniello et al., 2009; Inui et al., 2006; Quertermous, 2007; Scott et al., 2007). Pertinent to this study is the involvement of Aplnr Aplnr in endothelial cell maturation during develop- ment and the fact that it appears to be expressed at a higher Please cite this article in press as: Jackson et al., Modulation of APLNR Signaling Is Required during the Development and Maintenance of the Hematopoietic System, Stem Cell Reports (2021), https://doi.org/10.1016/j.stemcr.2021.02.003 Please cite this article in press as: Jackson et al., Modulation of APLNR Signaling Is Required during the Development and Maintenance of the Hematopoietic System, Stem Cell Reports (2021), https://doi.org/10.1016/j.stemcr.2021.02.003 level in endothelial cells of the AGM region, implying that it could be related to their hemogenic potential both in mouse (Kidoya et al., 2015; Kidoya et al., 2008, 2010) and human embryos (Crosse et al., 2020). In the adult bone marrow, endothelial cells that express APELIN, one of the APLNR li- gands, participate in vascular remodeling after irradiation and drive post-transplant recovery through feedback from HSPCs (Chen et al., 2019). The expression proﬁle of the genes encoding APELIN and its ligands (APELIN and APELA) during early mouse embryonic development indicates that this signaling pathway is also active in the mesoderm and its derivatives (D’Aniello et al., 2009; Devic et al., 1999). In differentiating human PSCs APLNR is expressed in Mixl1-ex- pressing mesodermal progenitors, and the addition of APE- LIN increased the production of blast colonies, which are considered to be derived from a common precursor to endo- thelial and hematopoietic cells (Yu et al., 2012; Vodyanik et al., 2010). Our previous research has also implicated the APLNR pathway during hematopoietic development in vitro. INTRODUCTION Aplnr-tdTomato-expressing cells were ﬁrst detected at day 3 of differentiation in both ECAD+ and ECAD cells, but by day 5 virtually all the Aplnr-tdTomato-expressing cells were ECAD (Figure 1C). Thus, differentiating ESCs transi- tion from ECAD+APLNR to ECAD+APLNR+ phenotype, which then downregulate ECAD to become ECADAPLNR+. The highest proportion (20%) of the intermediate, double- positive cells was present around day 3. INTRODUCTION We showed that the expression of genes encoding APLNR and one of its ligands, APELIN, correlated with the increased production of HSPCs when the transcription fac- tor, HOXB4 was activated in differentiating mouse and hu- man PSCs (Jackson et al., 2012, 2016). Although all of these studies implicate a role for APLNR signaling during hemato- poietic development, the speciﬁc function of this pathway has not been addressed directly. To this end, we generated an Aplnr-tdTomato reporter mouse ESC (mESC) line and have shown that the Aplnr-tdTomato reporter marks a pop- ulation of differentiating mesoderm cells that has the poten- tial to form hematopoietic and endothelial lineages. We then generated an Aplnr-null mESC line and demonstrated that HSPCs production was signiﬁcantly impaired, implying that this signaling pathway is indeed required for their gen- eration. To assess the role of APLNR signaling in HSC matu- ration and maintenance, we added APELIN ligands to AGM explant cultures and observed a marked decrease in the number of long-term reconstituting HSCs and an increase in the differentiation of myeloid cells. that the Aplnr-tdTomato reporter faithfully mimicked Aplnr transcript expression by demonstrating the expression of Aplnr transcripts in ﬂuorescence-activated cell sorting (FACS)-sorted tdTomato-positive, but not tdTomato-nega- tive cells (Figure 1B). However, we noted that the expression of the Aplnr-tdTomato reporter did not correlate with the expression of a protein detected by a commercially available a-APLNR antibody (Figure S2). Real-time PCR analyses of FACS-sorted cells detected Aplnr transcripts in cells sorted based on expression of the Aplnr-tdTomato reporter but not a-APLNR antibody staining, suggesting that the a-APLNR antibodywasbindingnon-speciﬁcallytothecellsurface(Fig- uresS2AandS2B).Thiswassupportedbydemonstratingthat the a-APLNR antibody detected a protein in 293T cells that do not express Aplnr transcripts (Figure S2C). We were ﬁnally convincedthatthiscommercialantibodywasnot speciﬁc for APLNR when we demonstrated that it detected a protein in differentiating Aplnr-null ESCs (see below) (Figure S2D). These ﬁndings emphasized the requirement of the Aplr- tdTomato reporter mESC lines in deﬁning the potential of APLNR-expressing cells. level in endothelial cells of the AGM region, implying that it could be related to their hemogenic potential both in mouse (Kidoya et al., 2015; Kidoya et al., 2008, 2010) and human embryos (Crosse et al., 2020). In the adult bone marrow, endothelial cells that express APELIN, one of the APLNR li- gands, participate in vascular remodeling after irradiation and drive post-transplant recovery through feedback from HSPCs (Chen et al., 2019). 2 Stem Cell Reports j Vol. 16 j 1–14 j April 13, 2021 RESULTS (B) Quantitative RT-PCR of Aplnr expression in undifferentiated Aplnr-tdTomato reporter ESCs (day 0 [D0]), unsorted differentiated day 6 (D6) cells and day 6 cells sorted into Aplnr-tdTomato-positive (+) and -negative () populations (n = 3). Error bars represent standard deviation of data from three independent experiments. (C) Representative ﬂow cytometry plots of Aplnr-tdTomato and E-cadherin expression during differentiation from undifferentiated ESCs (D0) to day 5 of differentiation. (D) Flow cytometry of Aplnr-tdTomato ESCs at day 3 of differentiation stained with antibodies to FLK1 and PDGFRa and then analyzed after gating on either APLNR negative or APLNR high. (E) Representative ﬂow cytometry plots of Aplnr-tdTomato together with CD41, VE-cadherin, or FLK1 during differentiation from days 1 to 6 of differentiation. (F) Proportion of Aplnr-tdTomato-positive cells detected by ﬂow cytometry in undifferentiated (D0) cells and during a 14 day time course of differentiation. Error bars represent standard deviation of data from three independent experiments. Figure 1. Aplnr-tdTomato Reporter ESC Line Mimics the Activity of the Aplnr Locus during ESC Differentiation and Tracks a Transient Mesoderm Cell Population (A) CRISPR C di i G id RNA d i d h i i f h i di l f h A l (B) Quantitative RT-PCR of Aplnr expression in undifferentiated Aplnr-tdTomato reporter ESCs (day 0 [D0]), unsorted differentiated day 6 (D6) cells and day 6 cells sorted into Aplnr-tdTomato-positive (+) and -negative () populations (n = 3). Error bars represent standard deviation of data from three independent experiments. p p (C) Representative ﬂow cytometry plots of Aplnr-tdTomato and E-cadherin expression during differentiation from undifferentiated ESCs (D0) to day 5 of differentiation. ( ) y (D) Flow cytometry of Aplnr-tdTomato ESCs at day 3 of differentiation stained with antibodies to FLK1 and PDGFRa and then analyzed after gating on either APLNR negative or APLNR high. g g g g (E) Representative ﬂow cytometry plots of Aplnr-tdTomato together with CD41, VE-cadherin, or FLK1 during differentiation from days 1 to 6 of differentiation. different levels of Aplnr-tdTomato were isolated by ﬂow cy- tometry (Figure S3). The differentiation potential of FACS- sorted cells was assessed using CFU-C assays (Figure 2A). He- matopoietic activity was found almost exclusively within in the Aplnr-tdTomato-expressing cell population with virtu- ally no CFU-Cs generated from the sorted Aplnr-tdTo- mato-negative cell population. RESULTS To conﬁrm that the Aplnr-tdTomato reporter is expressed in the emerging multipotent mesoderm, we show that most of the cells expressing a high level of Aplnr-tdTomato at day 3 co-expressed high levels of FLK1 and that around half of these also express PDGFRa (Figure 1D). As differen- tiation progressed, Aplnr-tdTomato was observed in cells expressing the endothelial cell markers (VE-cadherin and FLK1), with the majority of VE-CAD+ and FLK1+ cells expressing the reporter at day 4 (Figure 1E). Hematopoietic cells expressing CD41 were ﬁrst detected in this differenti- ation protocol at day 6 and the Aplnr-tdTomato reporter Aplnr-tdTomato Reporter Marks Mesodermal Cells in Differentiating ESCs To deﬁnethephenotypeofcells expressingtheAPLNRrecep- tor during hematopoietic differentiation of ESCs we gener- ated an Aplnr-tdTomato reporter mESC line using CRISPR- Cas9-mediated genome editing (Figure 1A). mESCs were transfected with the Aplnr targeting vector, Cas9 plasmid, and speciﬁc gRNAs. Thirty-ﬁve G418-resistant colonies were selected and 18 of these were screened by genomic PCR and Southern blotting (Figure S1A). We conﬁrmed 2 Stem Cell Reports j Vol. 16 j 1–14 j April 13, 2021 Please cite this article in press as: Jackson et al., Modulation of APLNR Signaling Is Required during the Development and Maintenance of the Hematopoietic System, Stem Cell Reports (2021), https://doi.org/10.1016/j.stemcr.2021.02.003 Please cite this article in press as: Jackson et al., Modulation of APLNR Signaling Is Required during the Development and Maintenance of the Hematopoietic System, Stem Cell Reports (2021), https://doi.org/10.1016/j.stemcr.2021.02.003 Please cite this article in press as: Jackson et al., Modulation of APLNR Signaling Is Required during the Development and Maintenance of the Hematopoietic System, Stem Cell Reports (2021), https://doi.org/10.1016/j.stemcr.2021.02.003 A B C D E F Figure 1 Aplnr-tdTomato Reporter ESC Line Mimics the Activity of the Aplnr Locus during ESC Differentiation and Tracks a A B C A C B B E E D D F D F F Figure 1. Aplnr-tdTomato Reporter ESC Line Mimics the Activity of the Aplnr Locus during ESC Differentiation and Tracks a Transient Mesoderm Cell Population (A) CRISPR-Cas9 gene-editing strategy. Guide RNAs were designed to cut the genomic region of chromosome 2 immediately after the Aplnr coding sequence. Schematic of targeting vector that was used to insert the tdTomato reporter gene at this site, followed by a T2A sequence and NeoR cassette (not shown). RESULTS There was a signiﬁcantly higher overall number of CFU-Cs generated from cells ex- pressing a high level of Aplnr-tdTomato compared with cells expressing low levels or that were negative for Aplnr- was detected in a proportion of these cells (Figure 1E). Over- all, the highest proportion (around 70%) of Aplnr-tdTo- mato cells was observed at day 4 and this gradually declined as differentiation proceeded (Figure 1F). Hematopoietic Activity Is within the Cell Populations that Express High Levels of the Aplnr-td-Tomato Reporter Aplnr-tdTomato mESCs were differentiated into embryoid bodies (EBs) for 5 days, dissociated, and cells expressing Stem Cell Reports j Vol. 16 j 1–14 j April 13, 2021 3 Please cite this article in press as: Jackson et al., Modulation of APLNR Signaling Is Required during the Development and Maintenance of the Hematopoietic System, Stem Cell Reports (2021), https://doi.org/10.1016/j.stemcr.2021.02.003 Please cite this article in press as: Jackson et al., Modulation of APLNR Signaling Is Required during the Development and Maintenance of the Hematopoietic System, Stem Cell Reports (2021), https://doi.org/10.1016/j.stemcr.2021.02.003 A B C D E Figure 2. Aplnr-tdTomato Marks Mesoderm Fated to Become Hemogenic Endothelium (A) Number of CFU-Cs generated from 105 unsorted differentiating ESCs (day 6) and cell populations sorted biased a high (Hi), low (Lo), or negligible (Neg) level of tdTomato expression (n = 3; p < 0.05). Statistically signiﬁcant difference in number of CFU-Mix (p = 0.003) and CFU-GM (p < 0.03) colonies from Aplnr-tdTomato high and low cells. No difference was observed in the number of uni-lineage BFU-E and CFU-M (see Figure S3 for FACS sorting strategy). (B) Representative ﬂow cytometry plots of differentiating Aplnr-tdTomato ESCs (day 6) co-stained with antibodies against KIT, TIE2, VE- CAD, FLK1, and CD41. Backgating of cells expressing high, low, or negligible (Neg) levels of Aplnr-tdTomato demonstrates that cells expressing TIE2, FLK1, VE-CAD (VC), KIT, and CD41 are primarily found in the Aplnr-tdTomato-high cell population. (C) Quantiﬁcation of ﬂow cytometry data in (B) showing the percentage of cell type within each of the subpopulations deﬁned by the level of Aplnr-tdTomato expression (n = 3; p < 0.05). (D) Immunohistochemistry using an aCD31 antibody of differentiating ESCs after FACS and culturing on OP9 stromal cells in the presence of VEGF, demonstrating the endothelial potential differentiating cells expressing high and low levels of Aplnr-tdTomato. Two replicate experiments are shown. Scale bar represents 100 mm (340 magniﬁcation). RESULTS (E) Percentage of embryoid bodies (EBs) with associated beating cardiomyocytes in EBs generated from unsorted day 6 differentiating ESCs ll t thi t th t t d b d th i f hi h (Hi) l (L ) li ibl (N ) l l f A l tdT t A B A A B C C D E E D Figure 2. Aplnr-tdTomato Marks Mesoderm Fated to Become Hemogenic Endothelium (A) Number of CFU-Cs generated from 105 unsorted differentiating ESCs (day 6) and cell populations sorted biased a high (Hi), low (Lo), or negligible (Neg) level of tdTomato expression (n = 3; p < 0.05). Statistically signiﬁcant difference in number of CFU-Mix (*p = 0.003) and CFU-GM (p < 0.03) colonies from Aplnr-tdTomato high and low cells. No difference was observed in the number of uni-lineage BFU-E and CFU-M (see Figure S3 for FACS sorting strategy). (B) Representative ﬂow cytometry plots of differentiating Aplnr-tdTomato ESCs (day 6) co-stained with antibodies against KIT, TIE2, VE- CAD, FLK1, and CD41. Backgating of cells expressing high, low, or negligible (Neg) levels of Aplnr-tdTomato demonstrates that cells expressing TIE2, FLK1, VE-CAD (VC), KIT, and CD41 are primarily found in the Aplnr-tdTomato-high cell population. (C) Quantiﬁcation of ﬂow cytometry data in (B) showing the percentage of cell type within each of the subpopulations deﬁned by the level of Aplnr-tdTomato expression (n = 3; p < 0.05). p p ( ; p ) (D) Immunohistochemistry using an aCD31 antibody of differentiating ESCs after FACS and culturing on OP9 stromal cells in the presence of VEGF, demonstrating the endothelial potential differentiating cells expressing high and low levels of Aplnr-tdTomato. Two replicate experiments are shown. Scale bar represents 100 mm (340 magniﬁcation). d bodies (EBs) with associated beating cardiomyocytes in EBs generated from unsorted day 6 differentiating ESC t were sorted based on the expression of high (Hi), low (Lo), or negligible (Neg) levels of Aplnr-tdTomat (E) Percentage of embryoid bodies (EBs) with associated beating cardiomyocytes in EBs generated from unsorted day 6 differentiating ESCs or cells at this stage that were sorted based on the expression of high (Hi), low (Lo), or negligible (Neg) levels of Aplnr-tdTomato expression. pressing TIE2, FLK1, VE-CAD (VC), KIT, and CD41 are pri- marily found in the Aplnr-tdTomato-high cell population (Figure 2B). 4 Stem Cell Reports j Vol. 16 j 1–14 j April 13, 2021 Aplnr Is Required for Hematopoietic Cell Production from ESCs Aplnr Is Required for Hematopoietic Cell Production from ESCs To conﬁrm that APLNR signaling is required during the dif- ferentiation of hematopoietic cells from PSCs, we gener- ated Aplnr-null mESCs using a CRISPR-Cas9 strategy. Aplnr is a single-exon transcript with a coding region of 1,131 base pairs (bp), so our strategy involved excising the com- plete coding region using guide RNAs directed to the 50 and 30 ends (Figure S4A). pSPCas9-2A-mCherry-U6-gRNA plasmids containing pre-selected gRNAs (see the Experi- mental Procedures) were transfected into E14 mESCs. Cells that had been successfully transfected were sorted based on mCherry expression 24–48 h after transfection and then plated at low density. Individual mESC clones were isolated and genomic DNA was screened by PCR using primers that spanned the deleted region. The PCR assay was designed to amplify a 589 bp product from the deleted, knockout (KO) allele and a 1,700 bp amplicon from the wild-type (WT) allele. The KO allele was identiﬁed in 7 out of 30 mCherry-positive clones (Figure S4B). This assay did not distinguish between heterozygous and homozygous clones because the smaller 589 bp amplicon associated with the KO allele would likely be ampliﬁed preferentially over the larger WT allele. To differentiate between functionally het- erozygous and homozygous mESC clones, we sequenced the amplicons and found that, in the majority of clones that had one deleted Aplnr allele, the second Aplnr allele had an insertion or a deletion that resulted in a frameshift mutation (data not shown). Quantitative real-time PCR Activation of the APLNR Pathway Has No Signiﬁcant Effect on the Production of Hematopoietic Progenitors in Differentiating Mouse and Human PSCs g As HSPC production is impaired when Aplnr is deleted, we hypothesized that their production might be increased when the APLNR pathway is activated by known APLNR li- gands, such as APELIN. However, when APELIN was added to the serum-free cytokine-based hematopoietic differenti- ation of mESCs, we observed no effect on the number or phenotype of CFU-Cs (Figure 4A). This was somewhat un- expected given that previous studies had reported promo- tion of the production of blast colonies when Apelin was added to differentiating human ESCs (Yu et al., 2012). RESULTS A subset of these cells also expressed the he- matopoietic markers CD41 and KIT, implying that APLNR is expressed at the transitional state from an endothelial to hematopoietic phenotype (Figures 2B and 2C). tdTomato. The number of multilineage (CFU-Mix) colonies and CFU-GM colonies was signiﬁcantly higher from cells expressing a high level of Aplnr-tdTomato compared with the cells expressing a low level of Aplnr-tdTomato, whereas there was no signiﬁcant difference in the production of sin- gle lineage colonies (Figure 2A). Aplnr-tdTomato-expressing cells were plated onto irradi- ated OP9 cells, cultured in the presence of VEGF for Backgating of cells expressing high, low, or negligible (Neg) levels of Aplnr-tdTomato demonstrated that cells ex- 4 Stem Cell Reports j Vol. 16 j 1–14 j April 13, 2021 Please cite this article in press as: Jackson et al., Modulation of APLNR Signaling Is Required during the Development and Maintenance of the Hematopoietic System, Stem Cell Reports (2021), https://doi.org/10.1016/j.stemcr.2021.02.003 Please cite this article in press as: Jackson et al., Modulation of APLNR Signaling Is Required during the Development and Maintenance of the Hematopoietic System, Stem Cell Reports (2021), https://doi.org/10.1016/j.stemcr.2021.02.003 Please cite this article in press as: Jackson et al., Modulation of APLNR Signaling Is Required during the Development and Maintenance of the Hematopoietic System, Stem Cell Reports (2021), https://doi.org/10.1016/j.stemcr.2021.02.003 10 days, and then immuno-stained for the endothelial marker, CD31, to assess their potential to form endothelial cells (Figure 2D). Cells expressing high and low levels of Aplnr-tdTomato generated endothelial structures consist- ing of CD31+ cells but no such structures were produced by Aplnr-tdTomato-negative cells. To assess whether the Aplnr-tdTomato reporter was marking all mesoderm pro- genitors or a subpopulation committed to hematopoietic and endothelial lineages, we assessed the potential of sorted cells to differentiated into another mesodermal cell type. EBs were dissociated at day 6, sorted based on the expression of Aplnr-tdTomato, and then assessed for their potential to generate cells of the cardiac lineage. Although sorting reduced the overall production of beating cardio- myocytes compared with unsorted cells, there was no cor- relation between the level of Aplnr-tdTomato expression and the potential to generate cardiac cells suggesting that cardiomyocytes could be generated from mesoderm that did not express Aplnr-tdTomato (Figure 2E). Taken together, these data support our hypothesis that the Aplnr-tdTomato reporter marks mesoderm and that Aplnr expression is spe- ciﬁcally required for the efﬁcient endothelial and hemato- poietic differentiation. RESULTS was used to select clones in which the targeting events re- sulted in ablation of Aplnr transcripts and could therefore be considered as functionally null at the Aplnr locus (Figure S4C). Aplnr-null mESCs could be maintained as undifferenti- ated ESCs in the presence of leukemia inhibitory factor (LIF) in a comparable manner with controls (data not shown). However, when induced to differentiate and as- sessed in hematopoietic colony assays, signiﬁcantly lower numbers of hematopoietic CFU-C colonies (CFU-M, CFU- GM, and CFU-Mix) were formed from two independently derived Aplnr-null ESCs compared with control WT ESCs at day 6 of differentiation (Figure 3A). Flow cytometry ana- lyses conﬁrmed that the Aplnr-null ESCs produced a lower proportion of CD41+VE-cadherin+ hematopoietic progeni- tor cells (HPCs) (Figures 3B and 3C). When subjected to a macrophage-speciﬁc differentiation protocol, the proportion of mature F4/80+CD11B+CD16+ macrophages generated from Aplnr-null ESCs was signiﬁ- cantly lower than controls (Figure 3D). To conﬁrm that the reduced hematopoietic differentiation was due to the absence of Aplnr and not simply a consequence of ESC clonal variation, we introduced an APLNR-expressing plasmid into the Aplnr-null ESC clones and demonstrated that the production of F4/80+CD11b+ macrophages was increased in the presence of the exogenous Aplnr (Fig- ure 3D). These data indicate that the APLNR pathway is required for the development of HPCs and for the differen- tiation of the myeloid lineage. Aplnr Is Required for Hematopoietic Cell Production from ESCs To conﬁrm that the effects we had observed were not species speciﬁc we replicated our ﬁndings using both human ESCs and induced pluripotent stem cells (iPSCs), again demonstrating that the addition of APELIN peptides had no effect on CFU-C formation (Figure 4B). This apparent discrepancy might be partly explained by the fact that two of the proposed ligands (APELIN and APELA) for APLNR are expressed in differentiating ESCs (Figure S5). This could suggest that the APLNR signaling pathway is already activated and that any further addition of ligand Stem Cell Reports j Vol. 16 j 1–14 j April 13, 2021 5 Please cite this article in press as: Jackson et al., Modulation of APLNR Signaling Is Required during the Development and Maintenance of the Hematopoietic System, Stem Cell Reports (2021), https://doi.org/10.1016/j.stemcr.2021.02.003 Please cite this article in press as: Jackson et al., Modulation of APLNR Signaling Is Required during the Development and Maintenance of the Hematopoietic System, Stem Cell Reports (2021), https://doi.org/10.1016/j.stemcr.2021.02.003 A B C D Figure 3. Targeted Deletion of Aplnr Results in Impaired Hematopoietic Differentiation (A) Number of hematopoietic CFU-C colonies generated from 105 differentiated (day 6) control ESCs and two independently derived Aplnr- null (Null1 and Null2) ESC clones. CFU-M (macrophage), CFU-GM (granulocye/macrophage), CFU-GEMM (multilineage, including gran- ulocyte, erythroid, macrophage, and megakaryocytes), 20 EB-ery (secondary embryoid bodies with associated erythroid cells) (p < 0.05). Error bars represent standard deviation of data from four independent experiments. (B) Representative ﬂow cytometry analyses of day 6 differentiated control ESCs and two independently derived Aplnr-null ESC clones (Null1 and Null2) using antibodies to VE-CAD and CD41. (C) Quantiﬁcation of ﬂow cytometry analyses (B) showing the percentage of VE-CAD+CD41+ cells at day 6 of differentiation from control and two Aplnr-null ESC clones (p < 0.05). Error bars represent standard deviation of data from three independent experiments. (D) Representative ﬂow cytometry of cells generated following the macrophage differentiation protocol of control (wild type), two Aplnr- null ESC lines (Null1 and Null2), and the Null1 and Null2 ESCs that were transfected with an APLNR-expressing plasmid (+APLNR). A B C D Figure 3. Targeted Deletion of Aplnr Results in Impaired Hematopoietic Differentiation 5 B C A C A B C D D D Figure 3. Targeted Deletion of Aplnr Results in Impaired Hematopoietic Differentiation 5 Figure 3. Aplnr Is Required for Hematopoietic Cell Production from ESCs Targeted Deletion of Aplnr Results in Impaired Hematopoietic Differentiation (A) Number of hematopoietic CFU-C colonies generated from 105 differentiated (day 6) control ESCs and two independently derived Aplnr- null (Null1 and Null2) ESC clones. CFU-M (macrophage), CFU-GM (granulocye/macrophage), CFU-GEMM (multilineage, including gran- ulocyte, erythroid, macrophage, and megakaryocytes), 20 EB-ery (secondary embryoid bodies with associated erythroid cells) (p < 0.05). Error bars represent standard deviation of data from four independent experiments. (B) Representative ﬂow cytometry analyses of day 6 differentiated control ESCs and two independently derived Aplnr-null ESC clones (Null1 and Null2) using antibodies to VE-CAD and CD41. (C) Quantiﬁcation of ﬂow cytometry analyses (B) showing the percentage of VE-CAD+CD41+ cells at day 6 of differentiation from control and two Aplnr-null ESC clones (*p < 0.05). Error bars represent standard deviation of data from three independent experiments. (D) Representative ﬂow cytometry of cells generated following the macrophage differentiation protocol of control (wild type), two Aplnr- null ESC lines (Null1 and Null2), and the Null1 and Null2 ESCs that were transfected with an APLNR-expressing plasmid (+APLNR). RNA sequencing of mouse AGM tissue (GEO: GSE143637) (Vink et al., 2020), human AGM tissue (GEO: GSE151877; sample GSM4592621) (Crosse et al., 2020) and differenti- ating human iPSCs (Database: EMBL EBI Arrayexpress; E-MTAB-9295 (Fidanza et al., 2020). Aplnr/APLNR is ex- pressedatarelativelyhighlevelinmostofthecellsassociated with endothelial cell clusters in both mouse and human AGM and in differentiating human iPSCs (Figures 5B–5D). RNA encoding the ligand APELIN was expressed in more restricted subpopulations of endothelial cells in all datasets, whereas RNA encoding APELA was barely detectable (Figures 5B–5D). Interestingly, within HSPC clusters, RNA encoding APLNR and APELIN (but not APELA) was detected in mouse but not human AGM tissue. APLNR transcripts were detected in some iPSC-derived HSPCs, supporting the idea that APLNR could be expressed in cells at the EHT transition. RNA sequencing of mouse AGM tissue (GEO: GSE143637) (Vink et al., 2020), human AGM tissue (GEO: GSE151877; sample GSM4592621) (Crosse et al., 2020) and differenti- ating human iPSCs (Database: EMBL EBI Arrayexpress; E-MTAB-9295 (Fidanza et al., 2020). Aplnr/APLNR is ex- pressedatarelativelyhighlevelinmostofthecellsassociated with endothelial cell clusters in both mouse and human AGM and in differentiating human iPSCs (Figures 5B–5D). RNA encoding the ligand APELIN was expressed in more restricted subpopulations of endothelial cells in all datasets, whereas RNA encoding APELA was barely detectable (Figures 5B–5D). 6 Stem Cell Reports j Vol. 16 j 1–14 j April 13, 2021 Aplnr Is Required for Hematopoietic Cell Production from ESCs Interestingly, within HSPC clusters, RNA encoding APLNR and APELIN (but not APELA) was detected in mouse but not human AGM tissue. APLNR transcripts were detected in some iPSC-derived HSPCs, supporting the idea that APLNR could be expressed in cells at the EHT transition. fails to exert an observable functional effect. Interestingly, when APELIN ligands were included in the CFU-C assay, an increase in macrophage colonies was observed, implying that activation of the pathway might drive the differentia- tion of HPCs into myeloid lineages (Figure S6). Genes expressing APLNR and Its Ligands Are Expressed at the Site of HSC Emergence In Vivo Expressed at the Site of HSC Emergence In Vivo To assess the role of the APLNR pathway in HSPC mainte- nance and differentiation, we turned to the mouse AGM explant system that is known to support both the matura- tion and maintenance of deﬁnitive HSPCs (Taoudi et al., 2008). We ﬁrst demonstrated that the genes encoding APLNR and its ligands, APELIN and APELA, were expressed in the region of the developing embryo at E9.5 and E11.5 when deﬁnitive HSCs ﬁrst emerge (Figure 5A). Low, but detectable levels of expression were also observed in the yolk sac and fetal liver. These data support the hypothesis that the APLNR signaling pathway is active during the emer- gence of HSCs in vivo. To further deﬁne which cell types within the AGM express the genes encoding APLNR and its ligands we analyzed published datasets from single-cell Activation of the APLNR Pathway Reduces the Production of Transplantable HSCs in Aggregate Cultures To assess whether activation of the APLNR pathway could inﬂuence the production, maintenance, or differentiation 6 Stem Cell Reports j Vol. 16 j 1–14 j April 13, 2021 Please cite this article in press as: Jackson et al., Modulation of APLNR Signaling Is Required during the Development and Maintenance of the Hematopoietic System, Stem Cell Reports (2021), https://doi.org/10.1016/j.stemcr.2021.02.003 Please cite this article in press as: Jackson et al., Modulation of APLNR Signaling Is Required during the Development and Maintenance of the Hematopoietic System, Stem Cell Reports (2021), https://doi.org/10.1016/j.stemcr.2021.02.003 Please cite this article in press as: Jackson et al., Modulation of APLNR Signaling Is Required during the Development and Maintenance of the Hematopoietic System, Stem Cell Reports (2021), https://doi.org/10.1016/j.stemcr.2021.02.003 A B Figure 4. Aplnr Is Required for Hematopoietic Cell Production from ESCs (B) Number of CFU-Cs generated in differentiating human ESCs (H1) (n = 8) and human iPSCs (SFCi55) ( APELIN ligands added (no A) and after the addition of APELIN 13 (A13) or APELIN 36 (A36). Error bars rep from three independent experiments (A and B). contrast, when a high concentration (300 nM) of A13, A36, or A21 was added to the aggregate culture, the num- ber of successfully transplanted animals was reduced to 3/6, 1/10, and 2/6, respectively (Figure 5G). The effect of peptide addition was dose dependent, with the addi- tion of a lower concentration (100 nM) of all peptides having a less profound detrimental effect on the ability of cultured cells to reconstitute the hematopoietic system of irradiated recipient mice (Figure 5G). To deﬁne the timing of this detrimental effect, we isolated pre-HSC type I (VC+CD41loCD43+CD45) and pre-HSC type II cells (VC+CD41loCD43+CD45+) from day 11.5 AGM tis- sue and assessed the effect of A36 on their subsequent maturation into functional HSCs within aggregate cul- tures (Figure 5H). In control cultures, pre-HSC type I could be matured ex vivo into reconstituting HSCs, but in the presence of APELIN 36 resultant cells did not reconstitute irradiated mice. A comparable, but less pro- found effect of A36 was observed on pre-HSC type II when again reconstitution was signiﬁcantly reduced. These data demonstrate that activation of the APLNR pathway by the addition of ligands is detrimental to the generation and maintenance of functional HSCs in ex vivo cultures. HSCs, we added APLNR ligands, APELIN 13 (A13), APELIN 36 (A36), or APELA 21 (A21), which are known to support ex vivo maturation and expansion of deﬁnitive HSCs, to AGM reaggregation cultures (Figure 6A) (Rybtsov et al., 2014; Taoudi et al., 2008). The caudal region of E9.5 em- bryos that includes AGM tissue was dissected and placed in the aggregation culture in the presence of cytokines, with or without Apelin peptides, for 7 days. Resultant cells were assessed for CFU-C formation, ﬂow cytometry, and their ability to reconstitute lethally irradiated recipi- ents in vivo. The addition of either A13 or A36 peptides to the aggregate cultures resulted in a slight, but not signiﬁcant, increase in the total number of hematopoietic progenitors as assessed by CFU-C production (Figure 6B). Aplnr Is Required for Hematopoietic Cell Production from ESCs Addition of APELIN Ligands to Differentiating Mouse and Human PSCs Does Not Enhance Hematopoietic Colony Pro- duction (A) Number of CFU-Cs generated in differentiating mESCs with no APELIN ligands added (control) and after the addition of APELIN 13 (A13) or APELIN 36 (A36) at 30 and 100 nM (n = 3). (B) Number of CFU-Cs generated in differentiating human ESCs (H1) (n = 8) and human iPSCs (SFCi55) (n = 3) in control cultures with no APELIN ligands added (no A) and after the addition of APELIN 13 (A13) or APELIN 36 (A36). Error bars represent standard deviation of data from three independent experiments (A and B). A B Figure 4. Addition of APELIN Ligands to Differentiating Mouse and Human PSCs Does Not Enhance Hematopoietic Colony Pro- duction (A) Number of CFU-Cs generated in differentiating mESCs with no APELIN ligands added (control) and after the addition of APELIN 13 (A13) or APELIN 36 (A36) at 30 and 100 nM (n = 3). (B) Number of CFU-Cs generated in differentiating human ESCs (H1) (n = 8) and human iPSCs (SFCi55) (n = 3) in control cultures with no APELIN ligands added (no A) and after the addition of APELIN 13 (A13) or APELIN 36 (A36). Error bars represent standard deviation of data from three independent experiments (A and B) ELIN Ligands to Differentiating Mouse and Human PSCs Does Not Enhance Hematopoietic Colony Pro- Figure 4. Addition of APELIN Ligands to Differentiating Mouse and Human PSCs Does Not Enhance Hematopoietic Colony Pro- duction (A) Number of CFU-Cs generated in differentiating mESCs with no APELIN ligands added (control) and after the addition of APELIN 13 (A13) or APELIN 36 (A36) at 30 and 100 nM (n = 3). (B) Number of CFU-Cs generated in differentiating human ESCs (H1) (n = 8) and human iPSCs (SFCi55) (n = 3) in control cultures with no APELIN ligands added (no A) and after the addition of APELIN 13 (A13) or APELIN 36 (A36). Error bars represent standard deviation of data from three independent experiments (A and B). duction (A) Number of CFU-Cs generated in differentiating mESCs with no APELIN ligands added (control) and afte or APELIN 36 (A36) at 30 and 100 nM (n = 3). Aplnr Is Required for Hematopoietic Cell Production from ESCs Expression of Aplnr, Apelin, and Apela in Hematopoietic Tissue g p p , p , p p (A) Quantitative RT-PCR analyses of RNA isolated from the AGM region, yolk sac (YS), and fetal liver (FL) dissected from embryos at the indicated stage of development using primers to detect transcripts encoding APLNR and its ligands, APELIN and APELA (n = 2). (B–D) UMAP plots of single-cell sequencing data derived from mouse (B) and human (C) AGM tissue and differentiating human iPSCs (D) demonstrating Aplnr/APLNR, Apelin/APELIN, and Apela/APELA expression in endothelial cell clusters. Endothelial cell and HSPC clusters are highlighted with solid and dotted outlines, respectively. signaling pathway favors differentiation over self- renewal. have the potential to differentiate into hematopoietic and endothelial lineages and that deletion of the Aplnr gene impairs the in vitro production of HSPCs from mESCs. Our ﬁndings are consistent with previous studies that have reported a role for this signaling pathway in mesoderm progenitors in both mouse and human differ- entiating ESCs (Yu et al., 2012; Vodyanik et al., 2010; D’Aniello et al., 2013). Aplnr Is Required for Hematopoietic Cell Production from ESCs Flow cytometry analyses of cells derived from the CFU-C colonies demonstrated an increase in the proportion of mature myeloid cells expressing high levels of CD11b, a proportion of which also expressed Gr1 (Figures 6C and 6D). The increase in the production of mature myeloid cells in the presence of APLNR ligands coincided with a decrease in the production of immature (LIN/SCA+/ KIT+) HPCs (Figure 6E). To assess the effects of APLNR ligands on the produc- tion of functional reconstituting HSCs in ex vivo cultures, day 9.5 AGM cells (CD45.2) were cultured for 7 days in the presence of various concentrations of A13, A36, or A21, and then resultant cells were transplanted into irra- diated CD45.1/2 recipients. Almost all (8/9) recipients that received control cell aggregates cultured without Apelin peptides demonstrated successful HSC reconstitu- tion at 14 weeks after transplantation (>10% CD45+). In Taken together, the results of our study indicate that the APLNR pathway is required for the production of hematopoietic cells but that the activation of the APLNR pathway at a later stage negatively affects HSC function. The increased production of myeloid cells in the presence of APLNR ligands both in explant cultures and in differentiating human ESCs suggests that this Stem Cell Reports j Vol. 16 j 1–14 j April 13, 2021 7 Please cite this article in press as: Jackson et al., Modulation of APLNR Signaling Is Required during the Development and Maintenance of the Hematopoietic System, Stem Cell Reports (2021), https://doi.org/10.1016/j.stemcr.2021.02.003 Please cite this article in press as: Jackson et al., Modulation of APLNR Signaling Is Required during the Development and Maintenance of the Hematopoietic System, Stem Cell Reports (2021), https://doi.org/10.1016/j.stemcr.2021.02.003 A A A B C D Figure 5. Expression of Aplnr, Apelin, and Apela in Hematopoietic Tissue (A) Quantitative RT-PCR analyses of RNA isolated from the AGM region, yolk sac (YS), and fetal liver (FL) dissected from embryos at the indicated stage of development using primers to detect transcripts encoding APLNR and its ligands, APELIN and APELA (n = 2). (B–D) UMAP plots of single-cell sequencing data derived from mouse (B) and human (C) AGM tissue and differentiating human iPSCs (D) demonstrating Aplnr/APLNR, Apelin/APELIN, and Apela/APELA expression in endothelial cell clusters. Endothelial cell and HSPC clusters are highlighted with solid and dotted outlines, respectively. A B C D C B D D D B B C Figure 5. 8 Stem Cell Reports j Vol. 16 j 1–14 j April 13, 2021 DISCUSSION We previously identiﬁed the genes encoding APLNR and its ligand APELIN as the most highly upregulated genes when HOXB4 was activated in differentiating mESCs (Jackson et al., 2012). As this increase in expression correlated with an enhanced hematopoietic differentia- tion, we hypothesized that the APLNR signaling pathway could be associated with the production and/or mainte- nance of HSPCs. Here, we show that the Aplnr-tdTomato reporter marks differentiating mesodermal cells that We show that the cells expressing the highest level of the Aplnr-tdTomato reporter also express the highest level of endothelial markers. Our analyses of single-cell sequence data from mouse and human AGM tissue and differenti- ating iPSCs further conﬁrmed Aplnr/APLNR expression in endothelial cells. We noted that a proportion of Aplnr-tdTo- mato-positive cells also expressed hematopoietic markers 8 Stem Cell Reports j Vol. 16 j 1–14 j April 13, 2021 Please cite this article in press as: Jackson et al., Modulation of APLNR Signaling Is Required during the Development and Maintenance of the Hematopoietic System, Stem Cell Reports (2021), https://doi.org/10.1016/j.stemcr.2021.02.003 Please cite this article in press as: Jackson et al., Modulation of APLNR Signaling Is Required during the Development and Maintenance of the Hematopoietic System, Stem Cell Reports (2021), https://doi.org/10.1016/j.stemcr.2021.02.003 Please cite this article in press as: Jackson et al., Modulation of APLNR Signaling Is Required during the Development and Maintenance of the Hematopoietic System, Stem Cell Reports (2021), https://doi.org/10.1016/j.stemcr.2021.02.003 A B C D E F G Figure 6. Addition of Aplnr Ligands to AGM Reaggregate Explant Cultures Results in a Reduction in Transplantable HSCs (A) Schematic of experimental strategy for E9.5 dissection (as example) where the caudal region of the embryo was dissected, tissue dissociated, set up in reaggregate cultures in the presence of Apelin peptides for 7 days and then assayed by for colony formation (CFU-C), ﬂow cytometry, and transplantation into lethally irradiated mice. (B) CFU-C analyses of reaggregate cultures in the absence (0) or presence (100 or 300 nM) of APELIN 13, APELIN 36, or APELA 21. Error bars represent standard deviation of data from three independent experiments A A B B B C D D D C E F G F E F G G E Figure 6. DISCUSSION Addition of Aplnr Ligands to AGM Reaggregate Explant Cultures Results in a Reduction in Transplantable HSCs (A) Schematic of experimental strategy for E9.5 dissection (as example) where the caudal region of the embryo was dissected, tissue dissociated, set up in reaggregate cultures in the presence of Apelin peptides for 7 days and then assayed by for colony formation (CFU-C), ﬂow cytometry, and transplantation into lethally irradiated mice. (B) CFU-C analyses of reaggregate cultures in the absence (0) or presence (100 or 300 nM) of APELIN 13, APELIN 36, or APELA 21. Error bars represent standard deviation of data from three independent experiments. (C) Representative ﬂow cytometry plots of live cells obtained after 7 days of E9.5 aggregate culture in presence of APELIN 13, APELIN 36, or APELA 21 using antibodies against the myeloid markers, CD11B and GR1). (D) Quantiﬁcation of ﬂow cytometry in (C). Figure 6. Addition of Aplnr Ligands to AGM Reaggregate Explant Cultures Results in a Reduction in Transplantable HSCs Figure 6. Addition of Aplnr Ligands to AGM Reaggregate Explant Cultures Results in a Reduction in Transplantable HSCs (A) Schematic of experimental strategy for E9.5 dissection (as example) where the caudal region of the embryo was dissected, tissue dissociated, set up in reaggregate cultures in the presence of Apelin peptides for 7 days and then assayed by for colony formation (CFU-C), ﬂow cytometry, and transplantation into lethally irradiated mice. (B) CFU-C analyses of reaggregate cultures in the absence (0) or presence (100 or 300 nM) of APELIN 13, APELIN 36, or APELA 21. Error bars represent standard deviation of data from three independent experiments. (C) Representative ﬂow cytometry plots of live cells obtained after 7 days of E9.5 aggregate culture in presence of APELIN 13, APELIN 36, or APELA 21 using antibodies against the myeloid markers, CD11B and GR1). (D) Quantiﬁcation of ﬂow cytometry in (C). (legend continued on next page) Stem Cell Reports j Vol. DISCUSSION 16 j 1–14 j April 13, 2021 9 Please cite this article in press as: Jackson et al., Modulation of APLNR Signaling Is Required during the Development and Maintenance of the Hematopoietic System, Stem Cell Reports (2021), https://doi.org/10.1016/j.stemcr.2021.02.003 Please cite this article in press as: Jackson et al., Modulation of APLNR Signaling Is Required during the Development and Maintenance of the Hematopoietic System, Stem Cell Reports (2021), https://doi.org/10.1016/j.stemcr.2021.02.003 The differential requirements for APLNR signaling at pre- cise stages of HSC emergence and maintenance is compara- ble with that reported for other signaling pathways, such as the NOTCH signaling pathway, where stage-speciﬁc effects are observed in the production of adult HSCs in vivo (Souil- hol et al., 2016) and in HSPC production in vitro from ESCs (Huang et al., 2013). implying that it is expressed at the EHT transition stage. This is in keeping with single-cell sequencing analyses of human AGM implying that APLNR was expressed in hemo- genic endothelium (Crosse et al., 2020). Two further studies have reported the downregulation of Aplnr as endo- thelial cells acquire hematopoietic markers during EHT (Bruveris et al., 2020; Oatley et al., 2020). The slight differ- ence in the timing of Aplnr downregulation in cells that have acquired hematopoietic markers that we report might reﬂect the slightly longer half-life of tdTomato compared with Aplnr transcripts. Critical to the interpretation and the relevance of our ﬁndings to hematopoietic development and maintenance in vivo is the anatomical and temporal expression of APLNR ligands. Pertinent to this point is the ﬁnding, using Apln- CreER Rosa26-mTmG reporter mice, that APELIN is ex- pressed in a subpopulation of endothelial cells in adult bone. Conditional knockout of this endothelial cell popu- lation revealed their critical role in hematopoietic regener- ation following myeloablation (Chen et al., 2019). It is not clear which of the ligands are important in the activation of APLNR in different cell types and it is not known which of the many downstream intracellular pathways are respon- sible for exerting the ultimate biological effects. A condi- tional knockout approach for the APLNR receptor and the production of mice carrying the Aplnr-tdTomato re- porter will be instrumental in gaining a fuller understand- ing the complexity of this pathway during hematopoietic development and maintenance in vivo. (E) Proportion of LINSCA+KIT+ HPCs after 7 days of E9.5 aggregate culture in the presence of APELIN 13, APELIN 36, or APELA 21 peptides. (F) Proportion of CD45.2 donor cells present in the peripheral blood of lethally irradiated recipients 14 weeks after transplantation of cells derived from E9 ex vivo reaggregate cultures in the absence (0) or presence of 100 or 300 nM APELIN 13, APELIN 36, or APELA 21. Each data point represents one recipient animal. (G) Proportion of CD45.2 donor cells present in the peripheral blood of lethally irradiated recipients 14 weeks after transplantation of aggregate cultures that consisted of sorted pre-HSC type I and type II cells derived from E11 in coaggregate cultures in the absence (0) or presence of 100 or 300 nM APELIN 13 or APELIN 36. DISCUSSION We observed no change in the numbers or phenotype of CFU-C formation after the addition of APELIN ligands to mouse or human ESCs and iPSCs, whereas a previous study reported that Apelin promoted hematopoietic differentia- tion of human ESCs (Yu et al., 2012). We believe that this apparent discrepancy actually highlights the importance APLNR signaling at the transitional state between endothe- lial and hematopoietic fate because the positive effect of APELIN on hematopoietic differentiation of human ESCs was reﬂected in an increase in the number of bipotential blast colonies (Yu et al., 2012). We have shown that tran- scripts encoding APELIN ligands are present within differ- entiating mouse and human ESC/iPSC cultures, and it is possible that this would mask the effects of exogenous ligand addition. In AGM explant reaggregation cultures we show that addition of APLNR ligands results in a reduction in HSCs capable of long-term reconstitution and a concomitant in- crease in the production of more mature myeloid cells. This ﬁnding suggests that activation of the APLNR pathway is detrimental to the maintenance of HSCs and provides the ﬁrst indication that the precise modulation of APLNR signaling is critical in steady-state hematopoiesis. The in- crease in the production of myeloid cells by APELIN ligands in ex vivo cultures is consistent with our observation that the in vitro production of macrophages from ESCs is signif- icantly impaired in the absence of APLNR and that the addition of APELIN to the CFU-C assay resulted in an in- crease in macrophage colony formation. In future it would be interesting to assess whether the addition of APLNR li- gands affects cell cycle, the speed of which has been shown to be important for the emergence of functional deﬁnitive HSCs within the hematopoietic clusters the AGM region (Batsivari et al., 2017). EXPERIMENTAL PROCEDURES EXPERIMENTAL PROCEDURES mESC Maintenance and Differentiation The mESC line E14IV was maintained in Glasgow Minimum Essen- tial Medium (Gibco) supplemented with 10% fetal calf serum (FCS) (Lonza), 2 mM sodium pyruvate (Gibco), 4 mM L-glutamine (Gibco), 1% non-essential amino acids (Gibco), and 0.1 mM b-mer- captoethanol (Sigma) and 100 U/mL of LIF, as described previously (Jackson et al., 2012). Hematopoietic Differentiation of mESCs Two days before differentiation, mESCs were plated in N2B27 me- dium supplemented with 100 U/mL LIF and 10 ng/mL BMP4 to allow cells to adjust to serum-free conditions. To initiate differen- tiation, at day 0 cells were disaggregated with Accutase (Gibco) then resuspended in N2B27 containing 2.5 ng/mL Activin A, 10 ng/mL basic-helix-loop-helix (bFGF), and 5 ng/mL BMP4 and plated in ultra-low attachment plates (STEMCELL Technologies). Two days later, EBs were plated on gelatinized tissue culture plates in N2B27 containing 10 ng/mL bFGF, 5 ng/mL BMP4, and 15 ng/ (G) Proportion of CD45.2 donor cells present in the peripheral blood of lethally irradiated recipients 14 weeks after transplantation of aggregate cultures that consisted of sorted pre-HSC type I and type II cells derived from E11 in coaggregate cultures in the absence (0) or presence of 100 or 300 nM APELIN 13 or APELIN 36. 10 Stem Cell Reports j Vol. 16 j 1–14 j April 13, 2021 Please cite this article in press as: Jackson et al., Modulation of APLNR Signaling Is Required during the Development and Maintenance of the Hematopoietic System, Stem Cell Reports (2021), https://doi.org/10.1016/j.stemcr.2021.02.003 Please cite this article in press as: Jackson et al., Modulation of APLNR Signaling Is Required during the Development and Maintenance of the Hematopoietic System, Stem Cell Reports (2021), https://doi.org/10.1016/j.stemcr.2021.02.003 Please cite this article in press as: Jackson et al., Modulation of APLNR Signaling Is Required during the Development and Maintenance of the Hematopoietic System, Stem Cell Reports (2021), https://doi.org/10.1016/j.stemcr.2021.02.003 mLVEGF for 6 days, with a medium change on day 4. At day 6 the cells were harvested using Accutase (Gibco), counted, and single- cell suspensions were stained with antibodies for ﬂow cytometry and/or plated in CFU-C assays that were scored 10 days later. In experiments to assess the effects of activating the APLNR pathway, APELIN peptides were added from day 0, speciﬁcally, APELIN36(LVQPRGSRNGPGPWQGGRRKFRRQRPRLSHKGPMPF), its cleaved bioactive pyroglutamyl form (Pyr1), APELIN 13 (QRPRLSHKGPMPF), and APELA 21 (LYRHACPRRRCIPLHSRVPFP) (Phoenix Pharmaceuticals). Macrophage differentiation was car- ried out as described previously (Haideri et al., 2017). were cloned into TOPO blunt after a synthesized U6 promoter (IDT) before sub-cloning into the guide vector, pGL3-U6-sgRNA EGFP (Liu et al., 2018). Juo9 (subclone of E14) murine ESCs (2 3 106) were transfected in suspension with 3 mg AplnrT2ATdTomato HDR donor vector, 1 mg gRNA-A2 plasmid, 1 mgRNA-B2 plasmid, and 1 mg Cas9 Nicka- se(D10A)-GFP plasmid (Addgene no. Generation of Aplnr-tdTomato Reporter ESC Lines AplnrT2ATdTomato HDR donor vector was based on the backbone pBSK-2A-iCre-frtneofrt (a gift from Heiko Lickert), which was di- gested with Not1 and EcoR1 to accept two overlapping PCR frag- ments and a 1 kb 30 arm by Gibson assembly (NEB). Aplnr (NM 011784.3) is a single-exon gene and so the coding re- gion of approximately 1 kb acted as the 50 homology arm. A PCR fragment consisting of the Aplnr coding region and the T2A-encod- ing sequence was generated by ampliﬁcation of BAC BMQ407G118 using primers overlapping the backbone at the 50 end and introducing the T2A sequence at the 30 end. The T2ATdTo- mato sequence was generated by PCR of pTdTomato (Clontech). The integrity of the vector was tested by sub-cloning the entire expression cassette into CAG-IRES-Puro vector and the tdTomato signal conﬁrmed in transient transfected ESCs by ﬂow cytometry. Aplnr (NM 011784.3) is a single-exon gene and so the coding re- gion of approximately 1 kb acted as the 50 homology arm. A PCR fragment consisting of the Aplnr coding region and the T2A-encod- ing sequence was generated by ampliﬁcation of BAC BMQ407G118 using primers overlapping the backbone at the 50 end and introducing the T2A sequence at the 30 end. The T2ATdTo- mato sequence was generated by PCR of pTdTomato (Clontech). The integrity of the vector was tested by sub-cloning the entire expression cassette into CAG-IRES-Puro vector and the tdTomato signal conﬁrmed in transient transfected ESCs by ﬂow cytometry. Guide RNAs (gRNA) directed to the 30 end of the Aplnr sequence were predicted by the Zhang lab algorithm (https://zlab.bio). gRNAs directed to the sequence 30 to the Aplnr stop codon were identiﬁed and tested functionally using the SplitAx assay (Axton et al., 2017). In brief, a SplitAx vector consisting of the sequence encoding the N-terminal of GFP, the Aplnr sequence that covered the region that the gRNAs were directed to, and the sequence en- coding the C-terminal of GFP that was out of frame with the N-ter- minal sequence. After transfection of this SplitAx vector and gRNAs into HEK293 cells, gRNAs were selected if they successfully cut the SplitAx vector and resulted in reconstitution of an in-frame GFP. Transfection of gRNA A2 (TGGGTCAGACCCGCTGCACC) binding to CCTGGTGCAGCGGGTCTGACCCA and gRNA B2 (GGAGAAAGTACAGCCATGCT) binding to AGCATGGCTG- TACTTTCTCC resulted in a positive GFP signal in the SplitAx assay and were used for the subsequent gene targeting. Guides A2 and B2 Hematopoietic Differentiation of mESCs 48140) or Hu Cas9-GFP (Addgene no. 44720) using Xfect stem transfection reagent (Clon- tech) in 100 mL Xfect buffer and 2 mL Xfect polymer for 4 h. mESC medium was added to a volume of 2 mL and plated out overnight in gelatinized 6-well plates. Following overnight incubation, 40,000 GFP+ cells were FACS sorted and plated in a gelatinized 10 cm plate. G418 selection (400 mg/mL) was added the next day and, after 7 days, 28 G418-resistant colonies from the Cas9 D10A transfections and 80 colonies from the WT Cas9 transfections were generated. Thirty-fve colonies were picked and screened by PCR for correctly targeted 30 and 50 ends, which were then conﬁrmed by Southern blot. Animals Staged embryos were obtained by mating C57BL/6 (CD45.2/2), and the morning of discovery of the vaginal plug was designated as day 0.5. For culture and transplantation, embryonic day 9.5 (E9.5) (25–29 sp) and E11.0–E11.5 (>40 sp) samples were used. All experiments with animals were performed under a Project Li- cense granted by the Home Ofﬁce (UK), University of Edinburgh Ethical Review Committee, and conducted in accordance with local guidelines. signal conﬁrmed in transient transfected ESCs by ﬂow cytometry. Guide RNAs (gRNA) directed to the 30 end of the Aplnr sequence were predicted by the Zhang lab algorithm (https://zlab.bio). gRNAs directed to the sequence 30 to the Aplnr stop codon were identiﬁed and tested functionally using the SplitAx assay (Axton et al., 2017). In brief, a SplitAx vector consisting of the sequence encoding the N-terminal of GFP, the Aplnr sequence that covered the region that the gRNAs were directed to, and the sequence en- coding the C-terminal of GFP that was out of frame with the N-ter- minal sequence. After transfection of this SplitAx vector and gRNAs into HEK293 cells, gRNAs were selected if they successfully cut the SplitAx vector and resulted in reconstitution of an in-frame GFP. Transfection of gRNA A2 (TGGGTCAGACCCGCTGCACC) binding to CCTGGTGCAGCGGGTCTGACCCA and gRNA B2 (GGAGAAAGTACAGCCATGCT) binding to AGCATGGCTG- TACTTTCTCC resulted in a positive GFP signal in the SplitAx assay and were used for the subsequent gene targeting. Guides A2 and B2 Human ESC and iPSC Differentiation Human ESC and iPSC Differentiation Human ESCs and human iPSCs were maintained on CELLstart (In- vitrogen)-coated 6-well tissue culture plates (Costar) in StemPro medium comprising DMEM/F12 with GlutaMAX (Invitrogen), 1.8% BSA (Invitrogen), StemPro supplement (Invitrogen), 0.1 mM b-mercaptoethanol (Invitrogen), and 20 ng/mL human bFGF (Invitrogen), as described previously (Jackson et al., 2016; Yang et al., 2017). Cells were passaged mechanically when they reached about 80% conﬂuency using the EZPassage tool (Invitro- gen). In experiments to assess the effects of activating the APLNR pathway, APELIN peptides were added from day 0. The use of hu- man ESCs in haematopoietic differentiation experiments was approved by UK Stem Cell Bank. Generation of Aplnr-Null ESC Lines p gRNA sequences were identiﬁed (http://www.sanger.ac.uk/htgt/ wge/ﬁnd_crisprs). Four gRNAs with PAM sites after the ATG that had a low number of predicted off target binding sites and two guides with the PAM site after the TAA stop codon were selected. Linker nucleotides were added to the ends of the gRNA sequences and both strands synthesized (IDT). gRNAs were annealed and then ligated into the Bbs1 site of linearized pSPCAs9(Guide)-2A- mCherry vector. Combinations of 50 and 30 gRNAs were tested by transient transfection of mESCs and screened for excision of the coding region by PCR. Combinations with the highest level of excision in these transient transfections were used to generate clonal Aplnr-null ESC. ESCs (1 3 106 3 1014) were transfected with the two appropriate pSPCAs9(Guide)-2A-mCherry vectors us- ing Xfect (Clontech). Forty-eight hours after transfection cells were sorted for high levels of mCherry expression and plated onto 10 cm gelatinized dishes. Ten days later individual colonies were picked and screened using primers that spanned the coding region. Generation of Aplnr-tdTomato Reporter ESC Lines Statistics Data on histograms presented as average of at least three indepen- dent experiments ± SD and difference evaluated using t test or Prism software. CFU-C and Endothelial Assays A methylcellulose-based hematopoietic progenitor assay was routinely used to enumerate hematopoietic progenitors from both mouse (MethoCult GF M3434, STEMCELL Technologies) and human (MethoCult H4434, STEMCELL Technologies) differ- entiating PSC. Cells were collected from the differentiating PSC culture and prepared in single-cell suspensions. Methylcellulose medium (1.5 mL) was added into a 35 mm low attachment dish. For each cell treatment, two dishes were set up in parallel at densities of 1 3 105 and 5 3 104 for mouse cells and at 1 3 104 and 5 3 103 for human cells, then incubated at 37C, and then scored between 7–11 and 12–14 days for mouse and human cells, respectively. Colonies were classiﬁed based on the morphology using light microscopy. For endothelial assays, cell was placed on an OP9 cell layer in the presence of 50 ng/mL VEGF as described (Rybtsov et al., 2014). After 11 days, cultures were stained with anti-CD31 antibodies to assess endothelial colonies. Single-Cell RNA Sequencing Analysis The expression proﬁles of genes encoding APLNR and two if its ligands were analyzed in previously published single-cell RNA sequencing datasets. Speciﬁcally, the Aplnr expression proﬁle in FACS-sorted CD31+cKIThiGATA2med cells isolated from the AGM region of E11 embryos was generated using the online browser (https://gottgens-lab.stemcells.cam.ac.uk/DZIERZAK/) (GEO: GSE143637) (Vink et al., 2020). The APLNR expression pro- ﬁle in sorted CD34+ cells from the dissected AGM ventral region from CS16 human embryos was analyzed from a GEO down- loaded dataset (GEO: GSE1518767; sample GSM4592621) (Crosse et al., 2020). Data were analyzed using the R package Seurat 3.2.2, following the standard pipeline (https://www.cell.com/cell/ fulltext/S0092-8674(19)30559-8). In brief, data were normalized using SCTransform, followed by PCA, UMAP, clustering, and TSNE analysis using the ﬁrst 20 principal components. Expres- sion proﬁles in iPSC-derived hemato-endothelial cells were obtained from our previously published dataset (EMBL EBI Arrayexpress: E-MTAB-9295) and the plot was generated using our browser available at lab.antonellaﬁdanza.com (Fidanza et al., 2020). Data and Code Availability Accession numbers for the published RNA sequencing datasets that were analyzed in this study include GEO: GSE143637 (Vink et al., 2020), GEO: GSE1518767; sample GSM4592621 (Crosse et al., 2020), and EMBL EBI Arrayexpress: E-MTAB-9295 (Fidanza et al., 2020). FACS and Analysis Cell suspensions were stained with following antibodies: Ter119- v500 clone (Clone TER-119); anti-CD41-BV421 (brilliant violet 421) or Alexa Fluor 488 (clone MW30reg); anti-CD45 FITC (clone 30-F11); biotinylated anti-VE-cadherin (clone 11.D4.1) followed by incubation with streptavidin-APC (all purchased from BD Pharmingen or BioLegend). Anti-mouse VE-cadherin (VC) anti- body was biotinylated in-house using the FluoReporter Mini- Biotin-XX Protein Labeling Kit (Invitrogen). Cell populations were sorted using a FACSAria II sorter (BD) followed by purity checks. Type I pre-HSCs (Ter119VC+CD41+CD45-) and type II pre-HSCs (Ter119VC+CD41+CD45+) were sorted from E11 AGMs as described previously (Rybtsov et al., 2011). Dead cells were excluded using 7AAD staining and ﬂuorescence minus one control staining was used to deﬁne gating strategies. Data acqui- sition and analyses were performed using Fortessa (BD) and FlowJo software. HSC numbers were assessed using extreme limiting dilution analysis (ELDA) analysis (Hu and Smyth, 2009). Multilineage donor-derived hematopoietic contribution in recipient blood and organs was determined by staining with anti-CD45.1-V450, anti-CD45.2-V500, and lineage-speciﬁc anti-Mac1-ﬂuorescein iso- thiocyanate (FITC), Gr1-PE CD3e-APC, B220-PE-Cy7 monoclonal antibodies (BD Pharmingen). Ex Vivo Maturation of HSC Precursors Ex Vivo Maturation of HSC Precursors E9.5 and E11.5 embryos were dissected as described (Rybtsov et al., 2014). Tissues were incubated with collagenase/dispase solution (0.12 mg/mL) (Roche) at 37C as described (Taoudi et al., 2008), washed, and resuspended in PBS (Sigma) containing 3% FCS and then dissociated by pipetting. After dissociation (and sorting) 1 e.e. of the speciﬁc cell populations (e.g., type I or type II pre- HSCs) were coaggregated with 105 OP9 cells. Five to ten coaggre- gates (i.e., 5–10 e.e.) per experimental variant were cultured in Is- cove’s modiﬁed Dulbecco’s medium (Invitrogen), with 20% of pre-selected, heat-inactivated FCS, L-glutamine, and penicillin/ streptomycin supplemented with murine recombinant cytokines (SCF, IL3, and Flt3) each at 100 ng/mL (PeproTech) and various concentrations of APLNR ligand peptides, including APELIN 36 (LVQPRGSRNGPGPWQGGRRKFRRQRPRLSHKGPMPF), its cleaved bioactive pyroglutamyl form (Pyr1) APELIN 13 (QRPRLSHKGPMPF), and APELA 21 (LYRHACPRRRCIPLHSRVPFP) (Phoenix Pharmaceuticals). Coaggregates were cultured on ﬂoating 0.8 mm AAWP 25 mm nitrocellulose membranes (Milli- pore) for 6 days then dissociated using collagenase/dispase as described (Rybtsov et al., 2014). Single-Cell RNA Sequencing Analysis Long-Term Repopulation Assay and Blood Chimerism Analysis CD45.2/2 cells were injected into irradiated 2- to 3-month-old Bl/ 6J CD45.1/2 heterozygous recipients along with 100,000 CD45.1/ 1 nucleated bone marrow carrier cells per recipient. Recipients were g-irradiated using two doses (600 + 550 rad) separated by 3 h. For day 9.5 culture, one embryo equivalent (e.e.) of the 7 day cultured cells was injected. E11 AGM-sorted cells were injected af- ter 5 days of OP9-coaggregate culture at a dose of 1 e.e./recipient. Stem Cell Reports j Vol. 16 j 1–14 j April 13, 2021 11 Please cite this article in press as: Jackson et al., Modulation of APLNR Signaling Is Required during the Development and Maintenance of the Hematopoietic System, Stem Cell Reports (2021), https://doi.org/10.1016/j.stemcr.2021.02.003 Please cite this article in press as: Jackson et al., Modulation of APLNR Signaling Is Required during the Development and Maintenance of the Hematopoietic System, Stem Cell Reports (2021), https://doi.org/10.1016/j.stemcr.2021.02.003 Please cite this article in press as: Jackson et al., Modulation of APLNR Signaling Is Required during the Development and Maintenance of the Hematopoietic System, Stem Cell Reports (2021), https://doi.org/10.1016/j.stemcr.2021.02.003 Donor-derived chimerism was evaluated in peripheral blood at 6 and 14 weeks post transplantation. Erythrocytes were lysed using Pharm Lyse (BD Biosciences), and non-speciﬁc binding was blocked with an anti-CD16/32 (Fc-block) followed by staining with anti-CD45.1-APC (clone A20) and anti-CD45.2-PE (clone 104) monoclonal antibodies (eBioscience). Cell populations were identiﬁed as CD45.1-PE+ (donor), CD45.1-PE+CD45.2-APC+ (recipient), CD45.1-APC+ (carrier) using FACSCalibur or Fortessa (BD Biosciences). FACS and Analysis ACKNOWLEDGMENTS This work was funded by Bloodwise (to M.J.), the Wellcome Trust (to A.F. and A.H.T.), and the Medical Research Council (to S.R.). We thank Fiona Rossi and Claire Cryer for ﬂow cytometry. The graphical abstract was created using Biorender.com. Devic, E., Rizzoti, K., Bodin, S., Knibiehler, B., and Audigier, Y. (1999). Amino acid sequence and embryonic expression of msr/ Devic, E., Rizzoti, K., Bodin, S., Knibiehler, B., and Audigier, Y. (1999). Amino acid sequence and embryonic expression of msr/ apj, the mouse homolog of Xenopus X-msr and human APJ. Mech. Dev. 84, 199–203. apj, the mouse homolog of Xenopus X-msr and human APJ. Mech. Dev. 84, 199–203. Received: June 10, 2020 Ditadi, A., Sturgeon, C.M., and Keller, G. (2017). A view of human haematopoietic development from the Petri dish. Nat. Rev. Mol. Cell Biol. 18, 56–67. Revised: February 4, 2021 Revised: February 4, 2021 Accepted: February 5, 2021 Accepted: February 5, 2021 Published: March 4, 2021 Published: March 4, 2021 Dzierzak, E., and Medvinsky, A. (2008). The discovery of a source of adult hematopoietic cells in the embryo. Development 135, 2343– 2346. AUTHOR CONTRIBUTIONS M.J., A.H.T., R.A., A.F., S.R., M.K., and S.M. performed the experi- ments. M.J., A.F., and L.M.F. planned the experiments and wrote the manuscript. A.M. and T.B. provided intellectual input to the execution of the experiments and interpretation of the results. Dege, C., Fegan, K.H., Creamer, J.P., Berrien-Elliott, M.M., Luff, S.A., Kim, D., Wagner, J.A., Kingsley, P.D., McGrath, K.E., Fehniger, T.A., et al. (2020). Potently cytotoxic natural killer cells initially emerge from erythro-myeloid progenitors during mammalian development. Dev. Cell 53, 229–239.e7. SUPPLEMENTAL INFORMATION Supplemental Information can be found online at https://doi.org/ 10.1016/j.stemcr.2021.02.003. 12 Stem Cell Reports j Vol. 16 j 1–14 j April 13, 2021 Please cite this article in press as: Jackson et al., Modulation of APLNR Signaling Is Required during the Development and Maintenance of the Hematopoietic System, Stem Cell Reports (2021), https://doi.org/10.1016/j.stemcr.2021.02.003 Please cite this article in press as: Jackson et al., Modulation of APLNR Signaling Is Required during the Development and Maintenance of the Hematopoietic System, Stem Cell Reports (2021), https://doi.org/10.1016/j.stemcr.2021.02.003 Please cite this article in press as: Jackson et al., Modulation of APLNR Signaling Is Required during the Development and Maintenance of the Hematopoietic System, Stem Cell Reports (2021), https://doi.org/10.1016/j.stemcr.2021.02.003 G protein-coupled receptor APJ and its ligand apelin act down- stream of Cripto to specify embryonic stem cells toward the cardiac lineage through extracellular signal-regulated kinase/p70S6 kinase signaling pathway. 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https://openalex.org/W4387102639	https://sp.copernicus.org/articles/sp-osr7.pdf	English	null	7th edition of the Copernicus Ocean State Report (OSR7)	null	2,023	cc-by	139,156	7 OCEAN STATE REPORT COPERNICUS 7th edition of the Copernicus Ocean State Report \| 2023 7th edition 7th edition About the Copernicus Ocean State Report As part of the Copernicus Marine Service, the annual Copernicus Ocean State Report (OSR) launched in 2015 is the key tool of its ocean reporting framework. The OSR reports on the state, variability, and ongoing chang- es in the marine environment of the global ocean and the European regional seas over the past decades up to close to real time. Using observation-based (remote sensing, in situ) and ocean reanalysis data, the OSR provides a comprehensive 4-dimensional (latitude, longitude, depth, and time) analysis of the Blue, Green, and White Ocean. The OSR is intended to act as a reference, providing a unique ocean monitoring dashboard for the scientific community and for policy makers and others with decision making responsibilities. Editorial The Copernicus Ocean State Report (OSR) gathers an ensemble of peer-review publications that analyse the state, variability, and trend of the physics and biogeochemistry of the oce- an and sea ice (respectively the blue, green, and white ocean). Contributions to the OSR are short, cover the global ocean and European regional seas, and target a wide audience made up of the scientific community, policy makers, industries, and the general public. They are based on an in-depth analysis of Copernicus Marine products (and others) constituting mo- del reanalysis and satellite and in situ data available over several decades back in time up to 1 year before the publication of the OSR. Copernicus Marine products are regularly updated with the best available information and produced according to a coordinated and high-quality control protocol. All products and their documentation are openly available from https://marine.copernicus.eu/. This guarantees an assessment based on consistent and well-documented datasets. The Copernicus OSR is produced annually, and its prepara- tion is coordinated by the OSR team at Mercator Ocean International. This 7th issue of the Copernicus OSR gathers 14 peer review papers covering the European regional seas (9) and the global ocean (5) with contributions on the blue (10) and green (4). The main findings of OSR7 are summarized in Figures 1 and 2 and here below according to the topic they addressed: 1) identification and impact of extreme/unusual events (5), 2) identification of long-term trends (5), 3) new methodology/indicators for ocean monito- ring (2), and 4) analysis of ocean variability (2). A detailed description can be found in von Schuckmann et al. (2023). About State of the Planet State of the Planet (SP) is a journal dedicated to the publication of scientific syn- thesis reports and assessments on all subjects of the Earth and environmental sciences. In a rapidly changing world, expert-based assessments of academic findings curated for a wider audience to support decision making, science com- munication, education, and funder mandates are becoming more and more widespread. Such reports are extensive science community efforts offering timely, state-of-the-art insight into a specific field of the Earth sciences. State of the Planet is open to any reporting and assessment initiative by (inter-) govern- mental agencies, environmental services, learned societies or associations of researchers that aim to publish on a regular basis. publications@copernicus.org http://publications.copernicus.org publications@copernicus.org http://publications.copernicus.org This document was produced with funding by the European Union. Views and opinions expressed are however those of the author(s) only and the European Commission cannot be held responsible for any use which may be made of the information contained therein. Cover photo by Ivan Bandura on Unsplash von Schuckmann, K., Moreira, L., Le Traon, P.-Y., Grégoire, M., Marcos, M., Staneva, J., Brasseur, P., Garric, G., Lionello, P., Karstensen, J., and Neukermans, G. (Eds.): 7th edition of the Copernicus Ocean State Report (OSR7), Copernicus Publications, State Planet, 1-osr7, https://doi.org/10.5194/sp-1-osr7, 2023. von Schuckmann, K., Moreira, L., Le Traon, P.-Y., Grégoire, M., Marcos, M., Staneva, J., Brasseur, P., Garric, G., Lionello, P., Karstensen, J., and Neukermans, G. (Eds.): 7th edition of the Copernicus Ocean State Report (OSR7), Copernicus Publications, State Planet, 1-osr7, https://doi.org/10.5194/sp-1-osr7, 2023. Ocean variability Baker et al. (2023) investigate the variability of the South Atlantic meridional overturing circulation and the meridional heat transport, focusing on the period from 2013 to 2017. The study shows that the South Atlantic transport variability is still uncertain and recom- mends improved monitoring and modelling. Oikonomou et al. (2023) explore wave energy resources at the basin scale, along with the dominant wave regime. Results suggest that although the basin is characterized by low energy potential and large energy flux variability, it could serve as a deployment zone for low-power devices due to low peak period variability and high site accessibility levels. Monitoring Lorente et al. (2023) propose a new indicator for the monitoring of coastal upwelling intensi- ty tested in two pilot areas (i.e. the north-western Iberian Peninsula and the Bay of Biscay) in view of its application at larger scales (the coastal global ocean). Loisel et al. (2023) propose a new algorithm for the monitoring of suspended materials’ composition (organic and inorga- nic) from space. The algorithm is tested in the English Channel and the southern North Sea, with the perspective of being extended to the global coastal ocean. Long-term trend De Pascual-Collar et al. (2023) detect a long-term warming trend in waters below 150m in the Iberia–Biscay–Ireland region over 1993–2021. Aydogdu et al. (2023) show that the salinity in the upper 300m of the Mediterranean Sea has increased in the last 3 decades, with a basin average trend in the range from 0.0020 to 0.0110 psu per year depending on the product considered, with higher values in the eastern basin. Raudsepp et al. (2023) presented an overall decrease in ocean freshwater content in the Baltic Sea over the last 2 decades, with distinct regional patterns. Di Biagio et al. (2023) identify a potential regime shift that happe- ned over 1999–2021 in the regional circulation of the southern Adriatic Sea, with potential implications for the ventilation of the Ionian–Adriatic. Xi et al. (2023) find that the mean status, trends, and phenology of phytoplankton functional types in the Atlantic Ocean do not change significantly over 2002–2021, except for prokaryotes, which slightly decline. The study highlights an abrupt increase in diatoms during the years 2018 and 2019 in the North Atlantic. Extreme/Unusual events Peal et al. (2023) show that marine heatwaves have become more frequent and more ex- tended globally, whereas marine cold spells became less frequent over 1982 to 2021, ex- cept in the Southern Ocean, where the reverse is observed. Mayer et al. (2023) show a record-low biennial reduction in oceanic heat exchange across the Greenland–Scotland Rid- ge around the year 2018. This reduction amounted to 4%–9% of the usual heat exchange with respect to the reference period 1993–2020. Mourre et al. (2023) show that an intense and long-lasting storm in the Balearic Islands resulted in important modifications of the physical conditions of the region with intense upwelling, a reversal of the regional current system, and extreme cold surface temperatures. Renshaw et al. (2023) highlight unusual coccolithophore blooms in summer 2021 in Scottish waters and explain their occurrence due to unusual weather conditions. Gramcianinov et al. (2023) highlight that extreme wave events have impacted the eastern coast of South America and offshore regions and have created coastal hazards in the São Paulo area. https://sp.copernicus.org/articles/1-osr7 eISSN 2752-0706 Published by Copernicus Publications. Karina von Schuckmann, Lorena Moreira, and Pierre-Yves Le Traon Mercator Ocean International, Toulouse, France Published: 27 September 2023 cus Ocean State Report (OSR7) Changes in the state of the ocean affect the well-being of the planet, its ecosystems, and human societies (IPCC, 2019). The ocean is home to a vast array of plant and ani- mal species, many of which are still undiscovered (Census of Marine Life, 2010), and it is hence critical in support- ing global biodiversity and sustaining complex ecosystems (Pörtner et al., 2021; IPCC, 2022). The ocean supports vari- ous industries, such as ﬁshing, shipping, tourism, and renew- able and non-renewable resources, and provides job oppor- tunities, income, and economic growth (OECD, 2016; Euro- pean Commission, 2022). The ocean plays a vital role in reg- ulating Earth’s climate. It acts as a massive heat sink, absorb- ing and storing vast amounts of surplus heat accumulated in the Earth system from human activities (von Schuckmann et al., 2023). It absorbs signiﬁcant quantities of anthropogenic carbon through physical and biological processes, helping to mitigate climate change by reducing the greenhouse gas concentration in the atmosphere (Friedlingstein et al., 2022; Crisp et al., 2022). The ocean is intricately affected by cli- mate change. Rising sea levels, warming, deoxygenation and acidiﬁcation of the ocean, changing currents, and loss of sea ice and biodiversity all pose signiﬁcant risks to coastal com- munities, infrastructure, economies, and vulnerable ecosys- tems (IPCC, 2021, 2022). In addition, overexploitation and ocean pollution, including plastic waste and chemical con- taminants, can harm both marine life and human health, pos- ing risks through the food chain and direct exposure (e.g., Landrigan et al., 2020; Lamb et al., 2018). The degradation of the ocean and increased pressure on its functioning and services can lead to economic losses, unemployment, and reduced opportunities for sustainable development (OECD, 2016). Protecting and preserving the ocean is essential for ensuring a sustainable future, maintaining biodiversity, reg- ulating climate, supporting economies, and safeguarding hu- man health and well-being (IPCC, 2019; Pörtner et al., 2021). Monitoring and reporting on any change in the state of the ocean can help to achieve this goal, while at the same time it allows developing timely strategies for adaptation measures as well as to raise awareness, inform decision-making, en- courage action to protect and conserve the ocean, and hence transition towards sustainable ocean stewardship (e.g., von Schuckmann et al., 2020; Blunden and Boyer, 2022; IOC- UNESCO, 2022). The editors Marilaure Grégoire, Pierre Brasseur, Gilles Garric, Johannes Karstensen, Piero Lionello, Marta Marcos, Griet Neukermans, and Joanna Staneva. Chapter 1.1 – 7th edition of the Copernicus Ocean State Report (OSR7) Chapter 1.1 – 7th edition of the Copernicus Ocean State Planet, 1-osr7, 1, 2023 https://doi.org/10.5194/sp-1-osr7-1-2023 © Author(s) 2023. This work is distributed under the Creative Commons Attribution 4.0 License. ter 1.1 – 7th edition of the Copernicus Ocean State Report (O Introduction to the 7th edition of the Copernicus Ocean State Report (OSR7) n of the Copernicus Ocean State Report (OSR7) Karina von Schuckmann, Lorena Moreira, and Pierre-Yves Le Traon Mercator Ocean International, Toulouse, France Correspondence: Karina von Schuckmann (karina.von.schuckmann@mercator-ocean.fr) Karina von Schuckmann, Lorena Moreira, and Pierre-Yves Le Traon Mercator Ocean International, Toulouse, France Most recently, on 19 June 2023, the Treaty of the High Seas was adopted by consensus and standing ova- tion during the United Nations meeting in New York. This treaty, also known as BBNJ (Biodiversity Beyond National Jurisdiction), is key to protecting the ocean, promoting eq- uity and fairness, tackling environmental degradation, ﬁght- ing climate change, and preventing biodiversity loss in the high seas (An historic achievement: Treaty of the High Seas is adopted, 2023). Monitoring and reporting on any change in the state of the ocean can help to achieve this goal, while at the same time it allows developing timely strategies for adaptation measures as well as to raise awareness, inform decision-making, en- courage action to protect and conserve the ocean, and hence transition towards sustainable ocean stewardship (e.g., von Schuckmann et al., 2020; Blunden and Boyer, 2022; IOC- UNESCO, 2022). Most recently, on 19 June 2023, the Treaty of the High Seas was adopted by consensus and standing ova- tion during the United Nations meeting in New York. This treaty, also known as BBNJ (Biodiversity Beyond National Jurisdiction), is key to protecting the ocean, promoting eq- uity and fairness, tackling environmental degradation, ﬁght- ing climate change, and preventing biodiversity loss in the high seas (An historic achievement: Treaty of the High Seas is adopted, 2023). Regular reporting on the state, variability, and change of the ocean provides state-of-the-art science-driven ﬁndings to various stakeholders, including policymakers, scientists, in- dustries, and the general public. Ocean reporting involves several components as part of a so-called added value chain (von Schuckmann et al., 2020). Firstly, data collection from different methods including satellite observations (e.g., sea level, sea surface temperature (SST), sea surface salinity (SSS), ocean color, waves) and in situ measurements – au- tonomous, moored, and taken during research expeditions – are paramount for building data products from the global to regional scale (IOC, 2019). Reprocessed data are also used to feed ocean reanalyses that combine them with numeri- cal models through data assimilation schemes (Decade Col- laborative Centre for Ocean Prediction, DCC-OP, 2023). All these different products build the baseline of the Copernicus Marine Service and include information about ocean physics (blue ocean; e.g., temperature, salinity, currents, waves), bio- geochemistry and biodiversity (green ocean; e.g., chlorophyll CHAPTER1.1 CHAPTER1.1 K. von Schuckmann et al.: Introduction to the 7th edition of the Copernicus Ocean State Report 2 (see de Pascual-Collar et al. Karina von Schuckmann, Lorena Moreira, and Pierre-Yves Le Traon Mercator Ocean International, Toulouse, France in OSR7 Chap. 2), while devel- oping a multi-product approach based on data from obser- vations and ocean reanalysis systems for the Iberia–Biscay– Ireland region. Over the period 1993–2021, the results reveal that the upper near-surface layer of the ocean (0–150 m) is dominated by natural variations at the interannual to decadal scale and that the long-term ocean warming trend is emerg- ing in the intermediate to deep-ocean layers (> 150 m) in this region. This trend amounts to about 50 % of the global ocean warming trend and is strongly affected by interannual- to decadal-scale variations. Moreover, the study could ele- vate the major players of these changes that are linked to the Sub-Arctic Intermediate Water and Mediterranean Outﬂow Water variations, leveraging the role of major characteristics of the ocean in redistributing heat and hence triggering re- gional variations of ocean warming remotely, i.e., from the Mediterranean Sea and the North Atlantic Ocean. a, ocean pH), and sea ice parameters (white ocean; e.g., sea ice concentration, extent, or thickness), which are all rele- vant to understanding and reporting on the ocean state (Le Traon et al., 2019). Combined with scientiﬁc expertise, they provide the fundamental ingredients for the development of ocean monitoring indicators that are ﬁt for purpose for ocean reporting. At this higher level of the added value chain, the tools then help identify trends, patterns, and changes in the ocean environment triggered by climate change and other human-made pressures, climate variability, and ocean pro- cesses. This 7th edition of the Copernicus Ocean State Report covers a wide range of topics tackling variations, trends, and new tools for socioeconomic applications for Europe and on a global scale. The studies are built on the suite of Copernicus Marine products and are complemented with other products from the global community. In OSR7, a new scientiﬁc method has been developed for monitoring coastal upwelling, a process which is a major communicator between offshore waters and coastal ecosys- tems (see Lorente et al. in OSR7 Chap. 2). Any change in coastal upwelling can have implications for, for example, wa- ter quality, ﬁsheries, and aquaculture production in coastal areas (Sydeman et al., 2014). 1 The ocean in Europe During summer 2021, two unusual coccolithophore blooms were observed in Scottish waters, one in the Clyde Sea and the other by the east coast of the Shetland Islands. These blooms have attracted lot of public attention because they turned the sea a turquoise color (BBC News, 2023). The OSR7 is taking a close look at these events back-boned by the use of reanalysis and satellite data to examine the en- vironment that led to these blooms. Unusual weather condi- tions were a contributory factor in both cases, leading to high concentrations of nutrients in summer as cold temperatures restricted the regular spring bloom of diatoms in this area. These factors provide ideal conditions for coccolithophores to ﬂourish as temperatures and sunlight increase (see Ren- shaw et al. in OSR7 Chap. 4). Widespread routine monitor- ing of nutrient levels and phytoplankton components could greatly help in understanding future blooms. The OSR7 has also analyzed the interference of extreme events with ocean processes, like the coastal upwelling of the Balearic Islands (see Mourre et al. in OSR7 Chap. 4), and has demonstrated the implications of long-lasting storms of record strength on the ocean and coasts. In November 2021, an intense and long-lasting storm named “Blas” hit the area of the Balearic Islands, which resulted in intense upwelling along the northwestern coasts of the islands of Mallorca and Ibiza, as well as a reversal of the regional current system. These record implications have in turn re- sulted in extreme cold coastal surface temperatures up to 6 ◦C colder than usual. This study demonstrates the beneﬁts of op- erational oceanography for the characterization of extreme events through the provision of time series of high-resolution modeling results in coastal areas. The OSR7 explored dissolved oxygen variations in the Mediterranean Sea, more precisely in the southern Adriatic Sea, over the period 1999–2021, which is an essential un- dertaking to further enhance our understanding of marine ecosystem functioning and for assessing the marine environ- mental status (see Di Biagio et al. in OSR7 Chap. 3). The re- sults, derived from a regional ocean reanalysis system, high- light the critical role of heat ﬂuxes, ocean currents, and bio- logical production in driving dissolved oxygen variations in this area. A potential regime shift in the regional circulation was identiﬁed. Karina von Schuckmann, Lorena Moreira, and Pierre-Yves Le Traon Mercator Ocean International, Toulouse, France The method incorporates infor- mation on ocean currents, wind, sea level pressure, and sea surface temperature, and it has been developed based on var- ious data products ranging from direct coastal observations provided by a high-frequency radar (HFR), buoy measure- ments, and ocean reanalysis. Two pilot areas have been cho- sen, i.e., the northwestern Iberian Peninsula and the Bay of Biscay. This new indicator, the so-called coastal upwelling index, has been scientiﬁcally tested in OSR7, and its proof of concept revealed that this new method can be used for di- rect upwelling monitoring over any coastal area of the global ocean. 1 The ocean in Europe It can have substantial implications for the ventilation of the Ionian–Adriatic Ocean region and hence for marine organisms, and it needs to be further analyzed in future studies. Change in ocean salinity can have important implications for ocean density and ocean currents, the water cycle, and ocean biodiversity. The OSR7 has tackled an important indi- cator based on ocean salinity in the Mediterranean Sea during the last decades using Copernicus Marine reanalysis and ob- Ocean warming is one of the major indicators of monitor- ing the state and evolution of climate change (von Schuck- mann et al., 2016). The OSR7 is tackling this indicator aim- ing to regionalize capacities for reporting on ocean warming https://doi.org/10.5194/sp-1-osr7-1-2023 State Planet, 1-osr7, 1, 2023 CHAPTER1.1 n Schuckmann et al.: Introduction to the 7th edition of the Copernicus Ocean State Report K. von Schuckmann et al.: Introduction to the 7th edition of the Copernicus Ocean State Report 3 Figure 1. Overview of major outcomes for the European regional seas in the 7th edition of the Copernicus Ocean State Report. Figure 1. Overview of major outcomes for the European regional seas in the 7th edition of the Copernicus Ocean State Report. Figure 1. Overview of major outcomes for the European regional seas in the 7th edition of the Copernicu fraction of suspended material needed to monitor particle dynamics and biogeochemical cycles in coastal waters (see Loisel et al. in OSR7 Chap. 3). This new algorithm is based on a neural network approach and has been applied as a test bed to satellite observations from ocean color radiometry in the English Channel and the southern North Sea. The scien- tiﬁc evaluations as done in OSR7 have shown that this algo- rithm can be directly applied to all ocean color radiometry measurements, which enables routine monitoring of particu- late composition over the global coastal ocean. servation products (see Aydogdu et al. in OSR7 Chap. 2). The results show that, overall, salinity has increased in the upper (< 300 m) Mediterranean Sea at particularly strong rates in the eastern basin. A similar analysis has been performed for the Baltic Sea area (see Raudsepp et al. in OSR7 Chap. 2). Particularly, freshwater content has been calculated from the Copernicus Marine regional reanalysis, which allows mon- itoring the speciﬁc hydro-physical conditions of each sub- basin of the Baltic Sea. 1 The ocean in Europe Results show that the total freshwater content in the Baltic Sea shows a steady decrease on aver- age over the past 2 decades. However, at the regional scale, changes in freshwater content are not spatially uniform and are affected in the Baltic Sea by different processes, such as changes in ocean circulation, precipitation, river runoff, and sea ice formation. For example, the Gulf of Bothnia shows a freshwater content increase, and the freshwater content in the Baltic proper decreased over the past 2 decades. https://doi.org/10.5194/sp-1-osr7-1-2023 2 The ocean around the globe Climate change is causing extreme climate events to become more frequent and more severe (Seneviratne et al., 2021). Marine heatwaves and marine cold spells are prolonged; dis- crete periods of anomalously high or low ocean temperatures have wide-ranging impacts from dramatic shifts in biodiver- sity to changes in ﬁshery yields. In OSR7, global sea surface temperature measurements have been used to study the in- tensity and frequency of marine heatwaves and marine cold Monitoring coastal waters is of uppermost importance to provide insight into changes in these complex and highly variable environments. In OSR7, a new algorithm has been developed to estimate the organic and inorganic particulate https://doi.org/10.5194/sp-1-osr7-1-2023 State Planet, 1-osr7, 1, 2023 CHAPTER1.1 CHAPTER1.1 K. von Schuckmann et al.: Introduction to the 7th edition of the Copernicus Ocean State Report 4 Figure 2. Overview of major outcomes for the global ocean in the 7th edition of the Copernicus Ocean State Report. Figure 2. Overview of major outcomes for the global ocean in the 7th edition of the Copernicus Ocean State Report. spells over several decades from 1982 to 2021 across differ- ent categories from moderate (category 1) to extreme (cate- gory 4). The ﬁndings show that in large areas of the ocean it can be said with 99 % conﬁdence that marine heatwaves have become more frequent globally, whereas marine cold spells have become less frequent (see Peal et al. in OSR7 Chap. 2). An exception is the Southern Ocean where marine heatwaves are becoming slightly less frequent and marine cold spells are becoming more frequent. The most extreme events in cat- egories 3 and 4 follow this trend, but at a lower rate. This evolution for marine heatwaves is accompanied by an even faster trend with respect to their spatial extension, whereas the spatial extent of marine cold spells remained nearly con- stant. and near-real-time data have been used to investigate extreme wave indicators impacting offshore regions and the coasts in this area and have provided a comprehensive overview of the regional wave climate across timescales and space scales. In addition, national monitoring and warning system data in the São Paulo state area have been combined with Copernicus Marine data, and these results reveal that the increasing num- ber of coastal hazards in this area are strongly linked to the increasing number of extreme wave events from the coast to adjacent offshore regions. References Decade Collaborative Centre for Ocean Predic- tion (DCC-OP): https://oceandecade.org/actions/ decade-collaborative-centre-for-ocean-prediction-dcc-op/, last access: 12 June 2023. European Commission: The EU blue economy report 2022, Publications Ofﬁce of the European Union, Luxembourg, https://doi.org/10.2771/793264, 2022. Friedlingstein, P., O’Sullivan, M., Jones, M. W., Andrew, R. M., Gregor, L., Hauck, J., Le Quéré, C., Luijkx, I. T., Olsen, A., Pe- ters, G. P., Peters, W., Pongratz, J., Schwingshackl, C., Sitch, S., Canadell, J. G., Ciais, P., Jackson, R. B., Alin, S. R., Alkama, R., Arneth, A., Arora, V. K., Bates, N. R., Becker, M., Bellouin, N., Bittig, H. C., Bopp, L., Chevallier, F., Chini, L. P., Cronin, M., Evans, W., Falk, S., Feely, R. A., Gasser, T., Gehlen, M., Gkritza- lis, T., Gloege, L., Grassi, G., Gruber, N., Gürses, Ö., Harris, I., Hefner, M., Houghton, R. A., Hurtt, G. C., Iida, Y., Ilyina, T., Jain, A. K., Jersild, A., Kadono, K., Kato, E., Kennedy, D., Klein Goldewijk, K., Knauer, J., Korsbakken, J. I., Landschützer, P., Lefèvre, N., Lindsay, K., Liu, J., Liu, Z., Marland, G., Mayot, N., McGrath, M. J., Metzl, N., Monacci, N. M., Munro, D. R., Nakaoka, S.-I., Niwa, Y., O’Brien, K., Ono, T., Palmer, P. I., Pan, N., Pierrot, D., Pocock, K., Poulter, B., Resplandy, L., Robert- son, E., Rödenbeck, C., Rodriguez, C., Rosan, T. M., Schwinger, J., Séférian, R., Shutler, J. D., Skjelvan, I., Steinhoff, T., Sun, Q., Sutton, A. J., Sweeney, C., Takao, S., Tanhua, T., Tans, P. P., Tian, X., Tian, H., Tilbrook, B., Tsujino, H., Tubiello, F., van der Werf, G. R., Walker, A. P., Wanninkhof, R., Whitehead, C., Willstrand Wranne, A., Wright, R., Yuan, W., Yue, C., Yue, X., Zaehle, S., Zeng, J., and Zheng, B.: Global Carbon Budget 2022, Earth Syst. Sci. Data, 14, 4811–4900, https://doi.org/10.5194/essd-14-4811- 2022, 2022. Ocean phytoplankton prevails in the upper part of the ocean, where sunlight penetrates the water, and constitutes the base of the marine food web fueling ﬁsheries and regulat- ing key biogeochemical processes (Xi et al., 2021). There are different classes of phytoplankton – so-called phytoplankton functional types – which include haptophytes, prokaryotes, dinoﬂagellate, and diatoms. OSR7 investigated the mean sta- tus, trends, phenology, and most recent anomalies in the At- lantic Ocean (see Xi et al. in OSR7 Chap. 2). Their phenol- ogy ranges with different climate zones, reaching seasonal maximum blooms (Fig. 1). References this region are available. The ﬁndings have manifested a sur- prising discrepancy in seasonal to multi-annual circulation change between the observations and model results. This pre- vents a reliable estimate of how the meridional overturning circulation and heat transport are changing at the latitude that connects the Atlantic Ocean to the Southern Ocean. The mean and variation of real-world South Atlantic transports and the amplitude of their ﬂuctuations are uncertain. This in turn provides fundamental guidance for future strategies as well as investments in urgently needed sustained monitoring and improved modeling in this important area of the ocean for global climate. An historic achievement: Treaty of the High Seas is adopted: https://oceans-and-ﬁsheries.ec.europa.eu/news/ historic-achievement-treaty-high-seas-adopted-2023-06-19_en, last access: 20 June 2023. BBC News: Marine algae turned Scotland’s water bright blue, BBC News, https://www.bbc.com/news/uk-scotland-57720790, last access: 12 June 2023. Blunden, J. and Boyer, T.: State of the Climate in 2021, B. Am. Meteorol. Soc., 103, S1–S465, https://doi.org/10.1175/2022BAMSStateoftheClimate.1, 2022. g Given its crucial importance, circulation variations linked to the meridional overturning circulation have also been analyzed in the North Atlantic in OSR7 (see Mayer et al. in OSR7 Chap. 4). Particularly, focus had been set on the oceanic exchange across the Greenland–Scotland Ridge, which not only links to the meridional overturning circu- lation, but is also shaping Arctic climate. The results of this study have uncovered a record-low biennial reduction in oceanic heat exchange across the Greenland–Scotland Ridge around the year 2018. This reduction, amounting to 4 %–9 % of the usual heat exchange with respect to the reference pe- riod 1993–2020, occurred because of a reduction in the in- ﬂow of water from the Atlantic into the Arctic area through the Faroe–Shetland branch, which was exacerbated by the unusually cold temperatures of Atlantic Water arriving at the Greenland–Scotland Ridge. These low temperatures have been triggered by a stronger North Atlantic subpolar gyre varying naturally at interannual to decadal timescales. Census of Marine Life: First Census of Marine Life 2010: High- lights of a Decade of Discovery, edited by: Ausubel, J. H., Crist, D. T., and Waggoner, P. E., Census of Marine Life, ISBN 978-1- 4507-3102-7, 2010. Crisp, D., Dolman, H., Tanhua, T., McKinley, G. A., Hauck, J., Bastos, A., Sitch, S., Eggleston, S., and Aich, V.: How Well Do We Understand the Land-Ocean-Atmosphere Carbon Cycle?, Rev. Geophys., 60, e2021RG000736, https://doi.org/10.1029/2021RG000736, 2022. References The results also reveal that most phytoplankton functional types remained stable over the pe- riod 2002–2021, except for prokaryotes, which slightly de- clined over this period. The study also uncovered, for exam- ple, an abrupt increase in diatoms during the years 2018 and 2019 in the northern part of the basin. Arneth, A., Arora, V. K., Bates, N. 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D., Skjelvan, I., Steinhoff, T., Sun, Q., N., Pierrot, D., Pocock, K., Poulter, B., Resplandy, L., Robert- son, E., Rödenbeck, C., Rodriguez, C., Rosan, T. M., Schwinger, son, E., Rödenbeck, C., Rodriguez, C., Rosan, T. M., Schwinger, J., Séférian, R., Shutler, J. D., Skjelvan, I., Steinhoff, T., Sun, Q., J., Séférian, R., Shutler, J. D., Skjelvan, I., Steinhoff, T., Sun, Q., Sutton, A. J., Sweeney, C., Takao, S., Tanhua, T., Tans, P. P., Tian, X., Tian, H., Tilbrook, B., Tsujino, H., Tubiello, F., van der Werf, G. R., Walker, A. P., Wanninkhof, R., Whitehead, C., Willstrand Wranne, A., Wright, R., Yuan, W., Yue, C., Yue, X., Zaehle, S., Zeng, J., and Zheng, B.: Global Carbon Budget 2022, Earth Syst. Sci. Data, 14, 4811–4900, https://doi.org/10.5194/essd-14-4811- 2022, 2022. Disclaimer. Please note that this article has undergone editorial review only. IOC: The Global Ocean Observing System 2030 Strategy, IOC, Paris, IOC Brochure 2019-5, IOC/BRO/2019/5 rev.2, GOOS Re- port No. 239, 2019. IOC-UNESCO: State of the Ocean Report, pilot edition, Paris, IOC- UNESCO, IOC Technical Series, 172, 2022. Disclaimer. Please note that this article has undergone editorial review only. 2 The ocean around the globe The Southern Ocean region is a major player triggering changes in the global ocean circulation (Lee et al., 2023), and the OSR7 has addressed inter-basin transports in this re- gion (see Baker et al. in OSR7 Chap. 2). To allow for reliable monitoring of changes in the regional circulation in this sen- sitive area for global climate, the accuracy and feasibility of the available ocean products must be analyzed. The analy- sis in this issue of the ocean state report has investigated the variability of the South Atlantic meridional overturning cir- culation and the meridional heat transport focusing on the period from 2013 to 2017, i.e., when direct observations in Extreme events can induce major hazards along coastlines. The OSR7 has investigated extreme events and coastal haz- ards in the southwestern South Atlantic over the past quar- ter of a decade, which hosts the most economically impor- tant harbors in South America, high oil and gas production demands, and rich biodiversity (see Gramcianinov et al. in OSR7 Chap. 3). 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Chapter 1.2 – 7th edition of the Copernicus Ocean ter 1.2 – 7th edition of the Copernicus Ocean State Report (O Evaluation of operational ocean forecasting systems from the perspective of the users and the experts Ciliberti1, Enrique Alvarez Fanjul2, Jay Pearlman3, Kirsten Wilmer-Becker4, Pierre Bahurel2, Fabrice Ardhuin5, Alain Arnaud2, Mike Bell4, Segolene Berthou4, Laurent Bertino6, Arthur Capet7, Eric Chassignet8, Stefano Ciavatta2, Mauro Cirano9, Emanuela Clementi10, Gianpiero Cossarini11, Gianpaolo Coro12, Stuart Corney13, Fraser Davidson14, Marie Drevillon2, Yann Drillet2, Renaud Dussurget2, Ghada El Serafy15, Katja Fennel16, Marcos Garcia Sotillo1, Patrick Heimbach17,18, Fabrice Hernandez19, Patrick Hogan20, Ibrahim Hoteit21, Sudheer Joseph22, Simon Josey23, Pierre-Yves Le Traon2, Simone Libralato11, Marco Mancini24, Pascal Matte25, Angelique Melet2, Yasumasa Miyazawa26, Andrew M. Moore27, Antonio Novellino28, Andrew Porter29, Heather Regan6, Laia Romero30, Andreas Schiller31,32, John Siddorn33, Joanna Staneva34, Cecile Thomas-Courcoux2, Marina Tonani2, Jose Maria Garcia-Valdecasas1, Jennifer Veitch35, Karina von Schuckmann2 Liying Wan36 John Wilkin37 and Romane Zuﬁc2 ernicus Ocean State Report (OSR7) ean State Report (OSR7) 1Nologin Oceanic Weather Systems, Madrid, Spain Mercator Ocean International, Toulouse, France Institute of Electrical and Electronics Engineers, Paris, France 5Laboratoire d’Océanographie Physique et Spatiale (LOPS), IFREMER, Brest, France 6Nansen Environmental and Remote Sensing Center, Bergen, Norway 7Operational Directorate Natural Environment (OD Nature), Royal Belgian Institute of Natural Sciences (RBINS), Brussels, Belgium partment of Earth, Ocean and Atmospheric Science, Center for Ocean–Atmospheric Prediction Ocean and Atmospheric Science, Center for Ocean–Atmospheric Prediction Studies, Florida State University, Tallahassee, FL, USA rtment of Meteorology, Institute of Geosciences, Universidade Federal do Rio de Janeiro, Evaluation of operational ocean forecasting systems from the perspective of the users and the experts on of the Copernicus Ocean State Report (OSR7) Stefania A. Ciliberti1, Enrique Alvarez Fanjul2, Jay Pearlman3, Kirsten Wilmer-Becker4, Pierre Bahurel2, Fabrice Ardhuin5, Alain Arnaud2, Mike Bell4, Segolene Berthou4, Laurent Bertino6, Arthur Capet7, Eric Chassignet8, Stefano Ciavatta2, Mauro Cirano9, Emanuela Clementi10, Gianpiero Cossarini11, Gianpaolo Coro12, Stuart Corney13, Fraser Davidson14, Marie Drevillon2, Yann Drillet2, Renaud Dussurget2, Ghada El Serafy15, Katja Fennel16, Marcos Garcia Sotillo1, Patrick Heimbach17,18, Fabrice Hernandez19, Patrick Hogan20, Ibrahim Hoteit21, Sudheer Joseph22, Simon Josey23, Pierre-Yves Le Traon2, Simone Libralato11, Marco Mancini24, Pascal Matte25, Angelique Melet2, Yasumasa Miyazawa26, Andrew M. Moore27, Antonio Novellino28, Andrew Porter29, Heather Regan6, Laia Romero30, Andreas Schiller31,32, John Siddorn33, Joanna Staneva34, Cecile Thomas-Courcoux2, Marina Tonani2, Jose Maria Garcia-Valdecasas1, Jennifer Veitch35, Karina von Schuckmann2, Liying Wan36, John Wilkin37, and Romane Zuﬁc2 1Nologin Oceanic Weather Systems, Madrid, Spain 2Mercator Ocean International, Toulouse, France 3Institute of Electrical and Electronics Engineers, Paris, France 4Met Ofﬁce, Exeter, United Kingdom 5Laboratoire d’Océanographie Physique et Spatiale (LOPS), IFREMER, Brest, France 6Nansen Environmental and Remote Sensing Center, Bergen, Norway 7Operational Directorate Natural Environment (OD Nature), Royal Belgian Institute of Natural Sciences (RBINS), Brussels, Belgium 8Department of Earth, Ocean and Atmospheric Science, Center for Ocean–Atmospheric Prediction Studies, Florida State University, Tallahassee, FL, USA 9Department of Meteorology, Institute of Geosciences, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil 10Ocean Modeling and Data Assimilation Division, Fondazione Centro Euro-Mediterraneo sui Cambiamenti Climatici (CMCC), Bologna, Italy 11National Institute of Oceanography and Applied Geophysics – OGS, Trieste, Italy 12Istituto di Scienza e Tecnologie dell’Informazione “Alessandro Faedo”, Centro Nazionale delle Ricerche (CNR), Pisa, Italy 13Institute for Marine and Antarctic Studies, Oceans and Cryosphere, University of Tasmania, Hobart, TAS, Australia 14Oceanography Department, Fisheries and Oceans Canada, North Atlantic Fisheries Center, St. John’s, NL, Canada 15Unit of Marine and Coastal Systems, Deltares, Delft, Netherlands 16Department of Oceanography, Faculty of Science, Dalhousie University, Halifax, NS, Canada 17Oden Institute for Computational Engineering and Sciences, The University of Texas at Austin, Austin, TX, USA 18Jackson School of Geosciences, The University of Texas at Austin, Austin, TX, USA 19Institut de Recherche Pour le Développement (IRD), Marseille, France 20NOAA, National Centers for Environment Information, Stennis Space Center, Hancock County, Missisipi, USA 21Physical Sciences and Engineering Division, King Abdullah University of Science and Technology (KAUST), Thuwal, Saudi Arabia Stefania A. 1 Introduction (https://oceanpredict.org/un-decade-of-ocean-science/ foresea/, last access: 21 August 2023), DITTO (https://ditto-oceandecade.org/, last access: 21 August 2023), CoastPredict (https://www.coastpredict.org/, last access: 21 August 2023), Global Environ- ment Monitoring System for the Ocean and Coasts (GEMS Ocean (https://www.unep.org/explore-topics/ oceans-seas/what-we-do/ocean-and-coastal-observations, last access: 21 August 2023)), Ocean Best Practices (https://www.oceanbestpractices.org/, last access: 21 August 2023), and others. The Intergovernmental Oceanographic Commission (IOC) has an Ocean Decade Implementation Plan (UNESCO- IOC, 2021) that states seven outcomes required for the ocean we want, with the fourth outcome being “A pre- dicted ocean where society understands and can respond to changing ocean conditions.” To facilitate the achieve- ment of this goal, the IOC has endorsed Mercator Ocean International to implement the Decade Collaborative Center (DCC) for OceanPrediction (https://www.mercator-ocean. eu/oceanprediction/, last access: 21 August 2023), which is a cross-cutting structure that will work to develop global-scale collaboration between Decade Actions related to ocean pre- diction. To develop this architecture, the OceanPrediction DCC has implemented the Ocean Forecasting Co-Design Team (OFCT), which is composed of 43 international experts on all of the different aspects of the ocean forecasting value chain. The ﬁrst task of this group is to analyze the present status of ocean forecasting at a global level, in order to properly iden- tify the existing gaps before moving into the design phase. To have a predicted ocean, the OceanPrediction DCC understands that is critical to co-design ocean forecasting architecture that will permit different services to deliver as one and that could take advantage of the concept of digital twinning (European Union, 2022). This architecture will be designed to overcome the present-day limitations of our systems in terms of interoperability and tools sharing. This will translate into a new scenario for ocean forecasting, where more robust systems will be easier to implement thanks to a common set of agreed tools, standards, and best practices. This new architecture will serve as inspi- ration for the development targets of the different decadal actions related to ocean forecasting, such as ForeSea One of the ﬁrst steps in this process has been to explore the degree of satisfaction of both users and experts with re- spect to the existing ocean forecasting systems. This has been done by launching a series of surveys among the members of the OFCT and another one among the users of the forecast- ing services. This paper describes the ﬁndings derived from the analysis of these surveys. Published: 27 September 2023 Published: 27 September 2023 S. A. Ciliberti et al.: Evaluation of operational ocean forecasting systems S. A. Ciliberti et al.: Evaluation of operational ocean forecasting s 22Indian National Centre for Ocean Information Services (INCOIS), Pragathi Nagar, Nizampet, Hyderabad, Telangana 500090, India 23Marine Systems Modelling, National Oceanography Center, Southampton, UK 24Advanced Scientiﬁc Computing Division, Fondazione Centro Euro-Mediterraneo sui Cambiamenti Climatici (CMCC), Lecce, Italy 25Meteorological Research Division, Environment and Climate Change Canada, Québec, QC, Canada 26Application Laboratory, Japan Agency for Marine–Earth Science and Technology, Yokohama, Kanagawa, Japan 27Physical & Biological Sciences Division, Ocean Sciences Department Institute of Marine Sciences, Institute of Marine Sciences, University of California Santa Cruz, Santa Cruz, California, USA 28ETT – People and Technology, Genoa, Italy 29Science and Technology Facilities Council, Daresbury Laboratory, Hartree Centre, Daresbury, UK 30Lobelia Earth, Barcelona, Spain 31CSIRO Environment, Castray Esplanade, Hobart, Tasmania, Australia 32Institute for Marine and Antarctic Studies, University of Tasmania, Hobart, Australia 33Data, Science and Technology, National Oceanography Centre, Southampton, UK 34Institute for Coastal Systems, Helmholtz Centre Hereon Geesthacht, Geesthacht, Germany 35Egagasini Node, South African Environmental Observation Network (SAEON), Cape Town, South Africa 36National Marine Environmental Forecasting Center Beijing, China 37Department of Marine and Coastal Sciences, Rutgers, The State University of New Jersey, New Brunswick, NJ, USA Correspondence: Enrique Alvarez Fanjul (ealvarez@mercator-ocean.fr) P bli h d 27 S t b 2023 22Indian National Centre for Ocean Information Services (INCOIS), Pragathi Nagar, Nizampet Hyderabad Telangana 500090 India 23Marine Systems Modelling, National Oceanography Center, Southa 23Marine Systems Modelling, National Oceanography Center, Southampton, UK Rio de Janeiro, Brazil odeling and Data Assimilation Division, Fondazione Centro Euro-Mediterraneo g p y pp p y y 12Istituto di Scienza e Tecnologie dell’Informazione “Alessandro Faedo”, Centro Nazionale delle Ricerche (CNR), Pisa, Italy 13Institute for Marine and Antarctic Studies, Oceans and Cryosphere, University of Tasmania, Hobart, TAS, Australia 14Oceanography Department, Fisheries and Oceans Canada, North Atlantic Fisheries Center, St. John’s, NL, Canada 15Unit of Marine and Coastal Systems, Deltares, Delft, Netherlands 16Department of Oceanography, Faculty of Science, Dalhousie University, Halifax, NS, Canada 7 7Oden Institute for Computational Engineering and Sciences, The University of Texas at Austin, Austin, TX, USA , , 18Jackson School of Geosciences, The University of Texas at Austin, Austin, TX, USA 19Institut de Recherche Pour le Développement (IRD), Marseille, France Pour le Développement (IRD), Marseille, France 20NOAA, National Centers for Environment Information, Stennis Space Center, Hancock County, Missisipi, USA sical Sciences and Engineering Division, King Abdullah University of Science and Technology (KAUST), Thuwal, Saudi Arabia Published by Copernicus Publications. CHAPTER1.2 CHAPTER1.2 2 State Planet, 1-osr7, 2, 2023 (CMCC), Lecce, Italy 25Meteorological Research Division, Environment and Climate Change Canada, Québec, QC, Canada 26Application Laboratory, Japan Agency for Marine–Earth Science and Technology, 25Meteorological Research Division, Environment and Climate Change Canada, Québec, QC, Canada 26Application Laboratory, Japan Agency for Marine–Earth Science and Technology, Yokohama Kanagawa Japan Yokohama, Kanagawa, Japan g p 27Physical & Biological Sciences Division, Ocean Sciences Department Institute of Marine Sciences, Institute of Marine Sciences, University of California Santa Cruz, Santa Cruz, California, USA 28ETT P l d T h l G It l 27Physical & Biological Sciences Division, Ocean Sciences Department Institute of Marine Sciences, Institute of Marine Sciences, University of California Santa Cruz, Santa Cruz, California, USA 28 29Science and Technology Facilities Council, Daresbury Laboratory, Hartree Centre, Daresbury, UK 30Lobelia Earth, Barcelona, Spain 29Science and Technology Facilities Council, Daresbury Laboratory, Hartree Centre, Daresbury, UK 30Lobelia Earth, Barcelona, Spain 31CSIRO Environment, Castray Esplanade, Hobart, Tasmania, Australia 32Institute for Marine and Antarctic Studies, University of Tasmania, Hobart, Australia 33Data, Science and Technology, National Oceanography Centre, Southampton, UK 34Institute for Coastal Systems, Helmholtz Centre Hereon Geesthacht, Geesthacht, Germany 35Egagasini Node, South African Environmental Observation Network (SAEON), Cape Town, South Africa 36National Marine Environmental Forecasting Center Beijing, China 37Department of Marine and Coastal Sciences, Rutgers, The State University of New Jersey, New Brunswick, NJ, USA Correspondence: Enrique Alvarez Fanjul (ealvarez@mercator-ocean.fr) Correspondence: Enrique Alvarez Fanjul (ealvarez@mercator-ocean.fr) 3.1 Experts survey: analysis of the results All of the 43 OFCT experts have been invited to complete the diagnosis poll. An analysis of the provided rating values per EOV is given in Fig. 1. All of the 43 OFCT experts have been invited to complete the diagnosis poll. An analysis of the provided rating values per EOV is given in Fig. 1. The diagnosis poll focuses on addressing the evaluation of a nowcast and a short-/middle-term forecast (up to 10 d) to solve a proposed list of EOVs such as currents, sea level, or groups of EOVs that are generically related to ice, waves, biochemistry, and biological variables. The question to be an- swered was as follows: “Qualify, based on your knowledge and experience, from 1 (not satisﬁed) to 10 (extremely sat- isﬁed), our ability to provide a solution at each spatial scale (global, regional and coastal).” The evaluation is driven by four main OOFS properties, namely existence, quality, ac- cessibility, and timeliness, that are deﬁned as follows: exis- tence refers to the availability of sufﬁcient OOFS at the re- quired scale; quality certiﬁes that delivered product/service consistently functions well and provides useful results; ac- cessibility, as deﬁned by Information Technology Infrastruc- ture Library (ITIL, 2023), is considered to be the process of granting the right to use a service; and timeliness refers to the expected time needed to access a product/service. Timeliness can be measured as the time between when information is ex- pected and when it is readily available for use. For analyses, based on the scale of 1–10, the ratings were grouped into very satisﬁed (8–10), satisﬁed (6 to 8), moder- ately satisﬁed (4 to 6), unsatisﬁed (2–4), and very unsatisﬁed (0–2). The analysis reveals that experts are satisﬁed about the performances of blue and white ocean variables at global and regional scales (rates from 6.5 to 8, except for quality, with respect to currents and ice, which has a mean rating of 5.3). For the same variable, at coastal scale the degree of satisfac- tion is only moderate, with mean rates spanning from about 4.5 to 5.7. Wave forecasting is particularly well located on the rating scale, as mean rates above 7.0 are given for all OOFS properties, with maximum value of 7.9 for timeliness at global scale. When considering the coastal scale, the rates are above 6.0. Considering green ocean variables, the values in Fig. S. A. Ciliberti et al.: Evaluation of operational ocean forecasting systems 3 speciﬁc list of EOVs (e.g., currents, sea level, temperature, salinity, waves, ice variables, and biochemical and biological variables) analyzed according to speciﬁc OOFS properties (e.g., quality, reliability, timeliness, accessibility, and usabil- ity). 2 Methodology This work is based on the analysis of a series of surveys. Polls have been designed to champion speciﬁc categories – namely, experts and end-users – in order to provide a score for each type of application (e.g., from ports, coastal en- gineering and disaster risk reduction to ecosystem health) and for each type of Essential Ocean Variable (EOV) used (The Expert Team on Operational Ocean Forecasting Sys- tem, 2022; e.g., a reference list of the key EOVs per ocean universe in blue, green, and white has been provided), focus- ing on some speciﬁc Operational Ocean Forecasting Systems (OOFS) which have properties that are related to their avail- ability and quality (including the timeliness, reliability, and accessibility). Note that the questions on the polls for users and experts are not the same. For example, we consider that the differen- tiation between OOFS spatial scales does not make sense to the users. The methodology employed, based on polls, does not pretend to be mathematically precise, but it is providing signiﬁcant results on the perception on the status of OOFS, both from the perspective of users and developers, making it therefore a valuable reference for the identiﬁcation of gaps that will be carried out by the OFCT during a second step. A ﬁrst set of two surveys (named diagnosis and appli- cations) has been designed for the experts participating in the OFCT, with the main purpose being obtaining a highly skilled subjective view on the availability and quality of to- day’s OOFS at different spatial scales, ranging from global to coastal. 1 Introduction Section 2 introduces the sur- veys, while Sect. 3 presents the results. Section 4 establishes a discussion and identiﬁes some conclusions as part of the outlook for future exploitation. https://doi.org/10.5194/sp-1-osr7-2-2023 CHAPTER1.2 https://doi.org/10.5194/sp-1-osr7-2-2023 3.2 End-user survey: analysis of the results A total of 164 responses were received from end-users work- ing on all continents and have been evaluated; 70 % of them belong to public institutions, while 25.6 % are from private companies. These percentages are close to the ones corre- sponding to end-users downloading data from the Coperni- cus Marine Data Store (Le Traon et al., 2019). Figure 3 gives a comprehensive overview of end-user feedback by showing the absolute number of detected uses of EOVs in the differ- ent applications. Currents and sea level variables are the most demanded variables for applications related to coastal man- agement and disaster risk reduction (entries higher than 10 for each considered application), and temperature has ma- jor impact on applications that refer to ﬁsheries, aquaculture, ecological and biological uses (entries at around 10 for each application and up to 15 for the ﬁsheries one), together with biochemical and biological variables (entries lower than 10). Almost all of the proposed applications gain direct beneﬁts from the waves products. Lower entries are found for ice variables, mainly supporting navigation applications, and for salinity. Maybe the most unexpected and surprising result of this exer- cise is the large difference existing in the perception between users and experts. While users are always quite satisﬁed with the systems, experts are generally more critical, especially regarding those services related to the green ocean. These different perceptions are probably due to several factors. Ex- perts are considering the quality by validating with observa- tions, a process that can be more severe than the users’ per- ception, which is based on application experience. On top of that, and probably more importantly, an approximate indica- tion of the value of an EOV could be more than sufﬁcient to allow the satisfactory practical use of a system. For example, a port pilot could be satisﬁed by knowing if wave heights will or will not be over a given threshold, but they are not af- fected if the waves have one value or another over that thresh- old because operations will be canceled independent of how much the variable is exceeding the threshold. S. A. Ciliberti et al.: Evaluation of operational ocean forecasting systems S. A. Ciliberti et al.: Evaluation of operational ocean forecasting systems 4 Figure 1. The experts’ average rates (in blue) and standard deviations (in black) per OOFS property (E is for existence, Q is for quality, Ac is for accessibility, and T is for timeliness), considering a proposed list of EOVs (e.g., currents, sea level, ice, waves, biochemistry, and biology) at a given scale (e.g., global, regional, and coastal). The color scale corresponds to mean values. Figure 1. The experts’ average rates (in blue) and standard deviations (in black) per OOFS property (E is for existence, Q is for quality, Ac is for accessibility, and T is for timeliness), considering a proposed list of EOVs (e.g., currents, sea level, ice, waves, biochemistry, and biology) at a given scale (e.g., global, regional, and coastal). The color scale corresponds to mean values. Figure 1. The experts’ average rates (in blue) and standard deviations (in black) per OOFS property (E is for existence, Q is for quality, Ac is for accessibility, and T is for timeliness), considering a proposed list of EOVs (e.g., currents, sea level, ice, waves, biochemistry, and biology) at a given scale (e.g., global, regional, and coastal). The color scale corresponds to mean values. ing an average score between 7.5 and 8. Waves variables are also well positioned, with an average score above 7.5. Look- ing at all quality values, mean scores span from 6.5 to 7, with standard deviations lower than 2.0. This analysis con- ﬁrms that more than 90 % of the end-users are satisﬁed with today’s OOFS products. tion and proximity to the coast. There is quite a consensus as green ocean variables are considered to be very important for applications like ﬁsheries, aquaculture, and water quality (Fig. 2b–c). It is in fact quite striking that at a coastal scale almost all EOVs or families of EOVs are used for almost the vast majority of applications, demonstrating the relevance and the need for high-resolution ocean forecasting services. State Planet, 1-osr7, 2, 2023 3.1 Experts survey: analysis of the results 1 are clearly lower (from slightly satisﬁed to not satisﬁed at all), indicating a poor level of appreciation for biogeochemical solutions and a very poor one for systems dealing with bio- logical forecasts (quality values around 2.6). The analyzed results reveal also that standard deviation from the mean is particularly higher if the EOVs are eval- uated per each OOFS type at the coastal scale (values higher than 2.0), reﬂecting that the experts provided a wide range of ratings with respect to the OOFS capabilities. The only ex- ception is given by waves, whose quality has been positively evaluated (mean rates about 7.0 with a standard deviation of 1.1). The applications poll complements the diagnosis one by gathering the experts’ indications on which EOV is useful for serving a speciﬁc proposed list of applications that spans from coastal management, ports, energy, navigation, and ma- rine and ecosystem health to disaster risk reduction and aqua- culture. A third poll has been designed for end-users of ocean fore- casting services. The objective is to explore the satisfaction level perception with respect to what today’s OOFS offer. The survey has been disseminated through several channels, including the OceanPrediction DCC community, Ocean Pre- dict (https://oceanpredict.org/, last access: 25 August 2023), Global Ocean Observing System (GOOS), and others. As with the diagnosis poll, the end-users’ survey focuses on a The applications poll results are summarized in Fig. 2, which shows the aggregation of the answers given by ex- perts from a global to coastal scale, mapping the need for speciﬁc EOVs to serve a target application. From a qualita- tive point of view, the panels show that a wide range of EOVs are requested and that the demand increases with the resolu- https://doi.org/10.5194/sp-1-osr7-2-2023 State Planet, 1-osr7, 2, 2023 State Planet, 1-osr7, 2, 2023 State Planet, 1-osr7, 2, 2023 CHAPTER1.2 CHAPTER1.2 S. A. Ciliberti et al.: Evaluation of operational ocean forecasting systems 5 Figure 2. Schematic overview of survey outcomes related to the needs of different EOVs (x axis) for each application area (y axis) at (a) global, (b) regional, and (c) coastal scales. Figure 2. Schematic overview of survey outcomes related to the needs of different EOVs (x axis) for each application area (y axis) at (a) global, (b) regional, and (c) coastal scales. Figure 3. Histogram of the survey outcomes related to end-users’ answers, considering different EOVs (colors) being employed at each application area (x axis). Figure 3. Histogram of the survey outcomes related to end-users’ answers, considering different EOVs (colors) being employed at each application area (x axis). rvey outcomes related to end-users’ answers, considering different EOVs (colors) being employed at each ative maturity of the operational chains and the need to im- prove in all the aspects related to the prediction engines, in- cluding the acquisition of new data to be assimilated. due to several factors that can be present on some occasions, such as the lack of an appropriate resolution, poor atmo- spheric forcing at that scale, the lack of river data, and the complexity and importance of the nonlinear effect and inter- actions between EOVs near the coast. Take, for example, a sea level that has a striking low mean quality value of only 5.7 at the coast. The degradation could be due to lack of cou- pling with waves near the coast or to poor bathymetry. The importance of this skill’s degradation is reinforced with the analysis of the results from the applications survey, which demonstrates that it is precisely near the coast where ocean forecasting EOVs are more widely used. Nevertheless, there are some aspects that would require further investigation. It is striking that, when not satisﬁed, the experts tend to evaluate all aspects of the system in a negative way at the same time. There is no evident reason why biogeochemical or biological models behave so poorly in technical aspects such as timeliness. Maybe the difﬁculty in obtaining good, accurate solutions is biasing the criteria of the experts when evaluating these on more purely techni- cal aspects. This fact will be further investigated in the fu- ture. Another potentially biased result is the qualiﬁcation on the existence of systems. 3.2 End-user survey: analysis of the results On top of that, the experts know where to look to ﬁnd the problems in the systems, and they do so by scrutinizing all variables, oceano- graphic situations, and geographical regions of the domain of the system, while the end-users commonly have a perspec- tive limited into one particular aspect of the service. When the range of processes and results under scrutiny is larger, it is to be expected that more mishaps can be found. As done with the expert survey, a map providing an eval- uation of OOFS properties with respect to a subset of EOV is shown in Fig. 4, which displays the mean values and the associated standard deviation. It shows a general and positive level of perception of the overall OOFS capacity for provid- ing products – as demonstrated by mean rates that are gener- ally above 7 for timeliness, accessibility, and usability. Fig- ure 4 reveals that, in contrast with the experts’ opinion, bio- geochemical and biological variables are well appreciated, with score values ranging from 6 to 8. It is interesting to note that the temperature score is around 6.5, despite currents hav- To the trained eye, the evaluation of the experts is not sur- prising. It clearly shows that wave and sea level forecasting systems are quite mature and that more effort must be made with respect to the green ocean, probably at all points of the value chain, from observations to downstream services. Also, the progressive degradation of the system’s performance as we come closer to the coast is obvious. This is most likely https://doi.org/10.5194/sp-1-osr7-2-2023 CHAPTER1.2 S. A. Ciliberti et al.: Evaluation of operational ocean forecasting systems https://doi.org/10.5194/sp-1-osr7-2-2023 References Figure 4. Mean values (in blue) and standard deviations (in black) of the end-users’ evaluation of the Operational Ocean Forecasting Services (OOFS; e.g., quality, reliability, timeliness, accessibility, and usability), when considering a proposed list of EOVs (e.g., biogeochemical and biological variables, currents, ice, salinity, sea level, temperature, and waves). The color scale corresponds to the mean values. See the text for more details on the scoring. European Union: European Commission, Directorate – General for Research and Innovation, The digital twin ocean: an interactive replica of the ocean for better decision-making, Publications Of- ﬁce of the European Union, 2022, https://data.europa.eu/doi/10. 2777/343496 (last access: 21 August 2023), 2022. ITIL (The Information Technology Infrastructure Library): The Information Technology Infrastructure Library (ITIL), https:// www.itlibrary.org/, last access 7 July 2023. Le Traon, P. Y., Reppucci, A., Alvarez-Fanjul, E., Aouf, L., Behrens, A., Belmonte, M., Bentamy, A., Bertino, L., Brando, V. E., Brandt Kreiner, M., Benkiran, M., Carval, T., Ciliberti, S. A., Claustre, H., Clementi, E., Coppini, G., Cossarini, G., De Al- fonso Alonso-Muñoyerro, M., Delamarche, A., Dibarboure, G., Dinessen, F., Drevillon, M., Drillet, Y., Faugere, Y., Fernandez, V., Fleming, A., Garcia-Hermosa, M. I., Garcia Sotillo, M., Gar- ric, G., Gasparin, F., Giordan, C., Gehlen, M. Gregoire, M. L., Guinehut, S., Hamon, M., Harris, C., Hernandez, F., Hinkler, J. B., Hoyer, J., Karvonens, J., Kay, S., King, R., Lavergne, T., Lemiuex-Dudon, B., Lima, L., Mao, C., Martin, M. J., Masina, S., Melet, A., Nardelli, B. B., Nolan, G., Pascual, A., Pistoia, J., Palazov, A., Piolle, J. F., Pujol, M. I., Pequignet, A. C., Peneva, E., Perez Gomez, B., Peiti de la Velleon, L., Pinardi, N., Pisano, A., Pouliquen, S., Reid, R., Remy, E., Santoleri, R., Siddorn, J., She, J., Staneva, J., Stoffelen, A., Tonani, M., Vandenbul- cke, L., von Schuckmann, K., Volpe, G., Wettre, C., and Zachar- ioudaki, A.: From Observation to Information and Users: The Copernicus Marine Service Perspective, Front. Mar. Sci., 6, 234, https://doi.org/10.3389/fmars.2019.00234, 2019. areas where they are developing or using systems, including at least the system(s) that they deal with. This situation can be different in another regions, unbeknownst to the partici- pants. OceanPrediction DCC is developing a global map of existing OOFS that will illuminate this question. Disclaimer. Please note that this article has undergone editorial review only. Publisher’s note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. S. A. Ciliberti et al.: Evaluation of operational ocean forecasting systems Both users and experts are replying based on their experience and, obviously, the geographical It is also not surprising that in most cases, the lowest value for the experts is associated with quality and not with exis- tence, timeliness, or accessibility. This demonstrates the rel- https://doi.org/10.5194/sp-1-osr7-2-2023 State Planet, 1-osr7, 2, 2023 CHAPTER1.2 CHAPTER1.2 S. A. Ciliberti et al.: Evaluation of operational ocean forecasting systems S. A. Ciliberti et al.: Evaluation of operational ocean forecasting systems S. A. Ciliberti et al.: Evaluation of operational ocean forecasting systems 6 Figure 4. Mean values (in blue) and standard deviations (in black) of the end-users’ evaluation of the Operational Ocean Forecasting Services (OOFS; e.g., quality, reliability, timeliness, accessibility, and usability), when considering a proposed list of EOVs (e.g., biogeochemical and biological variables, currents, ice, salinity, sea level, temperature, and waves). The color scale corresponds to the mean values. See the text for more details on the scoring. visualization of the main results. The original draft of this work was prepared by EAF and SAC; then JP and KWB reviewed it. All co- authors kindly participated in the surveys. Competing interests. At least one of the (co-)authors is a mem- ber of the editorial board of State of the Planet. The peer-review process was guided by an independent editor, and the authors also have no other competing interests to declare. Disclaimer. Please note that this article has undergone editorial review only. Comparing global trends in marine cold spells and marine heatwaves using reprocessed satellite data ition of the Copernicus Ocean State Report (OSR7) Robert Peal, Mark Worsfold, and Simon Good Met Ofﬁce, FitzRoy Road, Exeter, Devon, EX1 3PB, UK Correspondence: Mark Worsfold (mark.worsfold@metofﬁce.gov.uk) Received: 28 July 2022 – Discussion started: 10 October 2022 Revised: 25 January 2023 – Accepted: 27 January 2023 – Published: 27 September 2023 Received: 28 July 2022 – Discussion started: 10 October 2022 Revised: 25 January 2023 – Accepted: 27 January 2023 – Published: 27 September 2023 rnicus Ocean State Report (OSR7) Abstract. Climate change is causing extreme climate events to become more frequent and more severe. Marine heatwaves (MHWs) and marine cold spells (MCSs) are prolonged, discrete periods of anomalously high or low ocean temperatures with wide-ranging impacts from dramatic shifts in biodiversity to changes in ﬁshery yields. Previous research has found that MHWs are increasing in frequency and intensity, but MCSs remain less well understood. an State Report (OSR7) We used sea surface temperature (SST) data to compare the global observed MCS and MHW intensities and trends in their frequency over the period 1982–2021. These events were also assigned a category from I (moderate) to IV (extreme). Our ﬁndings show that in large areas of the ocean it can be said with 99 % conﬁdence that MCSs have become less frequent and MHWs have become more frequent. In those regions, the occurrence of MCSs has typically reduced by one event every 5 years, while there is one extra MHW every 5 to 10 years. However, parts of the Southern Ocean go against these trends with MHWs becoming slightly less frequent and MCSs becoming more frequent. The trend of increasing numbers of MHWs and decreasing numbers of MCSs is mostly due to increases (and decreases) in category I and II events. Category III and IV MHWs are less common than the milder events, and a trend analysis demonstrated that across most of the ocean they are occurring at a slightly increased rate. However, the spatial extent of the ocean affected by these events is increasing at a faster rate. The occurrences of category III and IV MCSs are infrequent, and a trend analysis also found that their rates of occurrence, as well as the spatial extent of the ocean affected, remain near constant. Comparing global trends in marine cold spells and marine heatwaves using reprocessed satellite data Many of our results are in agreement with previous studies; what is signiﬁcant about this study is the fact that it uses a different, higher-resolution input SST dataset. The similarity of the results between the different research efforts strengthens the argument that these events are not just a feature of their input dataset. References In summary, during the last 3 decades, the evolution and improvement of ocean forecasting services has been dra- matic, and this exercise has proven that the existing systems are useful and accurate in the opinion of the users. Neverthe- less, the experts have clearly stated that further improvements are required, mainly when approaching the coast and for the systems dealing with the green ocean. The Ocean Decade Implementation Plan is giving us a framework to further de- velop the systems, and this is the objective of OceanPredic- tion DCC and its associated decadal actions, such as ForeSea, Ocean Best Practices, DITTO, CoastPredict, GEMS Ocean, and others. To measure the success of these initiatives, it could be interesting to repeat this exercise by the end of the decade. Lemiuex-Dudon, B., Lima, L., Mao, C., Martin, M. J., Masina, , Melet, A., Nardelli, B. B., Nolan, G., Pascual, A., Pistoia, J., lazov, A., Piolle, J. F., Pujol, M. I., Pequignet, A. C., Peneva, J., She, J., Staneva, J., Stoffelen, A., Tonani, M., Vandenbul- cke, L., von Schuckmann, K., Volpe, G., Wettre, C., and Zachar- ioudaki, A.: From Observation to Information and Users: The Copernicus Marine Service Perspective, Front. Mar. Sci., 6, 234, https://doi.org/10.3389/fmars.2019.00234, 2019. The Expert Team on Operational Ocean Forecasting Sys- tems: Implementing Operational Ocean Monitoring and Fore- casting Systems. Editors: Enrique Alvarez Fanjul, Stefa- nia Ciliberti, Pierre Bahurel, IOC-UNESCO, GOOS-275, https://doi.org/10.48670/ETOOFS, 2022. Code and data availability. The authors will make the software code and data available upon request to the corresponding author. UNESCO-IOC: The United Nations Decade of Ocean Science for Sustainable Development (2021–2030) Implementation plan – Summary, Paris, UNESCO (IOC Ocean Decade Series, 19), https://unesdoc.unesco.org/ark:/48223/pf0000376780 (last access: 21 August 2023), 2021. Author contributions. SAC and EAF developed the methodol- ogy and led the investigation. EAF supervised the work, which was conceptualized with PB. SAC curated the data and worked on the https://doi.org/10.5194/sp-1-osr7-2-2023 State Planet, 1-osr7, 2, 2023 State Planet, 1-osr7, 2, 2023 https://doi.org/10.5194/sp-1-osr7-2-2023 CHAPTER1.2 Chapter 2.1 – 7th edition of the Copernicus Ocean State Report (OSR7) State Planet, 1-osr7, 3, 2023 https://doi.org/10.5194/sp-1-osr7-3-2023 © Author(s) 2023. This work is distributed under the Creative Commons Attribution 4.0 License. Chapter 2.1 – 7th edition of the Copernicus Ocean ter 2.1 – 7th edition of the Copernicus Ocean State Report (O Published by Copernicus Publications. R. Peal et al.: Comparing global trends in marine cold spells and marine heatwaves 2 Table 1. Data used for report. Table 1. Data used for report. Product ref. no. Product ID and type Data access Documentation 1 SST_GLO_SST_L4_REP_OBSERVATIONS_ 010_011; satellite observations EU Copernicus Marine Service Product (2022) Quality Information Document (QUID): Worsfold et al. (2022a) Product User Manual (PUM): Worsfold et al. (2022b) SST, which can be deﬁned as the temperature from which the growth and decay of the diurnal heating develops each day (GHRSST, 2022). search into MCSs has focussed on certain regions including investigating drivers of shallow-water events in some coastal regions (Schlegel et al., 2017) and effects of MCSs on ﬁsh- eries in the North Sea (Wakelin et al., 2021). OSTIA uses the NEMOVAR variational data assimilation scheme (Mogensen et al., 2009) to combine satellite and in situ data with a background ﬁeld that is based on the previ- ous day’s analysis (Good et al., 2020). In areas of the ocean where there are no observations, if the sea ice concentra- tion is greater than 50 %, the SSTs will eventually relax to −1.8 ◦C and elsewhere to a 1985–2007 climatology. The ﬁeld of MHW and MCS research moves rapidly, but two research papers in particular are very relevant to this investigation. Both papers used the Optimum Interpolation Sea Surface Temperature (OISST) climate dataset produced by the National Oceanic and Atmospheric Administration (NOAA) and the same detection method (Sect. 2) over the period 1982–2020. As a result of RAM memory overﬂow issues encountered by the R programming language (unfortunately a fundamen- tal limitation of this language) when processing the full- resolution data, the OSTIA dataset was linearly regridded to 0.25◦resolution for the purpose of this investigation. Schlegel et al. (2021) provided a comprehensive review of previous studies into MCSs and the possible causes of MCSs, which were found to be often driven by anomalous winds, al- though other drivers were also identiﬁed. The trends in MCSs were also investigated for different MCS categories. Wang et al. (2022) calculated and compared the trends in MHWs and MCSs but did not delve into event categories. 2.2 Deﬁnition and detection of marine heatwaves and marine cold spells This investigation has some parallels with the ﬁrst two, but it is the ﬁrst to use the higher-resolution Operational Sea Surface Temperature and Ice Analysis (OSTIA) climate SST dataset (Table 1) produced for the Copernicus Marine En- vironment Monitoring Service (CMEMS), and it can there- fore provide an independent assessment of the occurrence of MHWs and MCSs. Fundamentally an MHW is detected whenever the SSTs ex- ceed a threshold value, but this threshold has a variety of interpretations depending on the investigation requirements. One of the simpler deﬁnitions is to use a chosen percentile (usually 99th) as a threshold SST value calculated from a chosen climatology period (Darmaraki et al., 2019; Frölicher et al., 2018; Laufkötter et al., 2020). For this study we are us- ing the Hobday deﬁnition of a heatwave (Hobday et al., 2016, 2018; Schlegel et al., 2021; Wang et al., 2022), which has gained wide acceptance in the MHW–MCS research com- munity. To detect an MCS, consider that each pixel of the dataset has the following mathematical elements: 2 Methods 2.1 Input SST dataset used 2.1 Input SST dataset used 1 Introduction ing events (NOAA, 2015). They also have a range of socio- economic impacts (Smith et al., 2021) such as altering ﬁshery yields (Mills et al., 2013; Smale et al., 2019; Cheung et al., 2021). Marine cold spells (MCSs), which are deﬁned equiv- alently to MHWs as a “discrete, prolonged anomalously cold water event at a particular location” (Schlegel et al., 2021), remain less well understood, but a growing body of research suggests that low-SST (sea surface temperature) events can also have signiﬁcant impacts on ecology including coral reef destruction (Lirman et al., 2011) and have links to extreme climate events on land (Duchez et al., 2016). Previous re- There is now overwhelming evidence that climate change will increase the frequency and intensity of extreme climate events such as marine heatwaves (MHWs) (IPCC, 2021; Frölicher et al., 2018; Oliver, 2019), which are deﬁned as “prolonged discrete anomalously warm water events” (Hob- day et al., 2016) and have attracted a growing amount of interest in recent years. These events often have dramatic and sometimes devastating impacts on local ecology includ- ing shifting local biodiversity (Wernberg et al., 2013, 2016; Frölicher and Laufkötter, 2018) and triggering coral bleach- Published by Copernicus Publications. CHAPTER2.1 CHAPTER2.1 2.1 Input SST dataset used Most previous MHW–MCS investigations have used the 0.25◦resolution NOAA OISST climate SST (version 2.1) dataset (for example Schlegel et al., 2021, and Wang et al., 2022), which represents the SST at 0.5 m depth (Huang et al., 2020). This is often called a SST depth product, which will increase during the day due to solar heating and cool at night (termed a diurnal cycle). A. a time series of SST values (thin black line in Fig. 1a and b) A. a time series of SST values (thin black line in Fig. 1a and b) B. the daily climatology using the period January 1982– December 2011 as the baseline (smooth, thicker black line in Fig. 1a and b) This investigation used the 0.05◦resolution CMEMS OS- TIA climate dataset for the period 1982–2021 (Table 1). OSTIA is formed from nighttime SSTs, and daytime SSTs whenever windy conditions mean that it can be assumed that the diurnal cycle is not affecting the daytime temperature (Good et al., 2020). Therefore, it represents the foundation C. the 10th percentile of the climatology data B, taken from an 11 d rolling window centred on each day (solid green line in Fig. 1a and b), which is the threshold used to determine if there is an MCS https://doi.org/10.5194/sp-1-osr7-3-2023 State Planet, 1-osr7, 3, 2023 State Planet, 1-osr7, 3, 2023 R. Peal et al.: Comparing global trends in marine cold spells and marine heatwaves 1a shows that the anomalous SST in the region may be classiﬁed as a category IV MCS with a duration of around 100 d peaking in early November. – Duration is the number of consecutive days where the SST exceeded the 90th percentile value for MHWs or was below the 10th percentile value for MCSs. Extreme low-SST events have also been linked to signiﬁ- cant global weather events, which is illustrated by the second case study (Fig. 1b). Duchez et al. (2016) showed that the 2015 European summer continental heatwave, 1 of the top 10 hottest in the last 65 years, as well as several other severe Eu- ropean summer heatwaves, were preceded by anomalously cold SSTs in the North Atlantic, but establishing causality (if any) is the subject of further research. – Maximum intensity is the maximum difference between the SST and the climatology value during the event (MHWs have positive intensity, and MCSs have nega- tive intensity). – Category describes the severity of an event by assigning a category to the maximum intensity value relative to the local climatology. The inset in Fig. 1b shows that a category III MCS pre- vailed over much of the region in 2015, with some areas clas- siﬁed as a category IV. Further time series analysis of a pixel within a category IV region shows an MCS with a duration of around 69 d peaking in late March. All these MHW and MCS characteristics are calculated for each individual grid cell separately; the neighbouring cells are not considered. In these examples, the Hobday category framework has been used to describe the severity of marine SST anomalies that have been linked with signiﬁcant environmental and eco- logical events. Use of this framework may help researchers to better communicate the severity of both low- and high-SST anomalies with public and local ofﬁcials, allowing the area to prepare for the effects of marine temperature extremes. The MHWs and MCSs and their characteristics were cal- culated from the SST dataset using the heatwaveR package (Schlegel and Smit, 2018; Hobday et al., 2016). An important consideration is that areas with persistently high SSTs do not necessarily suffer as many heatwaves. While this may seem counter-intuitive at ﬁrst, consider that MHWs are deﬁned rel- ative to the climatology. R. Peal et al.: Comparing global trends in marine cold spells and marine heatwaves can help describe the severity of MCSs, two case studies of known cold-water events were selected and investigated. D. the difference between the 10th percentile C and the cli- matology B. D. the difference between the 10th percentile C and the cli- matology B. The ﬁrst case study illustrates the effect of MCSs on ma- rine ecosystems (Fig. 1a). An investigation by Abram et al. (2003) linked the coral reef death off Sumatra in October 1997 to the combination of Indonesian wildﬁres exacerbated by El Niño and anomalously low SST caused by the Indian Ocean Dipole (IOD) which caused increased upwelling of cold, nutrient-rich water in the region. Such upwelling events are associated with increased biological activity, which can lead to large algal blooms (often called “red tides”) (Genin et al., 1995). These events can limit oxygen supplies to coral reefs but usually pose only a minor threat as the biological activity is usually iron limited (Johnson et al., 2001). How- ever, in October 1997 the ash cloud from the Indonesian for- est ﬁres dumped large quantities of iron on the ocean, causing one of the largest red tides the region had ever seen, leading to mass asphyxiation of the coral (Abram et al., 2003). A pixel is considered to have an MCS wherever the SST value A is below the 10th percentile threshold value C for at least 5 d. If there is a gap of less than 2 d between an MCS, they are considered to be one event. Furthermore, the difference value D then allows this MCS to then be categorized. A heatwave is considered category I (moderate) when the SST value A is less than the sum of the climatology value B and the difference value D (i.e. where A< = B + D) (below solid green line in Fig. 1a and b). A category II (strong) MCS would be where A< = B+2D and so on for severe and extreme events (dashed green lines in Fig. 1a and b). MHWs are deﬁned equivalently as a period of at least 5 d where the SST value is above the 90th percentile of the SST data from an 11 d rolling window centred on each day. Using the Hobday deﬁnition, events can be described by the following characteristics: This event was examined using the Hobday framework, and Fig. R. Peal et al.: Comparing global trends in marine cold spells and marine heatwaves Therefore, an area with historically high average SST values will present less opportunity for re- cent SSTs to exceed that high climatology threshold. This characteristic is valuable since researchers are often most in- terested in areas with signiﬁcant deviations from historical temperatures. CHAPTER2.1 CHAPTER2.1 R. Peal et al.: Comparing global trends in marine cold spells and marine heatwaves 3 https://doi.org/10.5194/sp-1-osr7-3-2023 3.2 A global perspective of MHW and MCS distribution and strength The average maximum intensity of events and the average number of yearly events for both MHWs and MCSs were calculated over the global domain for the period 1982–2021 to produce a map of the distribution of such events (Fig. 2). Figure 2a and b show that the mean maximum intensity of both types of events at most locations in the global ocean was 1 to 2 ◦C and between 2 and 4 ◦C in the Niño 3 region of the eastern Paciﬁc (deﬁned as 5◦N–5◦S, 90–150◦W) (Tren- berth, 1997). Moreover, other regions with higher-intensity 3 Results and discussions 3.1 Using the Hobday MCS framework to describe anomalous low-SST events 3.1 Using the Hobday MCS framework to describe anomalous low-SST events To highlight the consequences of cold spells and how the Hobday category framework (Hobday et al., 2016, 2018) https://doi.org/10.5194/sp-1-osr7-3-2023 State Planet, 1-osr7, 3, 2023 State Planet, 1-osr7, 3, 2023 CHAPTER2.1 CHAPTER2.1 R. Peal et al.: Comparing global trends in marine cold spells and marine heatwaves R. Peal et al.: Comparing global trends in marine cold spells and marine heatwaves 4 Figure 1. Signiﬁcant MCSs. (a) Sumatra 1997. Main ﬁgure: SST values and MCS threshold during late 1997 at lat 0.875, long 98.625. Inset: highest MCS category detected across the region during 1997 (the red cross denotes the location where the time series data were extracted). (b) North Atlantic 2015. Main ﬁgure: SST values and MCS threshold during early 2015 at lat 41.125, long −50.375. Inset: highest MCS category detected across the region during 2015 (the red cross denotes the location where the time series data were extracted). Figure 1. Signiﬁcant MCSs. (a) Sumatra 1997. Main ﬁgure: SST values and MCS threshold during late 1997 at lat 0.875, long 98.625. Inset: highest MCS category detected across the region during 1997 (the red cross denotes the location where the time series data were extracted). (b) North Atlantic 2015. Main ﬁgure: SST values and MCS threshold during early 2015 at lat 41.125, long −50.375. Inset: highest MCS category detected across the region during 2015 (the red cross denotes the location where the time series data were extracted). MHWs (sometimes exceeding 6 ◦C) also experienced MCSs of similar intensity. 3.2 A global perspective of MHW and MCS distribution and strength These regions of high-intensity MHWs– MCSs were also located in areas in which western boundary currents (WBCs) are found (NW Atlantic – Gulf Stream, SW Atlantic – Brazil Current, South Africa – Agulhas Current, Japan – Kuroshio Current; Le Traon and Morrow, 2001). WBCs are found at the western edges of ocean gyres and transport warmer waters towards the poles (Holbrook et al., 2019). 1982–2016 period. As mentioned earlier, Wang et al. (2022) compared the trends in MCSs and MHWs (using similar but different metrics). An important point to reiterate is that these previous studies had used the NOAA OISST dataset. Despite the differences between investigations, it is re- markable that the spatial distribution of the values in Fig. 2 are very similar to the results from the previous investiga- tions. The average number of heatwaves per year was calculated for each pixel of the dataset and plotted. Examining Fig. 2c and d it can be seen that in most locations, MHWs were slightly more common than MCSs, with between one to three events detected per year compared to one to two MCS per year. State Planet, 1-osr7, 3, 2023 3.3 An exploration of linear trends in MHW and MCS events for each category In order to determine whether there was a change in the num- ber of MHWs and MCSs per year, a linear trend was calcu- lated for each pixel of the global dataset and plotted (Fig. 3). Further plots were also generated for each event category, and contours were added wherever the trend exceeded a 99 % conﬁdence level. In order to determine whether there was a change in the num- ber of MHWs and MCSs per year, a linear trend was calcu- lated for each pixel of the global dataset and plotted (Fig. 3). Further plots were also generated for each event category, and contours were added wherever the trend exceeded a 99 % conﬁdence level. However, MHWs were rare in the Niño 3 region, occurring no more than once per year, while MCSs occurred between two and three times per year in the same region. Regions with a higher frequency of MCSs tended to coin- cide with regions where they were more intense. The north- west Atlantic, south-west Atlantic, region off South Africa, and north-west Paciﬁc off Japan all had up to three MCSs per year. Figure 3a shows that occurrences of MCSs in much of the Atlantic, Indian, and western Paciﬁc oceans are decreasing at a rate of up to one event every 5 years and that these de- creases exceed the 99 % conﬁdence interval for most of the region. Figure 3c shows a large decrease in category I (mod- erate) events at a rate of almost one event every 5 years at the locations which exceed the 99 % conﬁdence value. A smaller but still signiﬁcant decrease in category II (strong) events of Schlegel et al. (2021) carried out a similar investigation into MCS count and intensity for the period 1982–2020, and Oliver et al. (2018) also did the same for MHWs for the https://doi.org/10.5194/sp-1-osr7-3-2023 State Planet, 1-osr7, 3, 2023 CHAPTER2.1 R. Peal et al.: Comparing global trends in marine cold spells and marine heatwaves R. Peal et al.: Comparing global trends in marine cold spells and marine heatwaves 5 Figure 2. Comparison of global MHW and MCS mean maximum intensity and frequency of occurrence during the period 1982–2021, calculated from product 2.1.1. (a) Mean MCS maximum intensity and (c) average number of MCSs per year at each grid point. 3.3 An exploration of linear trends in MHW and MCS events for each category (b) Mean MHW maximum intensity and (d) average number of MHWs per year at each grid point. Figure 2. Comparison of global MHW and MCS mean maximum intensity and frequency of occurrence during the period 1982–2021, calculated from product 2.1.1. (a) Mean MCS maximum intensity and (c) average number of MCSs per year at each grid point. (b) Mean MHW maximum intensity and (d) average number of MHWs per year at each grid point. ﬁrmed by Wang et al. (2022), who also determined that rising SSTs were responsible for trends in MCSs. In addition, cli- mate change research has also found that, while SSTs are in- creasing on average, the southern Antarctic Ocean has cooled slightly (Auger et al., 2021; Rye et al., 2020; Armour et al., 2016). The observed spatial patterns in our results for MHWs and MCSs ﬁts into a wider picture of the ocean’s behaviour changing as a consequence of climate change. about one event every 20 years was also observed (Fig. 3e). Figure 3g and i show that category III and IV events are con- tinuing to occur at a roughly constant rate. Therefore, the overall decrease in MCS is largely due to decreases in the numbers of category I and II events. Figure 3b shows that MHWs in the central Atlantic, In- dian, and western Paciﬁc oceans are increasing by one event every 5 to 10 years, and this trend is signiﬁcant to the 99 % conﬁdence level in much of the region. Figure 3d and f show that this is largely due to increases in category I and II MHWs of up to one event per 10 years in much of the region. Fig- ure 3h and j show that the trends in category III and IV MHWs are close to zero. https://doi.org/10.5194/sp-1-osr7-3-2023 3.4 Changes in the affected ocean area over time To investigate whether there are any changes in how much of the ocean is affected by MHWs and MCSs, each pixel was examined to determine the most severe event that had occurred in each year. This information was used to produce a time series for the fraction of the ocean area affected by an event (Fig. 4). It is also notable that the southern Paciﬁc Ocean (in par- ticular, east of South America) goes against the general trends. Figure 3a shows that MCSs are becoming more fre- quent in the southern Paciﬁc Ocean by one event every 10–20 years, which is largely due to increases in category II events (Fig. 3e). Meanwhile MHWs are also becoming less frequent in this region by one event every 10–20 years (Fig. 3b), which is largely due to decreases in category I events (Fig. 3d). Figure 4 (left) shows that MCSs are now occurring in less of the ocean than historically. The amount of ocean where the highest MCS categories detected were category I and II has decreased from 41.8 % in 1982 to 25.3 % in 2021 and from 27.8 % in 1982 to 13.2 % in 2021 respectively, while the amount of ocean without any MCSs at all has increased from 26 % to 56.6 % in 2021. In contrast, category III and IV MCSs have been detected at a near constant ∼1 %–3 % of the ocean. This suggests that even in a warming world, ex- treme MCSs have so far remained consistently rare. Interestingly, the increasing number of MCSs (for all cate- gories) in the Southern Ocean was also observed by Schlegel et al. (2021), although their research did not determine whether events of a speciﬁc category were responsible for this behaviour. Oliver (2019) determined that rising average SSTs were the main driver of trends in MHWs, which was also con- The investigation by Schlegel et al. (2021) also found that category III and IV events remained steady and that the area https://doi.org/10.5194/sp-1-osr7-3-2023 State Planet, 1-osr7, 3, 2023 CHAPTER2.1 CHAPTER2.1 R. Peal et al.: Comparing global trends in marine cold spells and marine heatwaves R. Peal et al.: Comparing global trends in marine cold spells and marine heatwaves 6 e 3. Trends in the number of MCS and MHW events per year for (a) all MCSs, (b) all MHWs (c, e, g, i), category I–IV MC and category I–IV MHWs. 4 Conclusions of the ocean which experienced an MCS had decreased (61 % in 1982 to 25 % in 2020). However, their investigation dif- fered in that they determined that this was mainly due to a decrease in category II–IV events. In this study, we have compared global MHWs and MCSs in the period 1982–2021. We have revealed that the mean peak intensity of MHWs and MCSs at most locations was 1 to 2 ◦C and the mean peak intensity was highest (up to 6 ◦C for both kinds of event) in regions where western boundary currents dominate. We have also shown that in much of the ocean, MCSs have become less frequent by around one event every 5 years, while there is one extra MHW event every 5 to 10 years. Most of the changes are due to increases in cate- gory II–IV MHWs and decreases in category I and II MCSs. However, events in parts of the Southern Ocean go against these trends with MHWs becoming slightly less frequent and MCSs becoming more frequent. Category III and IV MHWs remain less common than the less severe events, and at in- dividual locations they are occurring at a slightly increased rate over time. However, these events are becoming more widespread. Category III and IV MCSs are rare but are con- tinuing to occur at near-constant rates in much of the ocean. Figure 4 (right) demonstrates that the area of ocean with no MHWs has seen a steady decline throughout the time pe- riod from 37.8 % in 1982 to 18.7 % in 2021, indicating that MHWs are occurring at more locations in the ocean. Break- ing down the results by category, it can be observed that category I events were initially the most common; the area dominated by these events increased from 25.9 % to 43.8 % between 1982 and 2006 before decreasing sharply to just 23.3 % in 2021. In the same period, the area of ocean where II was the highest MHW category increased from 23.6 % to 34.8 % in 2021, overtaking category I as the most common category. R. Peal et al.: Comparing global trends in marine cold spells and marine heatwaves R. Peal et al.: Comparing global trends in marine cold spells and marine heatwaves 7 Figure 4. The fraction of the area of the ocean where the highest (a) MCS and (b) MHW category detected was I, II, III, IV, or no events being detected. The year of an event is deﬁned as the year of the peak intensity. Figure 4. The fraction of the area of the ocean where the highest (a) MCS and (b) MHW category detected was I, II, III, IV, or no events being detected. The year of an event is deﬁned as the year of the peak intensity. 3.4 Changes in the affected ocean area over time The year of an event has been deﬁned as the year of peak intensity. Linear trends were calculated quares ﬁt, and the contour line bounds the region where the trends exceed the 99 % conﬁdence level of a two-tailed t test co onstant rate of occurrence. The X markers indicate the inside of the contour to aid interpretation. Areas with no shading are w were detected. Figure 3. Trends in the number of MCS and MHW events per year for (a) all MCSs, (b) all MHWs (c, e, g, i), category I–IV MCSs (d, f, h, j), and category I–IV MHWs. The year of an event has been deﬁned as the year of peak intensity. Linear trends were calculated using a least-squares ﬁt, and the contour line bounds the region where the trends exceed the 99 % conﬁdence level of a two-tailed t test compared to a constant rate of occurrence. The X markers indicate the inside of the contour to aid interpretation. Areas with no shading are where no events were detected. CHAPTER2.1 State Planet, 1-osr7, 3, 2023 https://doi.org/10.5194/sp-1-osr7-3-2023 https://doi.org/10.5194/sp-1-osr7-3-2023 State Planet, 1-osr7, 3, 2023 State Planet, 1-osr7, 3, 2023 CHAPTER2.1 State Planet, 1-osr7, 3, 2023 References Abram, N., Gagan, M., McCulloch, M., Chappell, J., and Han- toro, W.: Coral Reef Death during the 1997 Indian Ocean Dipole Linked to Indonesian Wildﬁres, Science, 301, 952–955, https://doi.org/10.1126/science.1083841, 2003. Armour, K., Marshall, J., Scott, J., Donohoe, A., and New- som, E.: Southern Ocean warming delayed by circumpolar up- welling and equatorward transport, Nature Geosci., 9, 549–554, https://doi.org/10.1038/ngeo2731, 2016. Auger, M., Morrow, R., Kestenare, E., Sallée, J., and Cowley, R.: Southern Ocean in-situ temperature trends over 25 years emerge from interannual variability, Nat. Commun., 12, 514, https://doi.org/10.1038/s41467-020-20781-1, 2021. Data availability. Publicly available datasets were used and are available from CMEMS (Table 1). Cheung, W. W. L., Frölicher, T. L., Lam, V. W. Y., Oyinlola, M. A., Reygondeau, G., Sumaila, U. R., Tai, T. C., Teh, L. C. L., and Wabnitz, C. C. C.: Marine high temperature extremes amplify the impacts of climate change on ﬁsh and ﬁsheries, Sci. Adv., 7, eabh0895, https://doi.org/10.1126/sciadv.abh0895, 2021. Author contributions. MW generated the initial datasets for the period 1982–2020 required for the investigation. RP processed the data to produce the results outlined above and wrote the ﬁrst draft of the report. When data for 2021 became available, MW then updated the results with the latest data and performed the necessary rewrites for the review and submission process. SG also reviewed and edited the report. Darmaraki, S., Somot, S., Sevault, F., and Nabat, P.: Past variability of Mediterranean Sea marine heatwaves, Geophys. Res. Lett., 46, 9813–9823, https://doi.org/10.1029/2019GL082933, 2019. Duchez, A., Frajka-Williams, E., Josey, S., Evans, D., Grist, J., Marsh, R., McCarthy, G., Sinha, B., Berry, D., and Hirschi, J.: Drivers of Exceptionally Cold North Atlantic Ocean Tem- peratures and Their Link to the 2015 European Heat Wave, Environ. Res. Lett., 11, 74004, https://doi.org/10.1088/1748- 9326/11/7/074004, 2016. Competing interests. The contact author has declared that none of the authors has any competing interests. EU Copernicus Marine Service Product: Global Ocean OSTIA Sea Surface Temperature and Sea Ice Reprocessed, Merca- tor Ocean International [data set], https://doi.org/10.48670/moi- 00168, 2022. Disclaimer. Publisher’s note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Frölicher, T. L. and Laufkötter, C.: Emerging risks from marine heat waves, Nat. Commun., 9, 650, https://doi.org/10.1038/s41467- 018-03163-6, 2018. Acknowledgements. 4 Conclusions Although more research is required to quantify the differences in the outputs from the OISST and OSTIA datasets and their relative conﬁdence levels, the similarity of the results strengthens the argument that these events are not just a feature of their input dataset. Financial support. This work was partly funded by the CMEMS SST Thematic Assembly Centre (21001-COP-TAC Lot 3). Review statement. This paper was edited by Johannes Karstensen and reviewed by two anonymous referees. It also demonstrates the potential of using OSTIA for future MHW–MCS investigations. Research carried out by Yang et al. (2021) showed that the full-resolution OSTIA was superior to OISST in resolving smaller oceanic features. Unfortunately, fundamental limitations in the R program- ming language prevented us from using the full-resolution OSTIA dataset in this investigation. However, this presents an excellent topic for a follow-up investigation using the full-resolution dataset (using a Python version of the MHW code) to determine whether this brings about improvements in MHW–MCS detection or delivers greater insights into their formation and behaviour. 4 Conclusions The amount of ocean where the highest category was III or IV initially remained at around 3 % and 6 % respec- tively until 2008, after which there was a sharp increase in both categories to 7.7 % for category III and 15.4 % for cate- gory IV in 2021. The spatial increase in these categories is es- pecially a cause for concern since these types of events tend to have more dramatic impacts compared to lower-category events (Hobday et al., 2018). The average global frequency and maximum intensity (Fig. 2) and the trend analysis for all categories of events (Fig. 3a and b) had a similar spatial distribution to the MCS results obtained by Schlegel et al. (2021) and Wang et al. (2022) as well as the MHW results obtained by Oliver et al. (2018). The temporal changes in the ocean area affected (Fig. 4) were also similar to the results from investigations carried out by Schlegel et al. (2021) and Hobday et al. (2018). It is also interesting to note that the same investigation by Hobday et al. (2018) also produced a graph displaying a similar behaviour (a reduction in “no events” and category I MHWs and an increase in other categories). The mechanisms that drive these trends in MHWs and MCSs are still under investigation, and further research would provide valuable insight into the changing nature of https://doi.org/10.5194/sp-1-osr7-3-2023 State Planet, 1-osr7, 3, 2023 CHAPTER2.1 CHAPTER2.1 R. Peal et al.: Comparing global trends in marine cold spells and marine heatwaves 8 check rather than a research investigation. More information can be obtained from the authors on request. MHWs and MCSs. Future research also should focus on im- proving understanding of how MCSs form and their impact on ocean ecosystems as well as on developing tools for pre- dicting when they will occur, allowing for early warnings of these events. Although more research is required to quantify the differences in the outputs from the OISST and OSTIA datasets and their relative conﬁdence levels, the similarity of the results strengthens the argument that these events are not just a feature of their input dataset. MHWs and MCSs. Future research also should focus on im- proving understanding of how MCSs form and their impact on ocean ecosystems as well as on developing tools for pre- dicting when they will occur, allowing for early warnings of these events. 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Wernberg, T., Bennett, S., Babcock, R., de Bettignies, T., Cure, K., Depczynski, M., Dufois, F., Fromont, J., Fulton, C., Hovey, https://doi.org/10.5194/sp-1-osr7-3-2023 State Planet, 1-osr7, 3, 2023 CHAPTER2.1 CHAPTER2.1 CHAPTER2.1 South Atlantic overturning and heat transport variations in ocean reanalyses and observation-based estimates n of the Copernicus Ocean State Report (OSR7) Jonathan Andrew Baker1, Richard Renshaw1, Laura Claire Jackson1, Clotilde Dubois2, Doroteaciro Iovino3, Hao Zuo4, Renellys C. Perez5, Shenfu Dong5, Marion Kersalé6, Michael Mayer7,4, Johannes Mayer7, Sabrina Speich8, and Tarron Lamont9,10,11 1Met Ofﬁce, Exeter, UK 2Mercator Ocean International, Toulouse, France 3Centro Euro-Mediterraneo sui Cambiamenti Climatici, Bologna, Italy 4European Centre for Medium-Range Weather Forecasts, Reading, UK 5Atlantic Oceanographic and Meteorological Laboratory, National Oceanic and Atmospheric Administration, Miami, USA 6Direction Générale de l’Armement, Ingénierie des projets, Paris, France 7University of Vienna, Vienna, Austria 8Laboratoire de Météorologie Dynamique–IPSL, Ecole Normale Supérieure, Paris, France 9University of Cape Town, Cape Town, South Africa 10Department of Environment, Forestry and Fisheries, Oceans and Coasts Research Branch, Cape Town, South Africa ernicus Ocean State Report (OSR7) ean State Report (OSR7) 11Bayworld Centre for Research and Education, Cape Town, South Africa Correspondence: Jonathan Andrew Baker (jonathan.baker@metofﬁce.gov.uk) Received: 1 August 2022 – Discussion started: 30 September 2022 Revised: 15 March 2023 – Accepted: 31 March 2023 – Published: 27 September 2023 Revised: 15 March 2023 – Accepted: 31 March 2023 – Published: 27 September 2023 Abstract. The variability in the South Atlantic Meridional Overturning Circulation (MOC) and meridional heat transport measured across 34.5◦S during 2013–2017 differs signiﬁcantly between observational and ocean re- analysis estimates. Variability in an ocean reanalysis ensemble and an eddy-resolving reanalysis is similar to an altimeter-based estimate but smaller than energy-budget and mooring-based estimates. Over 1993–2020, there is no long-term trend in the ensemble-mean overturning and heat transport, although there are inter-model differ- ences, whereas the altimeter-based and energy-budget estimate transports increase over this period. Time-mean overturning volume transport (and the depth of maximum overturning) across 34.5◦S in the ensemble and obser- vations are similar, whereas the corresponding mean heat transports differ by up to 0.3 PW. The seasonal cycle of these transports varies between estimates, due to differences in the methods for estimating the geostrophic ﬂow and the sampling characteristics of the observational approaches. The baroclinic, barotropic, and Ekman MOC components tend to augment each other in mooring-based estimates, whereas in other estimates they tend to counteract each other, so the monthly-mean, interannual, and seasonal MOC anomalies have a greater mag- nitude in the mooring-based estimates. Thus, the mean and variation in real-world South Atlantic transports and the amplitude of their ﬂuctuations are still uncertain. R. Peal et al.: Comparing global trends in marine cold spells and marine heatwaves R. 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Chapter 2.2 – 7th edition of the Copernicus Ocean ter 2.2 – 7th edition of the Copernicus Ocean State Report (O Published by Copernicus Publications. 1 Introduction The Meridional Overturning Circulation (MOC) modulates climate on seasonal to millennial timescales via its merid- ional transport of freshwater, heat, and carbon through the global ocean (Rahmstorf, 2015; Weijer et al., 2019; Buckley and Marshall, 2016). It is therefore important to understand how the Atlantic MOC (AMOC), which dominates the upper cell of the global MOC, is changing. Changes in overturning in the South Atlantic are particularly important because they play a crucial role in determining the pathways of the global overturning circulation (Baker et al., 2021, 2020; Xu et al., 2022; Nadeau and Jansen, 2020), while freshwater transports in the South Atlantic impact the stability of the AMOC (Gar- zoli and Matano, 2011; Hawkins et al., 2011; Weijer et al., 2019, 2002). Transport changes here could determine the rate at which the AMOC weakens in response to increased green- house gas emissions (Weijer et al., 2020; Collins et al., 2019), beyond the weakening that may already have occurred over the past century (Caesar et al., 2018; Rahmstorf, 2015; Thor- nalley et al., 2018). We aim to build upon these studies by comparing an en- semble of global ocean reanalyses (product refs. 1, 2, 3) di- rectly against the observation-based estimates available over the SAMBA (2013–2017) and the altimetry (1993–2020) time periods. We also compare the reanalyses with new energy-budget MHT estimates at 34.5◦S, which are analo- gous to an estimate at 26◦N in the North Atlantic of Mayer et al. (2022), which is well correlated with observed transports across the RAPID array. While SAMBA array studies have primarily focused on daily-to-seasonal variability, here we focus on monthly-to-interannual variability. All of the time series were averaged to represent monthly values prior to fur- ther analysis. Ocean reanalyses may provide realistic three-dimensional estimates of past changes in the South Atlantic overturning and heat transport (Mignac et al., 2018) and thus could be a useful tool to infer the nature and cause of past MOC and MHT variability. An earlier version of the reanalysis ensem- ble used in this study provides a good representation of the subtropical and subpolar North Atlantic overturning circula- tion (Jackson et al., 2018, 2019; Baker et al., 2022); thus, it may also accurately simulate changes in the South Atlantic. From September 2013 to July 2017, the expanded nine-site South Atlantic Meridional Overturning Circulation – Basin- wide Array (SAMBA) (Fig. J. A. Baker et al.: South Atlantic MOC and MHT variations in observation-based estimates 3 port estimates vary less than the nine-site SAMBA array es- timates (Kersalé et al., 2021, 2020). 1 Introduction 1c) has collected measurements from which both daily meridional transports of heat and vol- ume across 34.5◦S can be estimated (Kersalé et al., 2020, 2021). Volume transports were also estimated during 2009– 2010 using the two-site pilot conﬁguration of the SAMBA array (Meinen et al., 2018, 2013). These studies have im- proved our understanding of the variability in the overturn- ing circulation and meridional heat transport (MHT) in this region. The SAMBA array has improved mooring coverage since 2021 (Chidichimo et al., 2023), but data recorded after 2017 have yet to be incorporated into published AMOC or MHT estimates. South Atlantic overturning and heat transport variations in ocean reanalyses and observation-based estimates Ocean reanalyses are useful tools to identify and under- stand the source of these differences and the mechanisms that control volume and heat transport variability in the South Atlantic, a region critical for determining the global overturning pathways and inter-basin transports. Published by Copernicus Publications. CHAPTER2.2 CHAPTER2.2 CHAPTER2.2 J. A. Baker et al.: South Atlantic MOC and MHT variations in observation-based estimates 2 ER Table 1. Data products used in this study, including documentation where available. Product Product ID and type Data access Documentation ref. no. 1 GLOBAL_REANALYSIS_PHY_001_031 (C-GLORSv7 and GLORYS2V4) [1993–2020]; numerical models EU Copernicus Marine Service Product (2022a) Quality Information Document (QUID): Desportes et al. (2022) Product User Manual (PUM): Gounou et al. (2022) 2 ORAP6 global ocean reanalysis [1993–2020] Updated version of product ref. 1, EU Copernicus Marine Service Product (2022a). In progress for Copernicus Ma- rine implementation, Zuo et al. (2021) 3 GloRanV14 global ocean reanalysis [1993–2021] Updated version of FOAM/GloSea5 from product ref. 1, EU Copernicus Marine Service Product (2022a). In progress for Copernicus Ma- rine implementation 4 GLOBAL_MULTIYEAR_PHY_001_030 (GLORYS12V1) [1993–2019]; numerical models EU Copernicus Marine Service Product (2022b) QUID: Drévillon et al. (2022a) PUM: Drévillon et al. (2022b) 5 South Atlantic Meridional Overturning Circulation – Basin-wide Array (SAMBA) observations for 2013–2017 Data from the Southwest Atlantic MOC project (SAM) are funded by the DOC- NOAA Climate Program Ofﬁce – Ocean Observation and Monitoring Division and the Atlantic Oceanographic and Meteorological Laboratory. These data are freely available via http://www.aoml.noaa.gov/phod/research/moc/samoc/sam/ (last access: 1 December 2022). Kersalé et al. (2020) for MOC Kersalé et al. (2021) for MHT 6 Blended in situ and satellite altimeter estimates for 1993–2021 (Dong et al., 2021) Dong et al. (2021) 7 Energy-budget estimates of Mayer et al. (2022) [1993–2017] Atmospheric energy budgets using ERA5 available at https://doi.org/10.24381/cds.c2451f6b (Mayer et al., 2021b) TOA radiation data from the University of Reading: Liu and Allan (2020) Mayer et al. (2022) Liu et al. (2020) 8 Energy-budget estimates of Trenberth et al. (2019a) [2000–2016] Trenberth et al. (2019a) Trenberth et al. (2019b) 1-osr7, 4, 2023 https://doi.org/10.5194/sp-1-osr State Planet, 1-osr7, 4, 2023 CHAPTER2.2 2.1 Data We use an ensemble of eddy-permitting (1/4◦horizontal resolution) global ocean reanalyses. These are GloRanV14 (an improvement on GloSea5; MacLachlan et al., 2015), C- GLORSv7 (Storto et al., 2016), GLORYS2V4 (Lellouche et al., 2013), and ORAP6 (Zuo et al., 2021). Together, these four reanalyses form a new Copernicus Marine Environ- ment Monitoring Service (CMEMS) reanalysis ensemble, updating product ref. 1 (see Table 1). We also use an eddy- resolving (1/12◦) global ocean reanalysis: GLORYS12V1 (product ref. 4). Each reanalysis uses the NEMO (Nucleus for European Modelling of the Ocean) model, but the sea-ice model and data assimilation techniques differ. Each reanal- ysis is constrained by observations and is driven by atmo- spheric forcing from either ERA5 (Hersbach et al., 2020) or ERA-Interim (Dee et al., 2011) over the period 1993–2020, with GloRan extended to December 2021. They all assimilate satellite SLA, sea-ice concentrations, and in situ temperature and salinity, and they either assimilate satellite sea surface temperature (SST) or implement SST nudging. Since MOC and MHT estimates are currently only avail- able from SAMBA during 2013–2017, longer-term varia- tions must be inferred using model-based and alternative observation-based estimates (Garzoli et al., 2013; Goes et al., 2015; Dong et al., 2009; Mignac et al., 2018; Bias- toch et al., 2021; Caínzos et al., 2022). This includes trans- port estimates derived from satellite sea level anomalies (SLAs) and in situ data (Dong et al., 2015; Majumder et al., 2016). Although Majumder et al. (2016) found large differences between ocean reanalyses and their observation- based estimate from 2000–2014, ocean reanalyses agree bet- ter with observations than free-running models (Mignac et al., 2018). Dong et al. (2021) generated MOC and MHT es- timates over 1993–2021 from a synthetic method combining in situ and satellite data (updated from Dong et al., 2015) that agreed well with expendable bathythermograph (XBT)- derived MOC and MHT estimates in the South Atlantic. The MHT estimates from Dong et al. (2021), however, differed signiﬁcantly from energy-budget MHT estimates produced by Trenberth et al. (2019b). All of the aforementioned trans- We compare the MOC and MHT from the ensemble with the SAMBA-based estimates of Kersalé et al. (2020, 2021), the altimeter-based estimate of Dong et al. (2021), and the energy-budget MHT estimates of Trenberth et al. (2019b) and Mayer et al. (2022). The energy-budget estimates of Mayer et al. J. A. Baker et al.: South Atlantic MOC and MHT variations in observation-based estimates Figure 1. Vertical proﬁle of the overturning transport across 34.5◦S in (a) depth space and (b) density space, averaged over the 2013–2017 period of SAMBA observations, from September 2013 to July 2017. The reanalysis ensemble mean (red line; product refs. 1, 2, 3) and spread (light cyan background shading) are plotted, along with each ensemble member (blue, orange, cyan and purple lines), the GLORYS12V1 reanalysis (pink line; product ref. 4), the SAMBA estimate of Kersalé et al. (2020; black line; product ref. 5), and an altimeter-based estimate of Dong et al. (2021; green line; product ref. 6). The ensemble spread is deﬁned as 2 times the standard deviation across the ensemble members. (c) Map showing the location of the SAMBA moorings (red dots) along 34.5◦S. Figure 1. Vertical proﬁle of the overturning transport across 34.5◦S in (a) depth space and (b) density space, averaged over the 2013–2017 period of SAMBA observations, from September 2013 to July 2017. The reanalysis ensemble mean (red line; product refs. 1, 2, 3) and spread (light cyan background shading) are plotted, along with each ensemble member (blue, orange, cyan and purple lines), the GLORYS12V1 reanalysis (pink line; product ref. 4), the SAMBA estimate of Kersalé et al. (2020; black line; product ref. 5), and an altimeter-based estimate of Dong et al. (2021; green line; product ref. 6). The ensemble spread is deﬁned as 2 times the standard deviation across the ensemble members. (c) Map showing the location of the SAMBA moorings (red dots) along 34.5◦S. radiative ﬂuxes from CERES-EBAF (Loeb et al., 2018) with a backward extension (Liu et al., 2020) and atmo- spheric energy-budget quantities from ERA5 (see Mayer et al., 2021a, for methods). These are combined with ocean heat content (OHC) tendencies from ocean reanalyses to infer the MHT. Mayer et al. (2022) use OHC tendencies from ORAP6 (“Mayer_ORAP6” in ﬁgures); here we use an additional (un- published) ORAS5-based estimate (“Mayer_ORAS5”), us- ing OHC tendencies from ORAS5 (Zuo et al., 2019), the same as that used in the Trenberth et al. (2019b) estimate. For further details, see the Supplement. We note that energy- budget estimates may accumulate errors at southern latitudes, since they are integrated southward from high, northern lati- tudes (Dong et al., 2021). estimates (Table 1). J. A. Baker et al.: South Atlantic MOC and MHT variations in observation-based estimates For the reanalysis, the MHT is calcu- lated by integrating the product of monthly-mean model ve- locity and temperature (scaled by density and speciﬁc heat coefﬁcient) across the whole section with a zero-net-volume transport constraint applied. Each observational product ap- plies its own constraint to reference the ﬂow due to differ- ences in their geostrophic techniques. The altimeter-based dataset references the ﬂow to the time-mean YoMaHA ve- locities at 1000 m (Katsumata and Yoshinari, 2010; Lebedev et al., 2007) and uses a zero-net-mass transport constraint (Dong et al., 2021). Kersalé et al. (2020) use models to ref- erence the time-mean barotropic component at 1500 db, and bottom pressure measurements from the moorings provide the time-varying barotropic velocity component. We calculate the overturning proﬁles, the monthly-to- interannual variation, and the seasonal cycles of the upper- cell MOC and the total MHT in each dataset. We separate the transports into their Ekman and geostrophic components. In the reanalyses, the Ekman component is calculated using the ERA5 or ERA-Interim wind stress, and for MHT, the zonal- mean SST across the section is used, assuming SST is rep- resentative of the Ekman layer temperature. The geostrophic component is calculated as a residual of the total and Ekman transports. 2.1 Data (2022) cal- culate the net surface heat ﬂux using top-of-the-atmosphere State Planet, 1-osr7, 4, 2023 https://doi.org/10.5194/sp-1-osr7-4-2023 CHAPTER2.2 CHAPTER2.2 CHAPTER2.2 CHAPTER2.2 2.2 Methods Ensemble mean and spread and the time mean of the altimeter-based and Mayer energy-budget estimates are cal- culated over 1993–2020 and over the 2013–2017 SAMBA observational period. We calculate monthly-mean MOC across 34.5◦S in depth coordinates, using commonly ap- plied methods (e.g. Frajka-Williams et al., 2019), integrat- ing monthly-mean velocity from coast to coast and from the surface down to the seaﬂoor with a zero-net-volume trans- port constraint applied. Without this constraint, the ensemble mean has a net southward transport through the section over the observational period of 1.14 Sv (as do the individual re- analyses), and GLORYS12V1 has a net southward transport of 3.1 Sv, but the constraint only has a small impact on MOC We also calculate the baroclinic and barotropic compo- nents of the ensemble’s geostrophic MOC. We use thermal wind balance and the model’s geopotential height anomalies to estimate the baroclinic velocities (see, e.g., Perez et al., 2011), integrating these from the deep ocean to the surface. The reference level is set ∼1000 m above the ocean ﬂoor, https://doi.org/10.5194/sp-1-osr7-4-2023 State Planet, 1-osr7, 4, 2023 CHAPTER2.2 J. A. Baker et al.: South Atlantic MOC and MHT variations in observation-based estimates 5 above the unphysically large zonal gradients in geopotential height anomaly that exist in the deepest layers of the model. Thus, the reference level depth varies spatially (∼2000 to ∼4000 m deep) due to the bathymetry, but it is constant in time. The reference velocity is not required to calculate the baroclinic MOC anomalies, so we set the baroclinic ve- locity to 0 at the reference level depth. A visual inspection conﬁrmed that the large month-to-month spatial variations in the baroclinic velocity ﬁeld are in good agreement with the associated changes in the total velocity ﬁeld. We tested the method using different reference level depths that gen- erated similar monthly-mean MOC anomalies (not shown). We calculate the baroclinic component of the MOC by in- tegrating the baroclinic velocities from the surface down to the depth of the time-mean total MOC maximum in each reanalysis (∼1250 m over 2013–2017). We calculate the barotropic component as a residual of the geostrophic and baroclinic MOC anomalies. The baroclinic and barotropic MOC anomalies in the reanalyses and in SAMBA estimates are not directly comparable because the reference levels dif- fer. 2.2 Methods However, our baroclinic MOC anomaly estimate in the reanalyses accounts for baroclinic velocity variations from around 1000 m above the ocean ﬂoor to the surface over which the velocities are greatest and have large monthly vari- ation. (crosses in Fig. 2c). Relative to the ensemble-mean val- ues (MOC: 16.56 Sv; MHT: 0.36 PW), the time-mean MHT range has a 75 % increase from its minimum to its maximum value (excluding the energy-budget estimates) compared to only a 20 % increase for the time-mean MOC range. These ranges are within the documented uncertainty in SAMBA (Table 2). The ensemble-mean MHT is similar to the energy- budget estimates based on Mayer et al. (2022) (Fig. 2c). While there is inter-model spread in the ensemble time-mean transports (crosses in Fig. 2b, d), the spread is smaller than the uncertainty in SAMBA (Table 2), although it is more comparable for the MHT than for the MOC. p Monthly-mean variability (i.e. the standard deviation) of MOC and MHT in the ensemble is similar to the altimeter- based estimate over 2013–2017 and 1993–2020, whereas variability observed from SAMBA is much greater (Figs. 2a, c and 3a–d; Table 2), with signiﬁcant differences (p < 0.05 in an F test for the equality of two variances). Similarly, the ensemble-mean time series is signiﬁcantly (p < 0.05) correlated with the altimeter-based estimate (r = 0.63 for MOC; r = 0.77 for MHT; both over 2013–2017), but it is not well correlated with SAMBA (r < 0.1). The monthly- mean SAMBA estimates (Fig. 3a, b) and the Mayer energy- budget MHT estimates have high-frequency variations of comparable magnitude (Fig. 3b, d and Table 2), although their variability is uncorrelated. Mayer_ORAP6 is weakly correlated with the ensemble mean and altimeter-based es- timate (r = 0.14 and r = 0.19 over 1993–2017 and r = 0.28 and r = 0.32 over 2013–2017, for the respective datasets). Mayer_ORAS5 has a higher correlation with the ensemble mean and altimeter-based estimate (r = 0.30 and r = 0.32 over 1993–2017 and r = 0.52 and r = 0.57 over 2013–2017, for the respective datasets). The GloRan reanalysis run with and without assimilating altimetry data (not shown) has a similar correlation with the altimeter-based estimate (r = 0.52 vs. r = 0.56 for MOC over 2013–2017). Thus, the strong correlation between ensemble mean and altimeter- based estimates is not dependent on directly assimilating al- timetry data. 2.2 Methods The experimental reanalysis does, however, still assimilate in situ and satellite temperature and salinity data, which would serve to constrain thermosteric and halosteric contributions, respectively, to sea level. In the 12-month run- ning mean estimates (Fig. 3e, f), the ensemble mean is only weakly correlated with the altimeter-based estimate (r = 0.24 for MOC; r = 0.25 for MHT), so their high monthly-mean correlation is largely due to similar seasonal variability. 3.1 MOC proﬁles and statistics of variability The ensemble of reanalyses captures the main structure of the observed overturning proﬁle (Fig. 1a). The depth and strength of the maximum overturning is similar among all estimates with a range of ∼15–18 Sv (Fig. 1a). The proﬁles diverge in the deeper ocean, with a weaker than observed lower overturning cell and southward ﬂow in the ensemble (i.e. the MOC decreases more gradually with depth). The re- analyses are less accurate at depth due to there being fewer observations to constrain the ﬂow. The overturning proﬁles of the ensemble and GLORYS12V1 in density space have no negative transport (i.e. no abyssal cell), and their MOC is stronger than in depth space (Fig. 1b). The temporal vari- ability in their upper MOC strength at 34.5◦S, however, is fairly insensitive to the vertical coordinate system used for integration (Fig. S1 in the Supplement). We therefore focus on the MOC in depth space because the reanalyses can then be directly compared with the observational estimates. The GLORYS12V1 reanalysis has a larger time-mean MOC and MHT than the ensemble mean (and GLO- RYS2V4). It has similar monthly-mean variability to the lower-resolution reanalyses: slightly larger than the ensem- ble mean but smaller than GLORYS2V4 (Table 2). It is also signiﬁcantly correlated with the ensemble mean (r = 0.80 for MOC and r = 0.84 for MHT over 1993–2019). Thus, fully resolving (as opposed to only permitting) eddies in the ocean reanalyses considered here is important to infer the time- We analyse the basic statistics of the variability in the maximum MOC strength and the MHT by looking at their time mean and standard deviation over 2013–2017 and 1993– 2020. The time-mean MOC estimates have a range of 15.5– 18.7 Sv, with the ensemble mean (labelled “mean” in ﬁgures) being only slightly weaker than the altimeter-based estimate and that observed across SAMBA (crosses in Fig. 2a). The time-mean MHT estimates have a range of 0.31–0.61 PW https://doi.org/10.5194/sp-1-osr7-4-2023 State Planet, 1-osr7, 4, 2023 CHAPTER2.2 CHAPTER2.2 J. A. Baker et al.: South Atlantic MOC and MHT variations in observation-based estimates 6 Table 2. Time mean and uncertainty (or ensemble spread) and monthly-mean variability and trends of the maximum MOC and the MHT across 34.5◦S, for the ensemble mean (product refs. 1, 2, 3), GLORYS12V1 (product ref. 4), SAMBA observations (product ref. 5), an altimeter-based estimate (product ref. 6), and energy-budget estimates (product refs. 7 and 8). J. A. Baker et al.: South Atlantic MOC and MHT variations in observation-based estimates 7 Figure 2. Whisker–box plots of the monthly-mean MOC (a, b) and MHT (c, d) across 34.5◦S over the SAMBA observational period (2013–2017), using the same products as in Fig. 1. Energy-budget estimates Mayer_ORAP6 and Mayer_ORAS5 (yellow; product ref. 7) are also used for the MHT. Reanalyses analysed are shown in (b) and (d) with a reduced scale to highlight the differences between models. Boxes represent the interquartile range (IQR) with the median (line) and mean (crosses) shown. Whiskers cover a range of values up to 1 IQR beyond the upper and lower quartiles, and diamonds are outlying values beyond this range. Note: the x-axis scale changes between panels (a) and (c), on the one hand, and (b) and (d), on the other. Figure 2. Whisker–box plots of the monthly-mean MOC (a, b) and MHT (c, d) across 34.5◦S over the SAMBA observational period (2013–2017), using the same products as in Fig. 1. Energy-budget estimates Mayer_ORAP6 and Mayer_ORAS5 (yellow; product ref. 7) are also used for the MHT. Reanalyses analysed are shown in (b) and (d) with a reduced scale to highlight the differences between models. Boxes represent the interquartile range (IQR) with the median (line) and mean (crosses) shown. Whiskers cover a range of values up to 1 IQR beyond the upper and lower quartiles, and diamonds are outlying values beyond this range. Note: the x-axis scale changes between panels (a) and (c), on the one hand, and (b) and (d), on the other. The 12-month running mean from SAMBA is entirely dif- ferent to other estimates (Fig. 3e, f), with a rapid increase in the MOC (∼14 Sv) and MHT (∼0.7 PW) from March 2014 to June 2016, followed by a rapid decline. Although an ex- tended time series is needed to determine longer timescale variations, the interannual variability captured by SAMBA over 2013–2017 exceeds that of other estimates. Only the Mayer MHT estimates have interannual variations of com- parable magnitude, but those variations occur before 2013 (Fig. 3f). mean transports across 34.5◦S, but it has minimal impact on the variation in the monthly-mean transports. The 12-month running mean MOC and MHT in the en- semble over 1993–2020 are relatively stable (Fig. 3e, f), with similar ensemble-mean values to those during 2013–2017 (Table 2) and no signiﬁcant trend over 1993–2020. J. A. Baker et al.: South Atlantic MOC and MHT variations in observation-based estimates However, the individual reanalyses have signiﬁcant (p < 0.05) trends in the MOC over 1993–2020 with differing sign and mag- nitude (Table 2). In contrast, only GloRan has a signiﬁcant (increasing) trend in MHT (∼0.042 PW per decade). GLO- RYS12V1 has no signiﬁcant trend in MOC or MHT. Hence, there is uncertainty in the long-term trends amongst the re- analyses. 3.1 MOC proﬁles and statistics of variability All volume transports are referenced to 0 at the surface. The time-mean MOC and monthly-mean variability calculated in the reanalyses using no net-zero-transport constraint is added in parentheses. Time-mean values are calculated over the 2013–2017 SAMBA observational period and over the full 1993–2020 ensemble period if available. Uncertainty in the ensemble mean is deﬁned as the standard error in the time-mean transport across the ensemble (note: this is smaller than the true uncertainty in the estimate). Monthly-mean variability (i.e. a measure of the deviation of monthly-mean data from the time mean) is deﬁned as the standard deviation of the monthly-mean transports over the time series. Methods used to calculate SAMBA observational uncertainty (Kersalé et al., 2021, 2020) are described in Meinen et al. (2013) and Kersalé et al. (2021). Trends that are statistically insigniﬁcant (p > 0.05) are labelled NS. Variable Statistic Ocean reanalyses South Atlantic MOC Energy-budget estimates estimates Ensemble GLORYS12V1 SAMBA Altimeter Trenberth Mayer ORAS5 Mayer ORAP6 Dong MOC Mean ± uncertainty (2013–2017) 16.56 ± 0.37 (16.29) 18.72 (18.02) 17.29 ± 5.0 18.69 – – – (Sv) Monthly-mean variability 2.67 (3.20) 2.90 (2.70) 11.35 3.25 – – – Mean ± uncertainty (1993–2020) 16.38 ± 0.66 (16.11) 19.23 (18.51) – 18.34 – – – Monthly-mean variability 3.00 (3.53) 3.30 (3.14) – 3.48 – – – Trends (Sv per decade) (1993–2020) 0.17 (NS) −0.08 (NS) – 0.66 – – – MHT Mean ± uncertainty (2013–17) 0.36 ± 0.03 0.44 0.50 ± 0.23 0.61 – 0.31 0.31 (PW) Monthly-mean variability 0.19 0.20 0.55 0.20 – 0.46 0.43 Mean ± uncertainty (1993–2020) 0.37 ± 0.04 0.49 – 0.58 0.33 (2000–2016) 0.33 (1993–2017) 0.34 (1993–2017) Monthly-mean variability 0.20 0.23 – 0.21 – 0.40 0.44 Trends (PW per decade) (1993–2020) −0.001 (NS) −0.007 (NS) – 0.036 −0.010 (2000–16) 0.086 (1993–2016) 0.094 (1993–2016) Variable Statistic Ensemble GloRanV14 C-GLORSv7 ORAP6 GLORYS2V4 MOC (Sv) Trends (Sv per decade) (1993–2020) 1.18 −0.32 0.41 −0.60 MHT (PW) Trends (PW per decade) (1993–2020) 0.042 −0.014 (NS) −0.012 (NS) −0.016 (NS) 7 4 2023 https://doi org/10 5194/s State Planet, 1-osr7, 4, 2023 CHAPTER2.2 CHAPTER2.2 J. A. Baker et al.: South Atlantic MOC and MHT variations in observation-based estimates 8 Figure 3. Time series of the monthly overturning (a, c, e) and heat transport (b, d, f) anomalies nominally across 34.5◦S, with monthly-mean values from September 2013 to July 2017 (a, b) and over 1993–2021 (c, d) and 12-month running mean values over 1993–2021 (e, f) in the four reanalyses of the ensemble, ensemble mean (red), GLORYS12V1 (pink), SAMBA observations (black), an altimeter-based estimate (green), and energy-budget estimates (yellow and brown, product ref. 8). Labels, shading, and product information as in Fig. 1. The horizontal grey dotted lines in (d) divide the y axis into two linear scales, with the y axis compressed above the line. Note: Trenberth energy-budget estimate is for latitude 33.5◦S. Figure 3. Time series of the monthly overturning (a, c, e) and heat transport (b, d, f) anomalies nominally across 34.5◦S, with monthly-mean values from September 2013 to July 2017 (a, b) and over 1993–2021 (c, d) and 12-month running mean values over 1993–2021 (e, f) in the four reanalyses of the ensemble, ensemble mean (red), GLORYS12V1 (pink), SAMBA observations (black), an altimeter-based estimate (green), and energy-budget estimates (yellow and brown, product ref. 8). Labels, shading, and product information as in Fig. 1. The horizontal grey dotted lines in (d) divide the y axis into two linear scales, with the y axis compressed above the line. Note: Trenberth energy-budget estimate is for latitude 33.5◦S. matological seasonal cycle between each estimate (Fig. 4) stem from their geostrophic differences (Fig. 4e, f) because the Ekman annual cycles are similar year to year (not shown) and for all estimates (Fig. 4c, d). Differences between esti- mates are clearer in the geostrophic component, peaking be- fore the ensemble mean in the altimeter-based estimate and after the ensemble mean in SAMBA. Thus, the Ekman and geostrophic components tend to counteract each other in the altimeter-based estimate and augment each other in SAMBA. This causes a greater increase in the magnitude of the total MOC and MHT seasonal cycles (relative to their geostrophic components) in SAMBA than it does in the altimeter-based estimate but a greater change in the seasonal cycle phase and shape in the altimeter-based estimate (cf. Fig. 4a, b and e, f). The relative contribution of the Ekman component to the total MOC and MHT in the ensemble is nonetheless sig- niﬁcantly greater than in SAMBA. 3.2 Seasonal cycles The altimeter-based estimate has signiﬁcant (p < 0.05) increases in MOC (∼0.66 Sv per decade) and MHT (∼0.036 PW per decade) over 1993–2020. The aforemen- tioned MHT trends are similar over 1993–2016 (Glo- RanV14: ∼0.047 PW per decade; altimeter: ∼0.032 PW per decade). There is a signiﬁcant increase in MHT over 1993– 2016 in both the ORAS5-based (∼0.086 PW per decade) and the ORAP6-based (∼0.094 PW per decade) Mayer esti- mates. The Trenberth estimate has a signiﬁcant but weak de- cline (∼−0.010 PW per decade) over 2000–2016; the Mayer estimates also decline over this period, but the trend is in- signiﬁcant. There is a predominantly annual cycle in the ensemble mean and altimeter-based transports, unlike the SAMBA seasonal cycle that has a stronger semi-annual variability (Fig. 3c, d). While we show the ensemble mean and altimeter-based sea- sonal cycles over 2013–2017 (Fig. 4), the seasonal cycles derived over the full record lengths are similar (not shown). The ensemble and altimeter-based overturning are weakest in austral summer, but the ensemble is strongest in May/June, peaking 2 months after the altimeter-based estimate (Fig. 4a, b). In contrast, the MOC in SAMBA is dominated by a semi- annual signal, with minima in April and September and max- ima in August and December. There are year-to-year vari- https://doi.org/10.5194/sp-1-osr7-4-2023 State Planet, 1-osr7, 4, 2023 State Planet, 1-osr7, 4, 2023 CHAPTER2.2 CHAPTER2.2 J. A. Baker et al.: South Atlantic MOC and MHT variations in observation-based estimates 9 Figure 4. Seasonal cycles of (a, c, e) the overturning and (b, d, f) the MHT anomalies across 34.5◦S, averaged over the SAMBA obser- vational period from September 2013 to July 2017. The exception is the energy-budget MHT estimate of Trenberth et al. (2019b), which is averaged over 2000–2016, and also the ORAS5-based Mayer energy-budget estimate, “Mayer_ORAS5_2000–16” (olive), which is averaged over the same period for comparison. The total (a, b), Ekman (c, d), and geostrophic (e, f) components of these transports are plotted. Labels, shading, and product information are as in Figs. 1 and 3. Figure 4. Seasonal cycles of (a, c, e) the overturning and (b, d, f) the MHT anomalies across 34.5◦S, averaged over the SAMBA obser- vational period from September 2013 to July 2017. The exception is the energy-budget MHT estimate of Trenberth et al. (2019b), which is averaged over 2000–2016, and also the ORAS5-based Mayer energy-budget estimate, “Mayer_ORAS5_2000–16” (olive), which is averaged over the same period for comparison. The total (a, b), Ekman (c, d), and geostrophic (e, f) components of these transports are plotted. Labels, shading, and product information are as in Figs. 1 and 3. the ensemble, with their sum equal to the geostrophic com- ponent (Fig. 5). By contrast, these components tend to aug- ment each other in SAMBA (Fig. 5), so their geostrophic sea- sonal cycle has variations of a greater magnitude. The baro- clinic component tends to dominate in both datasets, primar- ily controlling the phase of the geostrophic MOC seasonal cycle (Fig. 5). Although the barotropic component tends to counteract the baroclinic component in the ensemble, it has a notable effect on the phase of the geostrophic MOC sea- sonal cycle over 2013–2017 unlike over 1993–2020. Thus, while differences in the seasonality of the baroclinic MOC component account for most of the difference in the season- ality of the geostrophic MOC, differences in the barotropic component between the ensemble and SAMBA also play a role. this end-of-year peak relative to the magnitude of decrease from the preceding peak is smaller in the ensemble mean than in SAMBA, it is noteworthy, increasing by 52 % of the preceding decrease (and by 77 % in the seasonal cycle over 1993–2020) compared to 84 % in SAMBA. J. A. Baker et al.: South Atlantic MOC and MHT variations in observation-based estimates The altimeter- based estimate has no signiﬁcant increase in the geostrophic component in austral spring, and there is also no increase in the ensemble-mean MHT, unlike in SAMBA (Fig. 4f). J. A. Baker et al.: South Atlantic MOC and MHT variations in observation-based estimates In the ensemble mean (and in GLORYS12V1 and SAMBA), the geostrophic com- ponent of the MOC (Fig. 4e) has a second peak in Novem- ber or December (i.e. austral spring or summer) and thus has a semi-annual signal. Although the increase in the MOC to ations in the annual cycles of all estimates (not shown), with variations in phase, shape, and magnitude. In SAMBA, 4 years of observations are not long enough to examine the sensitivity of the seasonal cycle to changing the time period, but given the strong high-frequency variations, the seasonal cycle based on 4 years of data is unlikely to be robust. y y y The shape of the seasonal cycle in MHT is similar to that of the MOC for each estimate as expected given the high correlation between the monthly-mean MHT and MOC (r = 0.90, r = 0.91, and r = 0.96 for the ensemble mean, altimeter-based estimate, and SAMBA, respectively, over 2013–2017). The Mayer energy-budget estimates have sea- sonal cycles dominated by an annual signal, with a larger magnitude range than other estimates. They are similar to the Trenberth estimate but with greater month-to-month variability. However, when averaged over the 2000–2016 period used in the Trenberth estimate rather than 2013– 2017, they become smoother and closer to the ensemble (“Mayer_ORAS5_2000–16” in Fig. 4). Year-to-year variations in the annual cycles of each esti- mate over 2013–2017 (not shown) and differences in the cli- State Planet, 1-osr7, 4, 2023 https://doi.org/10.5194/sp-1-osr7-4-2023 CHAPTER2.2 CHAPTER2.2 J. A. Baker et al.: South Atlantic MOC and MHT variations in observation-based estimates J. A. Baker et al.: South Atlantic MOC and MHT variations in observation-based estimates 10 Figure 5. Seasonal cycles of the MOC anomalies across 34.5◦S, averaged over the (a, c, e) the SAMBA observational period from September 2013 to July 2017 and (b, d, f) the 1993–2020 period of the reanalyses. The geostrophic (a, b), baroclinic (c, d), and barotropic (e, f) components of these transports are plotted. Labels, shading, and product information are as in Figs. 1 and 3. Figure 5. Seasonal cycles of the MOC anomalies across 34.5◦S, averaged over the (a, c, e) the SAMBA observational period from September 2013 to July 2017 and (b, d, f) the 1993–2020 period of the reanalyses. The geostrophic (a, b), baroclinic (c, d), and barotropic (e, f) components of these transports are plotted. Labels, shading, and product information are as in Figs. 1 and 3. to dominate. Over 2013–2017, there is much larger spatial variability in the seasonal transport, with signiﬁcant contri- butions to the seasonal variations from the interior as well as from the boundaries. Therefore, differences in the MOC sea- sonality between datasets is likely caused by seasonal varia- tions in both the boundary currents and the interior baroclinic transports. A spatial analysis of the baroclinic transports in SAMBA could determine the regions responsible for the sea- sonality of this component and thus why it differs from the ensemble. mean variability in the ensemble and in SAMBA over 2013– 2017 (Fig. 6d, e), although the baroclinic variability is slightly higher in SAMBA (7.5 Sv vs. 5.3 Sv). Similarly, the interannual variability in the baroclinic and barotropic com- ponents has similar peak-to-trough magnitudes over 2013– 2017 in the ensemble and SAMBA (Fig. 6f). However, since the barotropic component counteracts the baroclinic compo- nent in the ensemble, the geostrophic and total MOC anoma- lies in the ensemble have much smaller monthly-mean and interannual variability than in SAMBA (Fig. 6a, b, f and Table 2). The monthly-mean and 12-month running mean baroclinic and barotropic components in the ensemble have even larger variability over 1993–2020, but these compo- nents counteract each other over the whole period (Fig. 6f). The altimeter-based estimate uses reference velocities at 1000 m depth that are constant in time. Thus, the barotropic component has no temporal variability, so the geostrophic MOC anomalies only account for baroclinic transport anomalies above 1000 m. J. A. Baker et al.: South Atlantic MOC and MHT variations in observation-based estimates Given the baroclinic com- ponent primarily determines the shape of the seasonal cycle in the ensemble and SAMBA, the fact the barotropic compo- nent is constant in the altimeter-based estimate may not sig- niﬁcantly impact its estimate of the MOC’s seasonal cycle phase. However, the magnitude of its monthly, interannual, and seasonal variability may be affected if temporal changes https://doi.org/10.5194/sp-1-osr7-4-2023 State Planet, 1-osr7, 4, 2023 3.3 Baroclinic and barotropic components We investigate possible causes of the difference in vari- ability between SAMBA and the ensemble by separating the geostrophic MOC anomalies into their baroclinic and barotropic components. The baroclinic and barotropic com- ponents of the MOC are not directly comparable between the ensemble and SAMBA due to differences in the reference level depth, but this probably has little impact on the differ- ences between these estimates (see Sect. 2.2). The seasonal cycles of these components largely counteract each other in We also analyse the monthly-mean and interannual vari- ations in the baroclinic and barotropic components of the MOC anomalies (Fig. 6). Both the baroclinic and the barotropic components of the MOC have similar monthly- https://doi.org/10.5194/sp-1-osr7-4-2023 State Planet, 1-osr7, 4, 2023 CHAPTER2.2 CHAPTER2.2 CHAPTER2.2 J. A. Baker et al.: South Atlantic MOC and MHT variations in observation-based estimates J. A. Baker et al.: South Atlantic MOC and MHT variations in observation-based estimates 11 Figure 6. Time series of (a–e) monthly-mean (2013–2017) and (f) 12-month running mean (1993–2019) MOC anomalies showing the geostrophic (blue), baroclinic (red), and barotropic (green) components in (a) the ensemble, (b) SAMBA, and (c–f) both the ensemble (solid lines) and SAMBA (dashed lines). Figure 6. Time series of (a–e) monthly-mean (2013–2017) and (f) 12-month running mean (1993–2019) MOC anomalies showing the geostrophic (blue), baroclinic (red), and barotropic (green) components in (a) the ensemble, (b) SAMBA, and (c–f) both the ensemble (solid lines) and SAMBA (dashed lines). ations that were previously under-resolved with only two mooring sites and are now better resolved but likely still aliased with nine sites. in the barotropic component are important, as suggested by the ensemble and SAMBA estimates of this component. The reference level depth used in the reanalyses (i.e. not in our baroclinic and barotropic component estimates but that im- plemented in the models and thus in the geostrophic esti- mate) is the ocean ﬂoor, closer to the depths used to estimate the time-varying barotropic component in SAMBA. Thus, differences in the reference level are unlikely to cause the dif- ferences in the geostrophic component between SAMBA and the ensemble. However, differences in the methods used to estimate the barotropic velocity at that reference level could cause some of the difference. 4 Discussion Seasonal variations in the baroclinic component of the MOC in the ensemble over 1993–2020 are caused by seasonal variations in both the eastern and western boundary volume transports, with variations in the western boundary tending https://doi.org/10.5194/sp-1-osr7-4-2023 State Planet, 1-osr7, 4, 2023 CHAPTER2.2 CHAPTER2.2 https://doi.org/10.5194/sp-1-osr7-4-2023 5 Conclusions To summarise, an ensemble of ocean reanalyses appears to be a useful tool to understand changes in the South Atlantic MOC and MHT and to identify differences between obser- vational estimates. Reanalyses also enable examination of variations prior to the SAMBA array record. Comparisons of reanalyses and observational estimates can be used together to reﬁne methodologies and sampling approaches and ulti- mately improve our understanding and estimations of ocean transports in the South Atlantic. While there is interannual variability in the reanalyses and altimeter-based estimate over 1993–2020, SAMBA observa- tions and some energy-budget MHT estimates have much larger interannual variability. The climatological seasonal cycles of the MOC and MHT vary considerably in phase and magnitude between estimates due to differences in the geostrophic ﬂow, with good agreement in the Ekman con- tributions among all datasets considered. Differences in the baroclinic component of the MOC are most important for determining the phase of the seasonal cycle in both the re- analyses and SAMBA, although the barotropic component also plays a role. The baroclinic, barotropic, and Ekman MOC components tend to augment each other in SAMBA, whereas they tend to counteract each other in the ensemble and altimeter-based estimate. Thus, in SAMBA the monthly- mean, interannual, and seasonal MOC anomalies have a greater magnitude than in the ensemble and altimeter-based estimate. This causes a large increase in the monthly-mean standard deviation of the total MOC in SAMBA. The baro- clinic and barotropic MOC anomalies also have more fre- quent monthly-mean ﬂuctuations in SAMBA. Data availability. All data products used in this paper are listed in Table 1, along with their corresponding documentation and online availability. Supplement. The supplement related to this article is available online at: https://doi.org/10.5194/sp-1-osr7-4-2023-supplement. Author contributions. JAB conceived the study, performed the analysis, and wrote the manuscript. All authors provided data for analysis, discussed the results, and edited or commented on the manuscript. Competing interests. The contact author has declared that none of the authors has any competing interests. q y Further insight into the cause of the similarities and dif- ferences between the ensemble, SAMBA and the altimeter- based estimate might be found by comparing the monthly- mean density proﬁles of these estimates. This could show how contributions from the baroclinic velocity to the geostrophic MOC anomalies vary between the datasets, in- cluding their spatial variations, and how these lead to differ- ences in seasonality. 5 Conclusions An ensemble of global ocean reanalyses from CMEMS pro- vides a useful estimate of the magnitude and variability in the South Atlantic MOC and MHT, although it differs substan- tially from estimates based on SAMBA array data at 34.5◦S, observed between 2013 and 2017. The ensemble is compared with several other estimates of the MOC and MHT, which differ in many aspects from – but also have similarities with – the reanalyses. We have shown that the monthly-mean MOC variability (i.e. standard deviation) is greater in SAMBA than in the ensemble and altimeter-based estimate, primarily because the Ekman, barotropic, and baroclinic components augment each other in SAMBA, whereas they tend to counteract each other in the ensemble and altimeter-based estimates. While the standard deviation provides an insight into the month- to-month ﬂuctuations, it does not determine the frequency of these ﬂuctuations. Both the baroclinic and barotropic compo- nents have more frequent monthly ﬂuctuations in SAMBA than in the ensemble (Fig. 5). These high-frequency varia- tions could be caused by ocean eddy variability and vari- The ensemble-mean (and 1/12◦GLORYS12V1 reanaly- sis) transports have no long-term trend over 1993–2020, al- though the trends in the individual reanalyses differ, and ob- servational estimates increase over this period. All estimates of the time-mean MOC are similar (∼15.5–18.7 Sv), but rel- ative to the ensemble-mean value there is greater spread in the MHT (0.31–0.61 PW), with the ensemble mean weaker than SAMBA observations. Monthly-mean MOC and MHT in the ensemble, the 1/12◦GLORYS12V1 reanalysis, and an altimeter-based estimate (Dong et al., 2021) vary signif- https://doi.org/10.5194/sp-1-osr7-4-2023 State Planet, 1-osr7, 4, 2023 State Planet, 1-osr7, 4, 2023 CHAPTER2.2 CHAPTER2.2 J. A. Baker et al.: South Atlantic MOC and MHT variations in observation-based estimates 12 J. A. Baker et al.: South Atlantic MOC and MHT variations in observation-based estimates J. A. Baker et al.: South Atlantic MOC and MHT variations in observation-based estimates icantly less than those from the SAMBA array. In contrast, energy-budget estimates of MHT (Mayer et al., 2022) have a large monthly-mean variability comparable to SAMBA. Both the monthly-mean MOC and MHT in the ensemble are signiﬁcantly correlated with the altimeter-based estimate across the whole 1993–2020 period (although most of the skill is from the seasonal cycle), whereas correlations with SAMBA estimates are not signiﬁcant. 5 Conclusions Similarly, the barotropic velocity (ver- tically averaged velocity) in the reanalyses can be compared with that used by the in situ altimetry and SAMBA methods to reference the ﬂow. We also suggest exploring the horizon- tal resolution of SAMBA moorings used on the boundaries since it may alias variability here, with too few sites over steeply sloping topography. The impact of array resolution on SAMBA could be inferred by recalculating the baroclinic and barotropic components of the MOC in the ensemble us- ing only a subset of their vertical density proﬁles. Reanaly- ses could therefore provide information on whether modiﬁ- cations to the observational density across the SAMBA array may provide more robust observational transport estimates. Use of the expanded set of moorings will also allow us to determine the importance of the aliasing of variability on the boundaries. Since the reanalyses are in reasonable agree- ment with altimeter-based estimates but not with SAMBA, this prompts closer inspection of the methodologies used to make the computations. Disclaimer. Publisher’s note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Acknowledgements. We thank two anonymous reviewers for their constructive comments that improved this study. Review statement. 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T., and Cheng, L.: Ocean Meridional Heat Transport Values, Version 1.0, UCAR/NCAR – GDEX, https://doi.org/10.5065/9v3y-fn61, 2019a. Zuo, H., Balmaseda, M. A., de Boisseson, E., Tietsche, S., Mayer, M., and de Rosnay, P.: The ORAP6 ocean and sea-ice reanalysis: description and evaluation, EGU General Assembly 2021, online, 19–30 April 2021, EGU21-9997, https://doi.org/10.5194/egusphere-egu21-9997, 2021. Trenberth, K. E., Zhang, Y., Fasullo, J. https://doi.org/10.5194/sp-1-osr7-4-2023 References D.: Temporal variability of the meridional overturning cir- culation at 34.5◦S: Results from two pilot boundary arrays in the South Atlantic, J. Geophys. Res.-Oceans, 118, 6461–6478, https://doi.org/10.1002/2013JC009228, 2013. Meinen, C. S., Speich, S., Piola, A. R., Ansorge, I., Campos, E., Kersalé, M., Terre, T., Chidichimo, M. P., Lamont, T., Sato, O. T., Perez, R. C., Valla, D., van den Berg, M., le Hé- naff, M., Dong, S., and Garzoli, S. L.: Meridional Overturn- ing Circulation Transport Variability at 34.5◦S During 2009– 2017: Baroclinic and Barotropic Flows and the Dueling Inﬂu- ence of the Boundaries, Geophys. Res. Lett., 45, 4180–4188, https://doi.org/10.1029/2018GL077408, 2018. Kersalé, M., Meinen, C. S., Perez, R. C., Piola, A. R., Spe- ich, S., Campos, E. J. D., Garzoli, S. L., Ansorge, I., Volkov, D. L., le Hénaff, M., Dong, S., Lamont, T., Sato, O. T., and van den Berg, M.: Multi-Year Estimates of Daily Heat Transport by the Atlantic Meridional Overturning Circulation at 34.5◦S, J. Geophys. Res.-Oceans, 126, e2020JC016947, https://doi.org/10.1029/2020JC016947, 2021. Lebedev, K., Yoshinari, H., Maximenko, N. A., and Hacker, P. W.: YoMaHa’07: Velocity data assessed from trajectories of Argo ﬂoats at parking level and at the sea surface, IPRC Technical Note No. 4, 16 pp., 2007. Mignac, D., Ferreira, D., and Haines, K.: South Atlantic meridional transports from NEMO-based simulations and reanalyses, Ocean Sci., 14, 53–68, https://doi.org/10.5194/os-14-53-2018, 2018. Lellouche, J.-M., Le Galloudec, O., Drévillon, M., Régnier, C., Greiner, E., Garric, G., Ferry, N., Desportes, C., Testut, C.-E., Bricaud, C., Bourdallé-Badie, R., Tranchant, B., Benkiran, M., Drillet, Y., Daudin, A., and De Nicola, C.: Evaluation of global Nadeau, L. P. and Jansen, M. F.: Overturning Circulation Pathways in a Two-Basin Ocean Model, J. Phys. Oceanogr., 50, 2105– 2122, https://doi.org/10.1175/JPO-D-20-0034.1, 2020. Y., Daudin, A., and De Nicola, C.: Evaluation of globa State Planet, 1-osr7, 4, 2023 https://doi.org/10.5194/sp-1-osr7-4-2023 CHAPTER2.2 Satellite monitoring of surface phytoplankton functional types in the Atlantic Ocean over 20 years (2002–2021) n of the Copernicus Ocean State Report (OSR7) Hongyan Xi1, Marine Bretagnon2, Svetlana N. Losa1,3, Vanda Brotas4, Mara Gomes4, Ilka Peeken1, Leonardo M. A. Alvarado1, Antoine Mangin2, and Astrid Bracher1,5 1Alfred Wegener Institute, Helmholtz Centre for Polar and Marine Research, Bremerhaven, Germany 2ACRI-ST, Sophia Antipolis CEDEX, France 3Shirshov Institute of Oceanology, Russian Academy of Sciences, Moscow, Russia 4MARE/ARNET – Marine and Environmental Sciences Centre, Faculdade de Ciências, Universidade de Lisboa, Campo Grande, Lisbon, Portugal 5Institute of Environmental Physics, University of Bremen, Bremen, Germany Hongyan Xi1, Marine Bretagnon2, Svetlana N. Losa1,3, Vanda Brotas4, Mara Gomes4, Ilka Peeken1, Leonardo M. A. Alvarado1, Antoine Mangin2, and Astrid Bracher1,5 1Alfred Wegener Institute, Helmholtz Centre for Polar and Marine Research, Bremerhaven, Germany 2ACRI-ST, Sophia Antipolis CEDEX, France 3Shirshov Institute of Oceanology, Russian Academy of Sciences, Moscow, Russia 4MARE/ARNET – Marine and Environmental Sciences Centre, Faculdade de Ciências, Universidade de Lisboa, Campo Grande, Lisbon, Portugal 5Institute of Environmental Physics, University of Bremen, Bremen, Germany rnicus Ocean State Report (OSR7) Correspondence: Hongyan Xi (hongyan.xi@awi.de) Correspondence: Hongyan Xi (hongyan.xi@awi.de) Received: 30 July 2022 – Discussion started: 30 September 2022 Revised: 24 January 2023 – Accepted: 17 March 2023 – Published: 27 September 2023 ean State Report (OSR7) Received: 30 July 2022 – Discussion started: 30 September 2022 Revised: 24 January 2023 – Accepted: 17 March 2023 – Published: 27 September 2023 Abstract. An analysis of multi-satellite-derived products of four major phytoplankton functional types (PFTs – diatoms, haptophytes, prokaryotes and dinoﬂagellates) was carried out to investigate the PFT time series in the Atlantic Ocean between 2002 and 2021. The investigation includes the 2-decade trends, climatology, phenology and anomaly of PFTs for the whole Atlantic Ocean and its different biogeochemical provinces in the surface layer that optical satellite signals can reach. The PFT time series over the whole Atlantic region showed mostly no clear trend over the last 2 decades, except for a small decline in prokaryotes and an abrupt increase in diatoms during 2018–2019, which is mainly observed in the northern Longhurst provinces. The phenology of diatoms, haptophytes and dinoﬂagellates is very similar: at higher latitudes bloom maxima are reached in spring (April in the Northern Hemisphere and October in the Southern Hemisphere), in the oligotrophic regions in winter time and in the tropical regions during May to September. In general, prokaryotes show opposite annual cycles to the other three PFTs and present more spatial complexity. J. A. Baker et al.: South Atlantic MOC and MHT variations in observation-based estimates T., and Cheng, L.: Observation-Based Estimates of Global and Basin Ocean Merid- ional Heat Transport Time Series, J. Climate, 32, 4567–4583, https://doi.org/10.1175/JCLI-D-18-0872.1, 2019b. Weijer, W., de Ruijter, W. P. M., Sterl, A., and Drijfhout, S. S.: Response of the Atlantic overturning circulation to South At- lantic sources of buoyancy, Glob. Planet. Change, 34, 293–311, https://doi.org/10.1016/S0921-8181(02)00121-2, 2002. State Planet, 1-osr7, 4, 2023 CHAPTER2.2 CHAPTER2.2 Chapter 2.3 – 7th edition of the Copernicus Ocean State Report (OSR7) Chapter 2.3 – 7th edition of the Co State Planet, 1-osr7, 5, 2023 https://doi.org/10.5194/sp-1-osr7-5-2023 © Author(s) 2023. This work is distributed under the Creative Commons Attribution 4.0 License. Chapter 2.3 – 7th edition of the Copernicus Ocean ter 2.3 – 7th edition of the Copernicus Ocean State Report (O 1 Introduction Phytoplankton diversity is very high, summarized in phytoplankton functional types (PFTs) as prokaryotes (cyanobacteria) and eukaryotes, including diatoms, hapto- phytes and dinoﬂagellates. Depending on area, season and size class, different PFTs can act as dominating organisms in the food web and, therefore, regulate the seasonality of the predators (Käse and Geuer, 2018). Diatoms, known as major siliciﬁers, have silica frustules that surround and pro- tect the cells and sink rapidly out of the surface layer of the ocean, contributing to the transport of carbon, nitrogen and silica to deeper waters (IOCCG, 2014). Haptophytes are another very abundant PFT in the global ocean occurring mainly in the middle-sized (2–20 µm) range. The prominent subgroup within haptophytes consists of coccolithophores, which have been considered a critical component of marine environments because of their dual capacity to ﬁx environ- mental carbon via biomineralization (calcium carbonate, cal- cite) and through photosynthesis (Reyes-Prieto et al., 2009). Dinoﬂagellates are also one of the largest groups of marine eukaryotes, although most species are, on average, smaller than the average of diatom species. The majority of dinoﬂag- ellate species are autotrophic and tend to thrive under sta- ble conditions. Due to their motility and ability to regulate their position in the water column, they can outcompete other phytoplankton and sometimes accumulate rapidly, resulting in a visible coloration of the water, known as harmful algal blooms (IOCCG, 2014). Prokaryotes as picophytoplankton are abundant in many ocean regions (notably at mid-latitudes to low latitudes but also others) and also account for a sub- stantial fraction of marine primary production, with the two taxa being Synechococcus and Prochlorococcus in tropical regions (Flombaum et al., 2013). Phytoplankton in the sunlit layer of the ocean act as the base of the marine food web fueling ﬁsheries and also regu- late key biogeochemical processes. Climate-induced changes causing temperature rise, ocean acidiﬁcation and ocean de- oxygenation stress the ocean’s contemporary biogeochem- ical cycles and ecosystems, thereby impacting the phyto- plankton communities (Gruber, 2011; Gruber et al., 2021; Bindoff et al., 2019). Related to this, the changing nutri- ent and light availability, particularly in the polar oceans, is also critical for the development of phytoplankton commu- nities (Käse and Geuer, 2018). Satellite monitoring of surface phytoplankton functional types in the Atlantic Ocean over 20 years (2002–2021) The PFT anomaly (in percent) of 2021 compared to the 20-year mean reveals mostly a slight decrease in diatoms and a prominent increase in haptophytes in most areas of the high latitudes. Both diatoms and prokaryotes show a mild decrease along coastlines and an increase in the gyres, while prokaryotes show a clear decrease at mid-latitudes to low latitudes and an increase on the western African coast (Canary Current Coastal Province, CNRY and Guinea Current Coastal Province, GUIN) and southwestern corner of North Atlantic Tropical Gyral Province (NATR). Dinoﬂagellates, as a minor contributor to the total biomass, are relatively stable in the whole Atlantic region. This study illustrated the past and current PFT state in the Atlantic Ocean and acted as the ﬁrst step to promote long-term consistent PFT observations that enable time series analyses of PFT trends and interannual variability to reveal potential climate-induced changes in phytoplankton composition on multiple temporal and spatial scales. Published by Copernicus Publications. CHAPTER2.3 CHAPTER2.3 CHAPTER2.3 H. Xi et al.: Atlantic PFT observations over 20 years 2 Table 1. Products used. Table 1. Products used. Table 1. Products used. Product Product ID and type Data access Documentation ref. no. 1 OCEANCOLOUR_GLO_BGC_L4_MY_009_104; EU Copernicus Quality Information Document (QUID): satellite observations Marine Service Garnesson et al. (2022); Product (2022) Product User Manual: Colella et al. (2022) 2 PFTs derived from SeaWiFS-/MODIS-/MERIS-merged Our own archive Xi et al. (2021) products for the period of January 2012 to April 2012; satellite observations 3 PFTs derived from Sentinel 3A OLCI product for Our own archive Xi et al. (2021) the period of May 2016 to December 2016; satellite observations 4 In situ PFT data; in situ observations Xi et al. (2023) Xi et al. (2023) 1 Introduction These algorithms use multi-spectral reﬂectance data from OC satellites and sea surface temperature data to estimate chl a concentration of six major phytoplankton groups. Here, we focus only on four PFTs, which on the whole account for the major part of the biomass in the Atlantic Ocean. Applied to multi-sensor merged products and Sentinel 3A Ocean and Land Colour Instrument (OLCI) data, the algorithms enable us to generate global PFT products, which have been avail- able from the EU Copernicus Marine Service since 2020 and are updated regularly, providing global chl a data with per- pixel uncertainty for diatoms, haptophytes, dinoﬂagellates, chlorophytes and phototrophic prokaryotes spanning the pe- riod from 2002 until today. Table 1): (1) merged remote sensing reﬂectance (Rrs) prod- ucts at 9 bands from SeaWiFS, MODIS and MERIS from July 2002 to December 2011; (2) merged Rrs products at 9 bands from MODIS and VIIRS from January 2012 to De- cember 2016; and (3) Rrs products at 11 bands from Sentinel 3A OLCI from January 2017 to December 2021 (Table 1; Xi et al., 2021). In this section, monthly PFT products with 25 km resolution in the open ocean (depth > 200 m) are used for spatiotemporal analysis in the Atlantic Ocean spanning the period from July 2002 to December 2021. p y Consistency of satellite data is checked with the follow- ing details. As the product developer we have addition- ally generated PFT retrievals from different sensor com- binations but with overlapping time periods. PFT prod- ucts from SeaWiFS-/MODIS-/MERIS-merged data and that from MODIS-/VIIRS-merged data have a 4-month overlap from January to April 2012 (product ref. no. 2 in Table 1), and MODIS-/VIIRS-derived PFTs are overlapped with the OLCI-derived PFTs since May 2016 (product ref. no. 3 in Table 1). To produce consistent PFT products over the last 2 decades for the Atlantic Ocean, we compare PFT retrievals within these overlapped periods to identify the systematic differences between two data sources and then set up the cor- rection functions through linear regressions by taking into account the per-pixel uncertainty. Sections 2.3 and 3.3 in Xi et al. (2021) may be referred to for a detailed description of the per-pixel uncertainty assessment of the PFT products. 1 Introduction In the past decades, satel- lite observations of ocean color (OC) information, espe- cially the surface chlorophyll a concentration (chl a) as a proxy for phytoplankton biomass, have been able to revolu- tionize our understanding of biogeochemical processes and provide insights into the changes in phytoplankton and in- ferred productivity driven by climate change (e.g., Antoine et al., 2005; Gregg and Rousseaux, 2014; McClain, 2009; Behrenfeld et al., 2016; Kulk et al., 2020). However, phyto- plankton biomass does not provide a full description of the complex nature of phytoplankton community and function. Phytoplankton composition varies across ocean biomes, and the different phytoplankton groups inﬂuence marine ecosys- tem and biogeochemical processes differently (Bracher et al., 2017). Continuous monitoring of phytoplankton com- position is important not only to understand the biogeo- chemical processes such as nutrient uptake and carbon and energy transfer, but also for ﬁsheries, ocean environment, water quality and even human health when certain species cause, for example, harmful algal blooms (Quéré et al., 2005; Bindoff et al., 2019; Bracher et al., 2022). PFTs have been the focus of various studies carried out worldwide as well as in the Atlantic Ocean, providing rich and valuable knowledge of PFT assessments in terms of their State Planet, 1-osr7, 5, 2023 https://doi.org/10.5194/sp-1-osr7-5-2023 CHAPTER2.3 H. Xi et al.: Atlantic PFT observations over 20 years 3 abundance, distribution, phenology, roles in the primary pro- duction, and relationship to other physical and biological pa- rameters (e.g., Head and Pepin, 2010; Brotas et al., 2013, 2022; Soppa et al., 2016; Brewin et al., 2017; Moisan et al., 2017; Bolaños et al., 2020; Yang et al., 2020). Informa- tion of phytoplankton composition with respect to the func- tional types and size classes can be retrieved by ocean color algorithms based on different types of input data. However, most of the studies focus on either a certain PFT (e.g., Lange et al., 2020), a short time period or a limited spatial coverage (e.g., Bracher et al., 2020; Brotas et al., 2022). A complete, systematic frame for the long-term monitoring of multiple PFTs on a wide scale is yet to be established. Previously, we have developed and further improved an approach (referred to as EOF-PFT) consisting of a set of empirical-orthogonal- function-based PFT algorithms thanks to a large global in situ PFT data set based on HPLC (high-performance liquid chromatography)-measured pigments (Xi et al., 2020, 2021). 2.2 In situ PFT data and matchup extraction To evaluate the satellite PFT products, we use in situ HPLC pigment data from past expeditions between 2009 and 2019 covering the whole Atlantic pole-to-pole region (65◦S to 80◦N), which included 11 expeditions from the North At- lantic to the Arctic Fram Strait (PS74, PSS76, PS78, PS80, PS85, PS93.2, PS99.1, PS99.2, PS106, PS107, PS121), four expeditions in the Atlantic Ocean (PS113, PS120, AMT28 and AMT29) and one expedition in the Southern Ocean (PS103). All pigment data were quality-controlled by apply- 1 Introduction Similar to how the OC-CCI chl a product was merged (Jack- son, 2020), one of the three sets of PFT products that has been veriﬁed to have the lowest uncertainties (produced on a pixel basis) and smallest biases when evaluated by in situ data will be chosen as the reference product. The other two sets of PFT products will be corrected to it. In the EOF-PFT approach development stage (Xi et al., 2020, 2021), we no- ticed that the PFT products derived from SeaWiFS-/MODIS- /MERIS-merged Rrs data show the lowest per-pixel uncer- tainties for nearly all the PFT quantities because their corre- sponding algorithms were trained based on a larger and more widely covered matchup data set between the satellite and in situ observations. Therefore, we take SeaWiFS-/MODIS- /MERIS-derived PFTs as a reference to correct the other two PFT data sets derived from MODIS-/VIIRS-merged data and OLCI data, respectively. In this section, we combine these PFT data sets of dif- ferent sensors, covering various lifespans and radiometric characteristics, into consistent long-term satellite PFT prod- ucts. The 2-decade quality-assured global PFT data sets for the Atlantic Ocean (50◦S to 50◦N, 60◦W to 10◦E) are derived by correcting the input-sensor-speciﬁc PFT prod- ucts using inter-sensor comparisons with uncertainty esti- mations, which then allow us to (1) evaluate Copernicus Marine Service PFT products and improve their continuity along the products derived from different satellite sensors and (2) analyze PFT time series in the last 2 decades in terms of climatology, trends, anomaly and phenology of multiple PFTs in the Atlantic Ocean and its different biogeochemical provinces (Longhurst, 2007). 2.3 Time series analysis We focus on preliminary explorations of the calibrated PFT products to reveal and understand the trends of and varia- tions in Atlantic PFTs in the last 2 decades. We derive the PFT time series of the whole Atlantic region, on different regional scales and also at the per-pixel level. For regional scales, PFT data of the Atlantic Ocean are partitioned into smaller regions using Longhurst’s geographic classiﬁcation system of biomes and provinces (Longhurst, 2007; Flanders Marine Institute, 2009). We determine the annual cycle (cli- matology) based on both pixel data and regional log-based mean values and derive anomalies to observe the interannual changes and detect trends reﬂected by the satellite observa- tions. Time series analysis is done both per pixel and for the whole region or province. We investigate the trends in the PFTs for the last 20 years using linear regression in the for- mat of Y = SX +I, where Y is the monthly PFT chl a either per pixel or of the regional log-based mean, X is the time on a monthly basis, S is the slope of the regression, and I is the intercept. Only trends with statistically signiﬁcant cor- relations of the regression (p < 0.05) are shown. Indicators of PFT phenology and the anomaly of 2021 (the last year of the considered time period) are also extracted in order to identify potential changes/shifts in PFTs. Abundance max- ima time, as one of the phenology indicators, is identiﬁed for each pixel by ﬁnding the month when the maximum PFT chl a occurred during the year. Anomaly in percent is deter- mined by computing the relative difference between the PFT state of 2021 and the average state of the last 2 decades (i.e., climatology). Figure 1. Scatterplots of monthly PFTs derived from SeaWiFS- g p y /MODIS-/MERIS-merged and MODIS-/VIIRS-merged Rrs data for the overlapping period January–April 2012. (a) Diatoms, (b) hap- tophytes, (c) prokaryotes and (d) dinoﬂagellates. The 1 : 1 line is shown in black and the linear regression line (using type II regres- sion with per-pixel uncertainty) in red. R2, slopes and offsets deter- mined in log-10 scale are also presented. merged data for the overlapped 4 months (January–April 2012). PFT retrievals from different satellite sensors show some differences but overall correlate well with each other (R2 > 0.82). 2.3 Time series analysis Type II linear regression between the retrievals from two satellite data sources is determined for each PFT by accounting for the per-pixel uncertainty. The slope and intercept values are then used to correct the MODIS- /VIIRS-derived PFTs to the SeaWiFS-/MODIS-/MERIS- derived ones so that they are overall consistent, though the pixelwise discrepancy still exists. The same is applied to the Sentinel 3A OLCI-derived PFTs by comparing them to the corrected MODIS-/VIIRS-derived PFTs for the over- lapped period April–December 2016 so that all PFT data from both MODIS/VIIRS and OLCI are now referenced to SeaWiFS-/MODIS-/MERIS-derived PFTs. Though R2 is slightly weaker (R2 between 0.77 and 0.83) compared to that from the MODIS-/VIIRS- versus SeaWiFS-/MODIS- /MERIS-derived PFTs (R2 between 0.82 and 0.98), OLCI- derived PFTs still showed overall good correlations to the corrected MODIS/VIIRS data, with regression slopes be- tween 0.83 and 1.03, despite prokaryote chl a retrievals from OLCI data being higher in general. H. Xi et al.: Atlantic PFT observations over 20 years 4 Figure 1. Scatterplots of monthly PFTs derived from SeaWiFS- /MODIS-/MERIS-merged and MODIS-/VIIRS-merged Rrs data for the overlapping period January–April 2012. (a) Diatoms, (b) hap- tophytes, (c) prokaryotes and (d) dinoﬂagellates. The 1 : 1 line is shown in black and the linear regression line (using type II regres- sion with per-pixel uncertainty) in red. R2, slopes and offsets deter- mined in log-10 scale are also presented. ing the method by Aiken et al. (2009). Diagnostic pigment analysis was carried out to determine the in situ PFT chl a concentrations with newly tuned weighting coefﬁcients by Alvarado et al. (2022), according to Vidussi et al. (2001) and Uitz et al. (2006), modiﬁed as in Hirata et al. (2011) and Brewin et al. (2015). In situ PFT data (product ref. no. 1 in Table 1) are then used to validate the corrected PFTs by ex- tracting matchups between daily 4 km PFT products from the Copernicus Marine Service and the in situ data. For each in situ measurement a matchup of 3×3 pixels around the in situ location on the same day was extracted. Averaged data based on 3 × 3 pixels were computed following the matchup pro- tocol as in Xi et al. (2020, 2021), including only matchups containing at least 50 % of valid pixels with a coefﬁcient of variation (CV) of the valid pixel values lower than 0.15. 2.1 PFT products from Copernicus Marine Service Satellite data used in this study are listed in Table 1. Mul- tiple PFT chl a products with per-pixel uncertainty have been available from the Copernicus Marine Service since May 2020, with updates in 2021 and 2022, and were de- rived from three sets of OC products (product ref. no. 1 in https://doi.org/10.5194/sp-1-osr7-5-2023 State Planet, 1-osr7, 5, 2023 CHAPTER2.3 H. Xi et al.: Atlantic PFT observations over 20 years H. Xi et al.: Atlantic PFT observations over 20 years 3 Results Validation was carried out by comparing the collocated satellite PFTs with the in situ PFTs using the extracted matchup data. Statistical results of the validation in Table 2 in general show acceptable agreement between the in situ and satellite-derived PFTs. Median percent differences (MD- PDs) are consistent with the median satellite PFT uncertain- 3.1 Inter-sensor corrections of PFT products and validation with in situ data 3.1 Inter-sensor corrections of PFT products and validation with in situ data Figure 1 shows the comparison between monthly PFTs from SeaWiFS-/MODIS-/MERIS-merged and MODIS-/VIIRS- https://doi.org/10.5194/sp-1-osr7-5-2023 State Planet, 1-osr7, 5, 2023 State Planet, 1-osr7, 5, 2023 State Planet, 1-osr7, 5, 2023 CHAPTER2.3 H. Xi et al.: Atlantic PFT observations over 20 years 5 Figure 2. PFT climatology based on monthly PFT chl a products from 2002 to 2021: (a) diatoms, (b) haptophytes, (c) prokaryotes and (d) dinoﬂagellates. Microscopic photos of the representative species for the four PFTs are presented (photo credit: Alfred We- gener Institute). ties (relative error in percent) estimated through Monte Carlo simulation and error propagation in Xi et al. (2021), and for dinoﬂagellates they are notably lower. A higher MDPD is found for prokaryotes due to a systematic overestimation of the picophytoplankton in the retrieval algorithms for all the three sets of satellite OC sensors; however, no signiﬁcant bias of satellite prokaryote products is detected between different sensors. Therefore the overestimation should have minor in- ﬂuence on the time series data of prokaryotes. In addition, a coarser evaluation by directly comparing the monthly satel- lite PFTs (which have better spatial coverage) to the in situ PFTs for the whole cruise track has shown that the PFT varia- tion regarding regional phytoplankton dynamics observed by the in situ PFT is very well revealed by satellite PFT maps (images not shown). These evaluations assure the quality of the satellite PFTs for time series analysis. 3.2 PFT climatology (2002–2021) Figure 2 shows the climatology (2002–2021) of the four PFTs generated using satellite monthly PFT products, depict- ing differences and similarities in terms of PFT chl a magni- tude and spatial variation among different PFTs. Diatoms, as major siliciﬁers, are typically large-celled (> 20 µm, though species with smaller cells also exist) and highly dependent on nutrient levels. They are sensitive to the global temperature, especially to the Equator–pole temperature gradient. The 2- decade climatology of diatoms in the Atlantic region shows a clearly higher abundance at high latitudes and in coastal regions and the lowest abundance (or even undetectable) in the vast subtropical gyres. Similarly, haptophytes, which are mostly classiﬁed as nanophytoplankton (2–20 µm), also have a higher abundance at high latitudes and in coastal regions but span a larger coverage than diatoms. Enhanced abun- dance level is also found in the equatorial belt. The low- est abundance of haptophytes is also found in the gyres, but their abundance is not as low as that of diatoms. Prokaryotes, commonly referred to as picophytoplankton (< 2 µm), show the highest abundance at mid-latitudes to low latitudes of the open Atlantic Ocean. Though spatially showing the lowest abundance in the gyres, prokaryotes are still the most dom- inant phytoplankton group in the majority of these regions. Dinoﬂagellates as a relatively minor contributor to the total biomass follow a similar distribution pattern to diatoms but are much lower in abundance at higher latitudes. Figure 2. PFT climatology based on monthly PFT chl a products from 2002 to 2021: (a) diatoms, (b) haptophytes, (c) prokaryotes and (d) dinoﬂagellates. Microscopic photos of the representative species for the four PFTs are presented (photo credit: Alfred We- gener Institute). varies from 0.017 to 0.026 mg m−3, and dinoﬂagellates, with the lowest mean chl a, are below 0.015 mg m−3. Despite dif- ferent magnitudes, diatoms, haptophytes and dinoﬂagellates present very similar annual cycles, with two biomass peaks in April and November, indicating the spring blooming es- pecially at high latitudes in both the Northern Hemisphere and Southern Hemisphere. In contrast, prokaryotes show a distinct biomass peak in June–July and a less prominent in- crease in December–January due to the suppressed growth of the other PFTs in these periods. H. Xi et al.: Atlantic PFT observations over 20 years H. Xi et al.: Atlantic PFT observations over 20 years 6 Table 2. Statistical validation results of satellite-derived PFT chl a (after inter-mission correction) as a function of in situ PFT chl a using least square ﬁt in logarithmic scale. N: number of matchups; R2: coefﬁcient of determination; MDPD: median percent difference; RMSD: root mean square difference; for a deﬁnition of equations of these terms please refer to Xi et al. (2020). Note that slope, intercept and R2 were calculated based on a logarithmic scale. Median uncertainties calculated based on satellite per-pixel PFT uncertainty (equivalent to relative error in percent, adapted from Xi et al., 2021) are also shown in the last column. N Slope Intercept R2 MDPD RMSD Median satellite PFT (%) (mg m−3) uncertainty (%) Diatoms 192 0.71 −0.27 0.76 60.5 0.30 57.3 Haptophytes 191 0.95 −0.007 0.41 58.9 0.18 41.5 Prokaryotes 187 0.71 0.12 0.36 185 0.06 86.5 Dinoﬂagellates 144 1.07 0.04 0.66 59.1 0.07 74.3 Figure 3. (a) Annual cycle of the four PFTs of diatoms, haptophytes, prokaryotes and dinoﬂagellates in the Atlantic Ocean (−50◦S to 50◦N, 60◦W to 10◦E); (b) 20-year time series from 2002 to 2021; and (c) per-pixel slope based on monthly chl a products of diatoms, (d) haptophytes, (e) prokaryotes and (f) dinoﬂagellates from 2002 to 2021 (where p < 0.05 is shown; slope unit: chl a mg m−3 per month). Figure 3. (a) Annual cycle of the four PFTs of diatoms, haptophytes, prokaryotes and dinoﬂagellates in the Atlantic Ocean (−50◦S to 50◦N, 60◦W to 10◦E); (b) 20-year time series from 2002 to 2021; and (c) per-pixel slope based on monthly chl a products of diatoms, (d) haptophytes, (e) prokaryotes and (f) dinoﬂagellates from 2002 to 2021 (where p < 0.05 is shown; slope unit: chl a mg m−3 per month). increase (2009–2010), but from 2011 onwards a continuous decline is again observed. The per-pixel trend of prokaryotes in Fig. 3e shows that the decreasing trend of prokaryotes is mainly found at low latitudes and particularly on the west coast of Africa (Canary Current Coastal Province, CNRY, and Guinea Current Coastal Province, GUIN; refer to Fig. 4 for Longhurst provinces). Haptophyte time series show the lowest abundance during 2013–2015, which is then elevated slightly from 2016 (Fig. 3b). 3.3 PFT trends during 2002–2021 in the Atlantic Ocean Following the climatology study, the annual cycles of the four PFTs over the whole Atlantic Ocean are also derived by extracting the mean biomass of the 2 decades for each month (Fig. 3a). Prokaryotes are clearly the most dominant group, showing the highest mean of the chl a (0.062–0.072 mg m−3) over the Atlantic all year round, followed by haptophyte chl a, which varies from 0.03 to 0.045 mg m−3. Diatom chl a Time series of the monthly PFT data averaged for the whole Atlantic are shown in Fig. 3b, depicting a signiﬁ- cant decrease (0.0001 chl a mg m−3 per month, p < 0.01) in prokaryote chl a, but no signiﬁcant trend is observed for the other three PFTs, although some interannual changes are vis- ible. Between the time window of 2003 and 2008, a slight decline in prokaryote chl a is observed, followed by a 2-year State Planet, 1-osr7, 5, 2023 https://doi.org/10.5194/sp-1-osr7-5-2023 State Planet, 1-osr7, 5, 2023 CHAPTER2.3 CHAPTER2.3 State Planet, 1-osr7, 5, 2023 H. Xi et al.: Atlantic PFT observations over 20 years A slight increasing trend of hap- tophytes on the pixel level is found at mid-latitudes to low latitudes, and a decrease is found near the coast at higher latitudes (Fig. 3d). Diatom chl a is rather stable until 2017, with an abrupt increase in 2018–2019 and then a decrease in 2020–2021 to the average level of the last 20 years (Fig. 3b). The per-pixel time series in Fig. 3c shows that a signiﬁcant decrease is found only on the west coast of Africa (CNRY), northwest of the North Atlantic (Northwest Atlantic Shelves Province, NWCS) and on the Patagonian coast. A very slight increasing trend of diatoms is presented in the gyres and equatorial region. Dinoﬂagellate chl a contributes a very mi- nor proportion to the total biomass (< 10 %) and has been relatively stable over the last 2 decades in the whole Atlantic region (Figs. 3b and f). Time series of diatom chl a in different Longhurst provinces of the Atlantic are further extracted in order to investigate whether the abrupt increase in diatoms during State Planet, 1-osr7, 5, 2023 https://doi.org/10.5194/sp-1-osr7-5-2023 CHAPTER2.3 H. Xi et al.: Atlantic PFT observations over 20 years 7 Figure 4. Time series of diatom chl a (mg m−3) in 11 Longhurst provinces in the Atlantic Ocean with bathymetric information based on ETOPO1 bathymetry (Amante and Eakins, 2009). Provinces according to Longhurst (2007) are NADR for North Atlantic Drift Province, NWCS for Northwest Atlantic Shelves Province, NASW for North Atlantic Subtropical Gyral Province (West), NASE for North Atlantic Subtropical Gyral Province (East), NATR for North Atlantic Tropical Gyral Province, CNRY for Canary Current Coastal Province, GUIN for Guinea Current Coastal Province, WTRA for Western Tropical Atlantic Province, ETRA for Eastern Tropical Atlantic Province, SATL for South Atlantic Gyral Province, SSTC for South Subtropical Convergence Province and SANT for Subantarctic Water Ring Province, respectively. Trend lines with slopes (chl a mg m−3 per month) and correlation coefﬁcients are shown for provinces with signiﬁcant trends (p < 0.05). Figure 4. Time series of diatom chl a (mg m−3) in 11 Longhurst provinces in the Atlantic Ocean with bathymetric information based on ETOPO1 bathymetry (Amante and Eakins, 2009). H. Xi et al.: Atlantic PFT observations over 20 years Provinces according to Longhurst (2007) are NADR for North Atlantic Drift Province, NWCS for Northwest Atlantic Shelves Province, NASW for North Atlantic Subtropical Gyral Province (West), NASE for North Atlantic Subtropical Gyral Province (East), NATR for North Atlantic Tropical Gyral Province, CNRY for Canary Current Coastal Province, GUIN for Guinea Current Coastal Province, WTRA for Western Tropical Atlantic Province, ETRA for Eastern Tropical Atlantic Province, SATL for South Atlantic Gyral Province, SSTC for South Subtropical Convergence Province and SANT for Subantarctic Water Ring Province, respectively. Trend lines with slopes (chl a mg m−3 per month) and correlation coefﬁcients are shown for provinces with signiﬁcant trends (p < 0.05). 2018–2019 took place in the whole Atlantic or only in some regions. Figure 4 presents large variability in the diatoms in different regions in terms of both magnitudes of chl a and temporal trends. In general, high latitudes and coastal re- gions, where diatom chl a is higher, present high interannual variation compared to the open ocean at lower latitudes. For instance, diatoms on the west coast of Africa (CNRY) have in general decreased in the last 2 decades except for a dramatic increase in late 2018 and spring 2019, followed by a 2-year decrease in 2020–2021. Despite an obvious elevation in di- atoms during 2018–2020, a signiﬁcant decline in diatoms in the last 2 decades is still found in the NWCS, consistent with the trend map shown in Fig. 3c. A slight increase is found in provinces in the gyres and equatorial region (North Atlantic Subtropical Gyral Province (West), NASW; NATR; West- ern Tropical Atlantic Province, WTRA; and South Atlantic Gyral Province, SATL), with very low diatom chl a (mean chl a < 0.02 mg m−3), and also in the Southern Ocean South Subtropical Convergence Province (SSTC). The prominent increase observed during 2018–2019 is mostly contributed by the North Atlantic Ocean CNRY, North Atlantic Sub- tropical Gyral Province (East) (NASE) and NASW. Other provinces such as in the gyres, with elevated diatom chl a since 2018, also contribute, but only slightly, to this increase due to much lower diatom chl a there compared to the other regions. H. Xi et al.: Atlantic PFT observations over 20 years 8 Figure 5. Occurrence month of PFT chl a maxima of 2021 in differ- ent provinces for (a) diatoms, (b) haptophytes, (c) prokaryotes and (d) dinoﬂagellates. Black lines indicate boundaries of Longhurst provinces as in Fig. 4. pixel for the four PFTs (Fig. 5). Diatoms in the North Atlantic (> 35◦N) reach the abundance maxima during late spring (April–May) but earlier (January–February) in the North At- lantic gyre. In the equatorial region the maxima months vary between May and August, with the Equator reaching the maxima the earliest (around May). In the Southern Hemi- sphere, diatoms reach maxima on average 6 months later than in the Northern Hemisphere, i.e., in the South Atlantic gyre in July–August (austral winter) and in October–December (late spring) in most of the Southern Ocean, which corresponds well with the maxima seasons in the Northern Hemisphere. Haptophytes and dinoﬂagellates show similar patterns to di- atoms but on average bloom 1 month later than diatoms, indi- cating PFT dominance succession between diatoms and hap- tophytes in late spring and early summer. Prokaryotes over the whole Atlantic show a more complex and distinct sea- sonal cycle compared to the other three PFTs. The per-pixel phenology map shows that prokaryotes reach an abundance maximum from autumn to winter at latitudes > 20◦N, and their maxima time is delayed with decreasing latitude. In other regions the per-pixel maxima month normally spans a wider time window of 3 to 5 months, such as in the equatorial region from January to May, the South Atlantic gyre from April to August, the western sector of the Southern Ocean from January to April and the eastern sector from November to January. Though geographically prokaryotes show more variation in the phenology, an overall inverse seasonal cycle is presented compared to diatoms, haptophytes and dinoﬂag- ellates, as depicted in Fig. 3a. Anomalies in percent of the four PFTs in 2021 compared to the average state of the last 2 decades are shown in Fig. 6. The diatom anomaly presents changes mainly at high lati- tudes and in gyres and some coastal regions (such as CNRY). The anomaly shows mostly lower diatom chl a at high lati- tudes, except for NWCS and the southeastern part of NADR, where diatom chl a is increased. In contrast to that, diatom chl a of 2021 in the gyres is generally higher (∼30 %) com- pared to the 20-year average state. H. Xi et al.: Atlantic PFT observations over 20 years Note that changes are shown in percent instead of the absolute values to enhance the visibility of small absolute changes, which in the gyres can be very sensitive, as diatom chl a is extremely low there (< 0.01 mg m−3). The haptophyte anomaly presents changes in similar regions to diatoms but reversely at high latitudes, especially in the Southern Ocean, where a more prominent increase and also larger coverage are observed. An increase in haptophytes in the area north of the Equator in WTRA is more signiﬁcant than diatoms. Different from diatoms and haptophytes, prokaryotes reveal a very slight decrease in 2021, mostly at low latitudes within 20◦N–20◦S, with higher prokaryote chl a on the west coast of Africa, espe- cially CNRY, whereas only a mild increase (< 20 %) is found at high latitudes. Dinoﬂagellates show the most stable state in 2021 among the four PFTs, with only a slight increase in chl a in the North Atlantic Ocean above 40◦N and a small decrease in CNRY. Figure 5. Occurrence month of PFT chl a maxima of 2021 in differ- ent provinces for (a) diatoms, (b) haptophytes, (c) prokaryotes and (d) dinoﬂagellates. Black lines indicate boundaries of Longhurst provinces as in Fig. 4. 3.4 PFT phenology and anomaly of 2021 The status of the PFTs in the Atlantic Ocean is investigated speciﬁcally in 2021 to reﬂect the Atlantic ecological state and changes for this year as compared to the previous years. To better understand the yearly transition and shifting be- tween different PFTs in the Atlantic Ocean, one of the phe- nology indicators, abundance maxima time, is mapped per- https://doi.org/10.5194/sp-1-osr7-5-2023 State Planet, 1-osr7, 5, 2023 CHAPTER2.3 CHAPTER2.3 H. Xi et al.: Atlantic PFT observations over 20 years 4 Discussion, conclusions and outlook A systematic time series analysis of PFTs in the Atlantic Ocean is carried out showing high potential of the Coper- nicus Marine Service satellite PFTs in monitoring the eco- logical state of the ocean at different scales. Due to differ- ent life spans and radiometric characteristics of satellite sen- sors, there are often inconsistencies and gaps between the same quantities retrieved from different sensors. Data conti- nuity and quality assurance are therefore necessary to pro- vide sound and continuous satellite observations enabling time series studies (Mélin and Franz, 2014; Sathyendranath et al., 2019). As preparatory work for such a study aiming at long-term monitoring of PFTs in the vast Atlantic Ocean, we applied a straightforward inter-mission bias correction as a preliminary trial using overlapped PFT products between the three sets of satellite data. Validation using in situ data shows no signiﬁcant biases of PFTs derived from different sensors, State Planet, 1-osr7, 5, 2023 https://doi.org/10.5194/sp-1-osr7-5-2023 CHAPTER2.3 H. Xi et al.: Atlantic PFT observations over 20 years 9 Figure 6. PFT anomaly in percent (%) of 2021 compared to the 20-year mean for (a) diatoms, (b) haptophytes, (c) prokaryotes and (d) dinoﬂagellates. Anomaly in percent is deﬁned as (PFT2021 − climatology)/climatology×100. Black lines indicate boundaries of Longhurst provinces as in Fig. 4. further explore a more generic method in the future for the global ocean. However, with these ﬁrst investigations this study paves the way to promote satellite PFT products into long-term time series studies. Satellite PFT products provide robust spatial distributions which are comparable to in situ data. The 20-year mean of the four PFTs has presented a trustworthy overview of how different PFTs vary and are distributed spatially in the sur- face layer of the Atlantic Ocean. Diatoms, haptophytes and dinoﬂagellates share similar geographic patterns, showing higher abundance at high latitudes and in coastal and equa- torial upwelling regions, where the nutrient level is generally high, and minimum abundance in the gyres, especially for diatoms and dinoﬂagellates. Prokaryotes are more dominant in the gyres and at low latitudes but contribute much less to the total biomass at high latitudes. The ﬁndings are consis- tent with previous studies of phytoplankton group and size classes (e.g., Hirata et al., 2011; Brewin et al., 2015; Losa et al., 2017) and are justiﬁed in detail in Xi et al. (2020). More recent studies by Bracher et al. (2020) and Brotas et al. 4 Discussion, conclusions and outlook (2022) based on in situ observations have also revealed similar PFT latitudinal distribution to our satellite observa- tions. Furthermore, Brotas et al. (2022) point out that di- noﬂagellates can be underestimated in the pigment approach, due to their pigment variability; some species do not have the diagnostic pigment peridinin, and there are several het- erotrophic species where the pigments are absent or strongly reduced. PFT time series of the last 2 decades are for the ﬁrst time generated from multi-satellite observations. For the whole Atlantic Ocean, no signiﬁcant trend was found for diatoms, haptophytes and dinoﬂagellates over the last 20 years, but a decline in prokaryotes was observed. However, the per-pixel trend maps revealed that regional trends are different from province to province, such as for diatoms; a signiﬁcant de- crease was found at latitudes above 40◦and on the west coast of Africa (CNRY). This was similarly found for haptophytes as well. There is a clear shift for prokaryotes in 2012: from 2003 to 2012, the average value is higher (0.064 mg m−3), and the seasonality is clearly deﬁned, whereas from 2013 to 2020, seasonal variations are softened, and the mean value is lower (0.053 mg m−3). The retreat of MERIS in 2012 should not inﬂuence the prokaryote data set very much for the following reasoning: ﬁrstly, such a decline was not found in other PFTs; secondly, MERIS observed more pix- els on the coast and at high latitudes, whereas we focus on the open ocean and have excluded the coastal regions with bathymetry < 200 m, and this study covers the Atlantic Ocean between 50◦N to 50◦S. The main reason might be the relatively lower retrieval accuracy of prokaryotes com- pared to other PFTs as discussed above in the validation. Our previous work showed that all of the retrieval models for the three sets of sensors have poorer performance for prokary- otic phytoplankton than for other PFT retrievals. This may cause weaker consistency of prokaryotes for the 2-decade pe- Figure 6. PFT anomaly in percent (%) of 2021 compared to the 20-year mean for (a) diatoms, (b) haptophytes, (c) prokaryotes and (d) dinoﬂagellates. Anomaly in percent is deﬁned as (PFT2021 − climatology)/climatology×100. Black lines indicate boundaries of Longhurst provinces as in Fig. 4. indicating that the inter-mission offset was effectively cor- rected. 4 Discussion, conclusions and outlook Retrievals of chl a of different PFTs are more up- scaled products compared to bulk satellite OC products such as total chl a, colored dissolved organic matter (CDOM) and absorption properties. It is especially still challenging to ac- curately retrieve prokaryotic phytoplankton because in the open ocean these dominate in the low-chl a areas, where the satellite signals are weaker. Therefore, higher uncertainties exist in these products (e.g., Brewin et al., 2017; Losa et al., 2017; Xi et al., 2021) as compared to uncertainties for other PFTs (see Table 2). In summary, our statistical results of PFT validation are comparable to the evaluations of satel- lite PFT products derived from different approaches, accord- ing to the Quality Information Documents (QUID) that have been published by the Copernicus Marine Service (Pardo et al., 2020; Garnesson et al., 2022). It is noteworthy that the bias correction only targeted the Atlantic Ocean and might not be applicable to other ocean regions, which leads us to State Planet, 1-osr7, 5, 2023 https://doi.org/10.5194/sp-1-osr7-5-2023 State Planet, 1-osr7, 5, 2023 CHAPTER2.3 CHAPTER2.3 10 H. Xi et al.: Atlantic PFT observations over 20 years riod even after inter-mission correction. Nevertheless, cover- age variability among different satellite missions should be taken into consideration in analyzing long-time-series stud- ies as the ability of the sensors to observe certain waters may differ (van Oostende et al., 2022). These ﬁndings in terms of 20-year trends still need to be evaluated further with both in situ measurements and numerical models, though avail- able matchup data between in situ and satellite data are very sparse, and disagreements between models and satellite ob- servations also exist (Gregg and Rousseaux, 2014). Indeed, a period of 20 years is not considered to be long enough for a robust trend analysis as the decadal variability might be too dominant, and for the Atlantic Ocean on average at least 35 years is needed to detect a climate-driven trend in chlorophyll concentration as indicated in Henson et al. (2010, 2016). Nonetheless, the 20-year-long time series provides the opportunity to observe interesting patterns, such as the di- atom increase during 2018–2019. Therefore, further inves- tigation of biophysical interactions and linkage to climate is necessary to ﬁnd evidence and interpret the ﬁndings ex- tracted from PFT time series. the structure and function of phytoplankton communities in the Southern Ocean. 4 Discussion, conclusions and outlook Diatoms and haptophytes as two ma- jor groups there may be severely subject to these changes. Interestingly, an increase in haptophytes and decrease in di- atoms indicated that the phytoplankton community structure has been altered in recent years, which cannot be easily cap- tured with observations other than satellite PFT time series. In summary, this study illustrates the past and current PFT state in the Atlantic Ocean and acts as the ﬁrst step to promote long-term PFT observations serving as ocean monitoring indicators (OMIs) implemented in the Coperni- cus Marine Service that enable time series analyses of PFT trends and interannual variability to reveal potential climate- induced changes in phytoplankton composition on multiple temporal and spatial scales. Data availability. Data and products used in this study, as well as their availabilities and documentations, are summarized in Ta- ble 1. In situ HPLC pigment and PFT data used for the vali- dation of Copernicus Marine Service PFT products are available on PANGAEA (https://doi.org/10.1594/PANGAEA.954738; Xi et al., 2023). Data availability. Data and products used in this study, as well as their availabilities and documentations, are summarized in Ta- ble 1. In situ HPLC pigment and PFT data used for the vali- dation of Copernicus Marine Service PFT products are available on PANGAEA (https://doi.org/10.1594/PANGAEA.954738; Xi et al., 2023). Phenology maps of the four PFTs correspond well with their mean annual cycles. Prokaryotes have distinct phenol- ogy compared to the other three PFTs, which present sim- ilar annual cycles and close bloom maxima time on a gen- eral scale despite haptophytes and dinoﬂagellates reaching biomass maxima a bit later than diatoms in some regions. This section has chooses only one coarse phenological index, the time of the maxima, using monthly satellite products, which has shown the capability of the Copernicus Marine Service satellite PFTs in revealing PFT shifting and growth state at a larger scale than traditional means that rely on ex- tensive ship-based measurements and long-term monitoring stations (e.g., Bracher et al., 2020; Nöthig et al., 2020; Yang et al., 2020). More phenological indices, including the PFT growth duration, biomass amplitude and start and ending dates, could be considered using higher-temporal-resolution products to fully understand the patterns of and interannual variability in the PFT phenology (Soppa et al., 2016). Author contributions. HX, AB and AM conceptualized the study. HX designed and carried out the experiments. State Planet, 1-osr7, 5, 2023 4 Discussion, conclusions and outlook MB provided support in satellite products and matchup data extraction. VB, MG and IP contributed to in situ data compilation for validation. SNL and LMAA helped with data analysis. HX drafted and revised the manuscript with contributions from all co-authors. Competing interests. The contact author has declared that none of the authors has any competing interests. Disclaimer. Publisher’s note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. The PFT anomaly of 2021 compared to the 20-year mean reveals mostly a slight decrease in diatoms above 40◦N/S (except for the southeastern part of NADR) and a signiﬁ- cant increase in haptophytes in most areas of the high lat- itudes, which corresponds well with the hypothesis of “At- lantiﬁcation” proposing that smaller phytoplankton are ex- panding to high latitudes (e.g., Nöthig et al., 2015; Neuker- mans et al., 2018; Oziel et al., 2020). A decline in silicate and nitrate concentrations might contribute to the decrease in diatoms in the North Atlantic as indicated in the Copernicus Ocean State Report 5 by von Schuckmann et al. (2021). In contrast to this, most changes in the Southern Ocean were found at latitudes higher than 40◦S, which is the region of the Great Calcite Belt. Deppeler and Davidson (2017) pointed out that climate-induced changes such as higher tem- perature and shallow mixed-layer depth are expected to alter Acknowledgements. We thank the European Union’s Horizon 2020 Research and Innovation Programme under grant agreement 810139 for the project Portugal Twinning for Innovation and Ex- cellence in Marine Science and Earth Observation – PORTWIMS and the Copernicus Marine Service Evolution project GLOPHYTS (grant no. 21036L05B-COP-INNO SCI-9000) for funding. Coper- nicus Marine Service is implemented by Mercator Ocean Interna- tional in the framework of a delegation agreement with the Eu- ropean Union. Astrid Bracher and Svetlana N. Losa were sup- ported by the DFG (German Research Foundation) Transregional Collaborative Research Center ArctiC Amplication: Climate Rel- evant Atmospheric and SurfaCe Processes, and Feedback Mecha- nisms (AC)3 (Project C03, grant no. 268020496 – TRR 172) and by the ESA S5P+Innovation Theme 7 Ocean Colour (S5POC) project (grant no. 4000127533/19/I-NS). Svetlana N. Losa’s work was also State Planet, 1-osr7, 5, 2023 https://doi.org/10.5194/sp-1-osr7-5-2023 CHAPTER2.3 11 H. Xi et al.: Atlantic PFT observations over 20 years Bindoff, N. L., Cheung, W. W. L., Kairo, J. G., Arístegui, J., Guinder, V. 4 Discussion, conclusions and outlook A., Hallberg, R., Hilmi, N., Jiao, N., Karim, M. S., Levin, L., O’Donoghue, S., Purca Cuicapusa, S. R., Rinke- vich, B., Suga, T., Tagliabue, A., and Williamson, P.: Chang- ing Ocean, Marine Ecosystems, and Dependent Communities, in: IPCC Special Report on the Ocean and Cryosphere in a Changing Climate, edited by: Pörtner, H.-O., Roberts, D. C., Masson-Delmotte, V., Zhai, P, Tignor, M., Poloczanska, E., Mintenbeck, K., Alegría, A., Nicolai, M., Okem, A., Pet- zold, J., Rama, B., and Weyer, N. M., Cambridge University Press, Cambridge, UK and New York, NY, USA, 447–587, https://doi.org/10.1017/9781009157964.007, 2019. partly made in the framework of the state assignment of the Fed- eral Agency for Scientiﬁc Organizations (FASO) Russia (theme FMWE-2021-0014). The contribution by Leonardo M. A. Alvarado was supported by the Federal Ministry of Economics and Tech- nology (BMWi) and German Aerospace Center (DLR) (grant no. 50EE1915) within the project TypSynSat. We also thank Gavin Til- stone (PML), Giorgio Dall’Olmo (OGS) and Robert Brewin (Uni- versity of Exeter) for AMT28 and AMT29 pigment data. Thanks to NASA, ESA and EUMETSAT for the SeaWiFS, MODIS, VIIRS, MERIS and OLCI data and especially the ACRI-ST GlobColour team for providing OLCI and merged ocean colour L3 products. The captain, crew and expedition scientists are also acknowledged for their support during the expeditions. We ﬁnally thank Sorin Con- stantin and the other two anonymous reviewers who helped to im- prove this work by providing constructive comments. Bolaños, L. M., Karp-Boss, L., Choi, C. J., Worden, A. Z., Graff., J. R., Haëntjens, N., Chase, A. P., Della Penna, A., Gaube, P., Morison, F., Menden-Deuer, S., Westberry, T. K., O’Malley, R. T., Boss, E., Behrenfeld, M. J., and Glovannoni, S. J.: Small phytoplankton dominate western North Atlantic biomass, The ISME Journal, 14, 1663–1674, https://doi.org/10.1038/s41396- 020-0636-0, 2020. Financial support. This research has been supported by the H2020 European Research Council (grant no. 810139), European Commission (EC) (grant no. 21036L05B-COP-INNO SCI-9000), the Deutsche Forschungsgemeinschaft (grant no. 268020496), Eu- ropean Space Agency (grant no. 4000127533/19/I-NS-S5P+I-OC), Federal Agency for Scientiﬁc Organizations (FASO) Russia (grant no. FMWE-2021-0014), and Federal Ministry of Economics and Technology (BMWi) and German Aerospace Center (DLR) (grant no. 50EE1915). Bracher, A., Bouman, H. A., Brewin, R. J. W., Bricaud, A., Bro- tas, V., Ciotti, A. M., Clementson, L., Devred, E., Di Cicco, A., Dutkiewicz, S., Hardman-Mountford, N. J., Hickman, A. E., Hieronymi, M., Hirata, T., Losa, S. N., Mouw, C. 4 Discussion, conclusions and outlook B., Or- ganelli, E., Raitsos, D. 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The dynamical role of upper layer salinity in the Mediterranean Sea tion of the Copernicus Ocean State Report (OSR7) Ali Aydogdu1, Pietro Miraglio1, Romain Escudier2, Emanuela Clementi1, and Simona Masina1 1Ocean Modeling and Data Assimilation Division, Fondazione Centro Euro-Mediterraneo sui Cambiamenti Climatici, Bologna, Italy 2Observations pour Les Systèmes D’analyse et de Prévision, Mercator Ocean International, Toulouse, France Ali Aydogdu1, Pietro Miraglio1, Romain Escudier2, Emanuela Clementi1, and Simona Masina1 1Ocean Modeling and Data Assimilation Division, Fondazione Centro Euro-Mediterraneo sui Cambiamenti Climatici, Bologna, Italy 2Observations pour Les Systèmes D’analyse et de Prévision, Mercator Ocean International, Toulouse, France Ali Aydogdu1, Pietro Miraglio1, Romain Escudier2, Emanuela Clementi1, and Simona Masina1 1Ocean Modeling and Data Assimilation Division, Fondazione Centro Euro-Mediterraneo sui Cambiamenti Climatici, Bologna, Italy 2Observations pour Les Systèmes D’analyse et de Prévision, Mercator Ocean International, Toulouse, France 1Ocean Modeling and Data Assimilation Division, Fondazione Centro Euro-Mediterraneo sui Cambiame Climatici, Bologna, Italy 2Observations pour Les Systèmes D’analyse et de Prévision, Mercator Ocean International, Toulouse, Fra Correspondence: Ali Aydogdu (ali.aydogdu@cmcc.it) Received: 25 August 2022 – Discussion started: 26 September 2022 Revised: 14 April 2023 – Accepted: 2 May 2023 – Published: 27 September 2023 Correspondence: Ali Aydogdu (ali.aydogdu@cmcc.it) Received: 25 August 2022 – Discussion started: 26 September 2022 Revised: 14 April 2023 – Accepted: 2 May 2023 – Published: 27 September 2023 Correspondence: Ali Aydogdu (ali.aydogdu@cmcc.it) Correspondence: Ali Aydogdu (ali.aydogdu@cmcc.it) Received: 25 August 2022 – Discussion started: 26 September 2022 Revised: 14 April 2023 – Accepted: 2 May 2023 – Published: 27 September 2023 Received: 25 August 2022 – Discussion started: 26 September 2022 Revised: 14 April 2023 – Accepted: 2 May 2023 – Published: 27 September 2023 us Ocean State Report (OSR7) Abstract. The Mediterranean Sea (MED) is a semi-enclosed basin with an excess amount of evaporation com- pared to the water inﬂux through precipitation at the surface and river runoff on the land boundaries. The deﬁcit in the water budget is balanced by the inﬂow in the Strait of Gibraltar and Turkish Straits System connecting the Mediterranean with the less saline Atlantic Ocean and the Black Sea, respectively. There is evidence that the Mediterranean region is a hotspot in a warming climate, which will possibly change the water cycle signiﬁcantly, but with large uncertainties. Therefore, it is inevitable to monitor the evolution of the essential ocean variables (EOVs) to respond to the associated risks and mitigate the related problems. References B., Dingle, J., Doerffer, R., Donlon, C., Dowell, M., Farman, A., Grant, M., Groom, S., Horseman, A., Jackson, T., Krasemann, H., Laven- der, S., Martinez-Vicente, V., Mazeran, C., Mélin, F., Moore, T. S., Müller, D., Regner, P., Roy, S., Steele, C., Steinmetz, F., Swinton, J., Taberner, M., Thompson, A., Valente, A., Züh- lke, M., Brando, V. E., Feng, H., Feldman, G., Franz, B. A., Frouin, R., Gould, R. W., Hooker, S. B., Kahru, M., Kratzer, S., Mitchell, B. G., Muller-Karger, F. E., Sosik, H. M., Voss, K. J., Werdell, J., and Platt, T.: An ocean-colour time se- ries for use in climate studies: The experience of the Ocean- Colour Climate Change Initiative (OC-CCI), Sensors, 19, 4285, https://doi.org/10.3390/s19194285, 2019. Xi, H., Peeken, I., Gomes, M., Brotas, V., Tilstone, G., Brewin, R. J. W., Dall’Olmo, G., Tracana, A., Alvarado, L. M. A., Murawski, S., Wiegmann, S., and Bracher, A.: Phytoplank- ton pigment concentrations and phytoplankton groups measured on water samples collected from various expeditions in the Atlantic Ocean from 71◦S to 84◦N, PANGAEA [data set], https://doi.org/10.1594/PANGAEA.954738, 2023. Yang, B., Boss, E. S., Haëntjens, N., Long, M. C., Behren- feld, M. J., Eveleth, R., and Doney, S. C.: Phytoplank- ton phenology in the North Atlantic: Insights from proﬁling ﬂoat measurements, Front. Mar. Sci., 7, 139, https://doi.org/10.3389/fmars.2020.00139, 2020. https://doi.org/10.5194/sp-1-osr7-5-2023 State Planet, 1-osr7, 5, 2023 CHAPTER2.3 CHAPTER2.3 Chapter 2.4 – 7th edition of the Copernicus Ocean State Report (OSR7) State Planet, 1-osr7, 6, 2023 https://doi.org/10.5194/sp-1-osr7-6-2023 © Author(s) 2023. This work is distributed under the Creative Commons Attribution 4.0 License. Chapter 2.4 – 7th edition of the Copernicus Ocean ter 2.4 – 7th edition of the Copernicus Ocean State Report (O The dynamical role of upper layer salinity in the Mediterranean Sea In this work, we evaluate the evolu- tion of the salinity content and anomaly between 0–300 m in the Mediterranean Sea during the last decades using the Copernicus Marine Service reanalysis and in situ objective analysis products. The results show an increasing mean salinity with a stronger trend in the eastern Mediterranean (EMED) basin. The spread of the products im- plies a larger variability in the western Mediterranean (WMED) basin, while the standard deviation is lower in the eastern side, especially in the Ionian and the Levantine basins. lantic Ocean and Black Sea waters ﬂow into the basin with an annual net inﬂow of 0.78 ± 0.05 Sv (Soto-Navarro et al., 2010) and 0.05 ± 0.04 Sv (Jarosz et al., 2013), respectively. These density contrasts contribute to the wind-driven circu- lation and generate a highly energetic anti-estuarine circu- lation (Cessi et al., 2014). The salinity of the Atlantic Wa- ters (AW) entering through the Straight of Gibraltar is about 36.2 psu. The salinity of the Dardanelles Strait can vary sig- niﬁcantly and it can be as low as 27 psu (Aydogdu et al., 2018; Sannino et al., 2017). Recently, Fedele et al. (2022) studied the characterisation of the Atlantic Waters and Lev- antine intermediate waters (LIW) from the Argo proﬁles in the last 20 years. Their conclusion is a clear saliniﬁcation and warming trend which characterised both AW and LIW over the last 2 decades. Skliris et al. (2018) argue that the Mediterranean basin saliniﬁcation is driven by changes in the regional water cycle rather than by changes in salt transports at the straits, as it is shown by the water mass transformation Published by Copernicus Publications. 1 Introduction The Mediterranean Sea (MED) is warming (Pisano et al., 2020). It is evaporating more and more (Skliris et al., 2018; Jordà et al., 2017) with marine heat waves increasing in in- tensity, duration and frequency (Juza et al., 2022; Dayan et al., 2022). The Mediterranean region is a hotspot with global warming (Tuel and El Tahir, 2020) that will likely alter the water cycle (Cos et al., 2022). Tracking the changes of the essential ocean variables (EOVs) is crucial in order to un- derstand the impact of climate change. Two of these EOVs are linked to the ocean salinity at the surface and subsur- face, which will be affected signiﬁcantly by the surface heat and freshwater ﬂuxes. The global water cycle modulating the ocean salinity is a key element of the Earth’s climate (Cheng et al., 2020). In the Mediterranean Sea, freshwater ﬂuxes through the land (rivers) and atmosphere (evaporation and precipitation) are balanced by two sea straits, namely Gibraltar and Dardanelles, from which the less saline At- Published by Copernicus Publications. CHAPTER2.4 2 Table 1. Products from Copernicus Marine Service used in this study. Product ref. no. Product ID and type Data access Documentation 1 MEDSEA_MULTIYEAR_PHY_006_004 (MEDREA24, multi-year product) [1987– 2021], numerical models EU Copernicus Marine Service Product (2022a) Quality Information Document (QUID): Escudier et al. (2022) Product User Manual (PUM): Lecci et al. (2022) 2 MEDSEA_MULTIYEAR_PHY_006_004 (MEDREA24, INTERIM product) [2021], numerical models EU Copernicus Marine Service Product (2022b) QUID: Escudier al. (2022) PUM: Lecci et al. (2022) 3 GLOBAL_REANALYSIS_PHY_001_031 (GREP) [1993–2019], numerical models EU Copernicus Marine Service Product (2022c) QUID: Desportes et al. (2022) PUM: Gounou et al. (2022) 4 INSITU_GLO_PHY_TS_OA_MY_013_052 (CORA) [1993–2020], in situ observations EU Copernicus Marine Service Product (2022d) QUID: Szekely (2022a) PUM: Szekely (2022b) 5 MULTIOBS_GLO_PHY_TSUV_3D_ MYNRT_015_012 (ARMOR3D) [1993– 2020], in situ observations and satellite observations EU Copernicus Marine Service Product (2021) QUID: Grenier et al. (2021) PUM: Guinehut (2021) Table 1. Products from Copernicus Marine Service used in this study. ocean reanalysis is reviewed in Storto et al. (2019b). The pe- riod covered by the GREP is 1993–2019. distribution in salinity coordinates. However, we will show that there is a bigger uncertainty compared to most of the basin in the radius of inﬂuence of both the Gibraltar and Dar- danelles straits. In Sect. 2, the data and methods used in this study are presented. In Sect. 1 Introduction 3, the results are shown and dis- cussed, while in Sect. 4, the conclusions are drawn. As observational products, the gridded reconstructions of CORA (Szekely et al., 2019) and ARMOR3D (Guinehut et al., 2012) are adopted. Both datasets are available between 1993 and 2020. In the CORA, the objective analysis is per- formed on measurements’ anomalies relative to a ﬁrst guess provided by monthly climatology for different decades in the World Ocean Atlas 2013 to accurately reproduce the climate tendencies; it is interpolated and centred on the 15th of each month. Instead, in ARMOR3D, the ﬁrst guess is adopted from World Ocean Atlas 2018. Both products use an objec- tive analysis method proposed by Bretherton et al. (1976). 2 Data and methods is normalised by the depth of the water column, which is constant in our case with z2 = 300 m and z1 = 0 m. SA = z2 z1 S −Sref z2 −z1 dz (2) SA = z2 z1 S −Sref z2 −z1 dz (2) psu (psu yr−1) MED WMED EMED MEDREA24 38.58 (0.0070) 38.15 (0.0055) 38.83 (0.0078) GREP 38.48 (0.0110) 38.01 (0.0111) 38.87(0.0106) CORA 38.61 (0.0020) 38.21(0.0019) 38.84 (0.0024) ARMOR3D 38.64 (0.0027) 38.24 (0.0020) 38.75(0.0032) Mean 38.57 (0.0056) 38.16 (0.0049) 38.87 (0.0061) Equation (2) is a modiﬁed version of the one proposed in Boyer et al. (2007), which uses the salinity as a proxy for the equivalent freshwater content. This method has been later adopted in various studies including, among the others, Hol- liday et al. (2020), with a density weight to account for baro- clinic properties of the water column. The formulation in Boyer et al. (2007) is based on a reference salinity. As an example, mean values of a basin (Aagaard and Carmack, 1989) is a widely used choice for Sref in global freshwater content calculations. However, it is argued that since a refer- ence value can be chosen arbitrarily, this would bring ambi- guity (Schauer and Losch, 2019) in computing the equivalent freshwater content. Therefore, in this study, we propose to evaluate the salinity content and anomaly following Eq. (2) by choosing Sref as a monthly climatology of each dataset computed from each product separately between 1993 and 2014. This period is chosen to be consistent with the Ocean Monitoring Indicators produced previously in the Mediter- ranean Sea and other Copernicus Marine domains. Further- more, the calculations are performed in the entire Mediter- ranean Sea (MED) as well as in its western (WMED) and eastern (EMED) subbasins, separated at the Sicily Strait. In Fig. 1, we present the monthly variation of the Sref as an example only, the one from MEDREA24, which shows a clear difference in the seasonality in the EMED and WMED, with a maximum in March and December, respectively. It is also evident that the Mediterranean monthly salinity refer- ence shows a seasonal cycle much similar to the one of the eastern basin (but with different magnitude) characterised by lower salinity during the summer period and larger values at the end of the year. 2 Data and methods In this study, the Copernicus Marine Service global and re- gional reanalyses as well as observational gridded products are used to explore the role of the salinity variability in the 0–300 m depth among different estimates as well as temporal and spatial anomalies against a mean. The investigation is performed in the entire Mediterranean Sea (MED) as well as in the eastern (EMED) and west- ern (WMED) basins, which have very different characteris- tics. The salinity mean (S) is computed using Eq. (1) as the monthly volume (V ) average of each product between 0 and 300 m depth, i.e. excluding the shelf areas close to the coast with a depth less than 300 m. The Mediterranean 1/24◦resolution regional reanaly- sis (hereinafter, MEDREA24; Escudier et al., 2021) from the Copernicus Marine Service is used as a regional high- resolution product. In this work, MEDREA24 and its in- terim extension until the end of 2021 are included. More- over, the 1/4◦resolution Global Reanalysis Ensemble Prod- uct (hereinafter, GREP) is also used. It consists of the global reanalysis from Mercator Ocean’s GLORYS2V4 (Lellouche et al., 2013), UK MetOfﬁce’s GLOSEA5v13 (MacLach- lan et al., 2014) using the Forecasting Ocean Assimilation Model (FOAM) system (Blockley et al., 2014), CMCC C- GLORSv7 (Storto et al., 2016) and ECMWF’s ORAS5 (Zuo et al., 2017). A study on the ocean heat content and steric sea level representation in the GREP ensemble can be found in Storto et al. (2019a). A more general status of the global S = 1 V V SdV (1) S = 1 V V SdV (1) The mean of different products and their standard deviation are evaluated in the common period 1993–2019. Besides this time frame, the CORA and ARMOR3D time series are avail- able until 2020, while the MEDREA24 time series is pro- vided up to 2021 (last 6 months extended in interim mode). The salinity anomalies are computed using Eq. (2). The difference of the salinity, S, with a reference salinity, Sref, State Planet, 1-osr7, 6, 2023 https://doi.org/10.5194/sp-1-osr7-6-2023 CHAPTER2.4 A. Aydogdu et al.: The dynamical role of upper layer salinity in the Mediterranean Sea 3 A. Aydogdu et al.: The dynamical role of upper layer salinity Table 2. The temporal mean salinity (in psu) and trend (in psu per year) in the 0–300 m between the common period 1993–2019 for separate products and their overall mean in Fig. 2. 2 Data and methods the early 2000s following the efforts in the Global Ocean Data Assimilation (GODAE) together with the Climate Vari- ability and Predictability Programme (CLIVAR) and the Global Climate Observing System/Global Ocean Observing System (GCOS/GOOS), the spread among different prod- ucts narrows. Possibly, the reanalyses are better constrained through data assimilation with this novel observation type (Johnson et al., 2022), which provides high-resolution and high-frequency temperature and salinity proﬁles all over the world’s oceans, while the observation-based gridded prod- ucts become more conﬁdent. The maximum spread between the period 1993–2019 occurs in the 1990s with a value of 0.096 psu, and it decreases to as low as 0.009 psu by the end of the 2010s. The mean salinity computed in the en- tire Mediterranean Sea from all products varies between ap- proximately 38.5 and 38.6 psu with a spatiotemporal mean of 38.57 psu (Table 2). In the western Mediterranean (Fig. 2b), the overall mean is centred around 38.16 psu with a larger spread – with a max- imum and minimum of 0.172 psu and 0.026, respectively – occurring in the early 2000s. An increase of the mean salin- ity in 2005 is evident from all the reanalysis products and, to a lesser extent, from the CORA dataset, for which one of the many possible reasons is the regime shift as discussed in Schroeder et al. (2016), corresponding to a major deep-water formation event at the beginning of the Western Mediter- ranean Transition (Zunino et al., 2012). https://doi.org/10.5194/sp-1-osr7-6-2023 A. Aydogdu et al.: The dynamical role of upper layer salinity in the Mediterranean Sea They are more conﬁned around the mean in the eastern basin, which may also explain the lower spread in this area as dis- cussed below in Fig. 3b. from the EN4 and MEDATLAS datasets, which show a trend of 0.0096 ± 0.0077 and 0.0088 ± 0.0092, respectively, in the ﬁrst 150 m while showing a trend of 0.0067 ± 0.0040 and 0.0067 ± 0.0036 between 150–600 m. The differences in trend in different products that we used are mainly due to the discrepancies at the beginning of the time series. The weak consistency among the reanalyses visible during the ﬁrst decade is likely due to the lack of observations not suf- ﬁcient to constrain the different models, which use differ- ent physics and initialisation (e.g. Masina and Storto, 2017). The reduction of the spread among the products evolves in parallel to the increase of the observational coverage after the advent of the Argo network. The observational products will be impacted from the scarcity of the observations in the 1990s, since they rely on statistical methods. The trend calcu- lated for each grid point from the MEDREA24, which is the analysed product covering the longest period, is presented in Fig. 2d. The dominant signal in the entire basin is posi- tive with a larger amplitude in the Balearic Sea, Ionian Sea, Adriatic Sea, Western Levantine and with a less evident sig- nal in the Gulf of Lions, Northern Aegean Sea and Eastern Levantine Basin. A small negative trend zone appears in the Alboran Sea. The trend in the entire analysed period is about 0.0049 psu yr−1 in the western basin. This is below the rate of the basin-wide trend, which is larger due to the trend in the eastern basin (0.0061 psu yr−1). Differences among dif- ferent products, especially the objectively analysed observa- tions and GREP, for the trend is larger in the western basin. They are more conﬁned around the mean in the eastern basin, which may also explain the lower spread in this area as dis- cussed below in Fig. 3b. it is less evident in the western basin. Along with its interim extension, MEDREA24 shows an increasing mean salinity until the end of 2021. All products present larger values after 2016 and a maximum in 2018. The spatial mean, computed between 1993–2014 from all products in the ﬁrst 300 m (Fig. A. Aydogdu et al.: The dynamical role of upper layer salinity in the Mediterranean Sea Figure 1. The monthly reference salinity Sref estimates calculated from the MEDREA24 in the period 1993–2014. The green, blue and red curves show the MED, WMED and EMED regions, respectively on its corresponding vertical axis. The same calculation is done for each product separately (not shown) to evaluate Eq. (2) to compute salinity anomaly. Figure 1. The monthly reference salinity Sref estimates calculated from the MEDREA24 in the period 1993–2014. The green, blue and red curves show the MED, WMED and EMED regions, respectively on its corresponding vertical axis. The same calculation is done for each product separately (not shown) to evaluate Eq. (2) to compute salinity anomaly. from the EN4 and MEDATLAS datasets, which show a trend of 0.0096 ± 0.0077 and 0.0088 ± 0.0092, respectively, in the ﬁrst 150 m while showing a trend of 0.0067 ± 0.0040 and 0.0067 ± 0.0036 between 150–600 m. The differences in trend in different products that we used are mainly due to the discrepancies at the beginning of the time series. The weak consistency among the reanalyses visible during the ﬁrst decade is likely due to the lack of observations not suf- ﬁcient to constrain the different models, which use differ- ent physics and initialisation (e.g. Masina and Storto, 2017). The reduction of the spread among the products evolves in parallel to the increase of the observational coverage after the advent of the Argo network. The observational products will be impacted from the scarcity of the observations in the 1990s, since they rely on statistical methods. The trend calcu- lated for each grid point from the MEDREA24, which is the analysed product covering the longest period, is presented in Fig. 2d. The dominant signal in the entire basin is posi- tive with a larger amplitude in the Balearic Sea, Ionian Sea, Adriatic Sea, Western Levantine and with a less evident sig- nal in the Gulf of Lions, Northern Aegean Sea and Eastern Levantine Basin. A small negative trend zone appears in the Alboran Sea. The trend in the entire analysed period is about 0.0049 psu yr−1 in the western basin. This is below the rate of the basin-wide trend, which is larger due to the trend in the eastern basin (0.0061 psu yr−1). Differences among dif- ferent products, especially the objectively analysed observa- tions and GREP, for the trend is larger in the western basin. 3 Results and discussion In the eastern Mediterranean (Fig. 2c), the overall mean is centred around 38.87 psu with a lower spread compared to the western basin, with a maximum and minimum of 0.086 psu and 0.003, respectively. The Copernicus Marine Service products described in the previous section allow the assessment of the salinity content of the Mediterranean Sea along with its anomalies and trends during the last decades. Overall, for the period between 1993–2019, we note that the observational products, gridded using optimal interpo- lation statistical techniques, show a higher average salinity compared to the reanalysis products that are dynamically in- tegrated and corrected through data assimilation. The spread is representing the offset of the products more than their vari- ability in the entire Mediterranean Sea, as well as in its east- ern and western subdomains. In Fig. 2, we present the time series of the mean salinity content in the ﬁrst 300 m derived from the analysed products (MEDREA24 in red, GREP ensemble mean in blue, GREP ensemble members in thin light blue, CORA in dark green, ARMOR3D in light green) and their overall mean (in black) and spread (shaded grey). During the early 1990s in the entire Mediterranean Sea (Fig. 2a), there is a large spread in salinity with the observa- tions showing a higher salinity, while the reanalysis products present relatively lower salinity. This is the case until 2005. Coinciding with the global coverage of the Argo proﬁlers in All the products show a positive trend between 1993–2019 (in parenthesis in Table 2). The trend in the mean of all prod- ucts is calculated as 0.0056 psu yr−1. This trend is consistent with the estimates between 1950–2002 of Skliris et al. (2018) https://doi.org/10.5194/sp-1-osr7-6-2023 State Planet, 1-osr7, 6, 2023 CHAPTER2.4 CHAPTER2.4 CHAPTER2.4 CHAPTER2.4 A. Aydogdu et al.: The dynamical role of upper layer salinity in the Mediterranean Sea A. Aydogdu et al.: The dynamical role of upper layer salinity in the Mediterranean Sea 3a), shows a gradual in- crease in the upper ocean integrated salinity from west to east. Minimum salinity occurs close to river mouths, such as in the North Adriatic Sea due to the freshwater input from the Po River, and on the pathways of the outﬂow of the Dar- danelles and Gibraltar straits. The Atlantic Waters, modiﬁed through its route, can be traced till the eastern basin from its low salinity. The spread deduced from all the products (Fig. 3b) implies that they agree more, meaning lower spread, in the Levantine and Ionian Seas and to a lesser degree be- tween the Balearic, Sardinia and Corsica islands. The spread is larger especially in the northern Aegean and Adriatic Sea and southwestern coast between the Gulf of Gabes and Strait of Gibraltar. This uncertainty or mismatch in the products is possibly due to the different volume ﬂuxes through the rivers and straits. In Fig. 4a–c, we show the time series of the salinity anomaly estimates in the western (Fig. 4a), eastern (Fig. 4b) and entire (Fig. 4c) basin from each product using Eq. (2). We recall that the salinity reference is computed for each product per se. Moreover, the salinity anomaly map in 2021 from MEDREA24 is depicted in Fig. 4d, computed against the overall mean between 1993–2014, which is shown in Fig. 3a. The anomalies have a larger range in the reanalysis prod- ucts. There is a negative anomaly within the ﬁrst decade in GREP and MEDREA24 which turns to positive ﬁrst in the western Mediterranean (Fig. 4a) and followed by the eastern For 2020, CORA and ARMOR3D products are available, and both continue to sustain the positive trend even though State Planet, 1-osr7, 6, 2023 https://doi.org/10.5194/sp-1-osr7-6-2023 CHAPTER2.4 CHAPTER2.4 A. Aydogdu et al.: The dynamical role of upper layer salinity in the Mediterranean Sea A. Aydogdu et al.: The dynamical role of upper layer salinity in the Mediterranean Sea 5 Figure 3. The maps of the (a) mean and (b) spread of inte- grated salinity in the period between 1993–2014 in 0–300 m com- puted from the GREP ensemble mean, CORA, ARMOR3D and MEDREA24 products. We refer to the text for the information on the data products used. The analysis is performed only if the water column is deeper than 300 m. Note that in (b), the colour scale is not linear to show the smaller standard deviation. Figure 2. A. Aydogdu et al.: The dynamical role of upper layer salinity in the Mediterranean Sea psu MED WMED EMED MEDREA24 0.026 0.027 0.025 GREP 0.042 0.056 0.034 CORA 0.001 −0.008 0.007 ARMOR3D 0.001 −0.009 0.008 Mean 0.018 0.017 0.019 eral agrees with the anomaly map to a large extent. However, we note that the spread on the pathway of the water entering from the Strait of Gibraltar and reaching the Levantine basin has a relatively larger spread compared to the deeper areas (see Fig. 3b). basin (Fig. 4b) after 2006. In the CORA and ARMOR3D, in- stead, there is a clear increase in the salinity anomaly in the eastern Mediterranean and the entire basin with a less evident positive trend in the western basin. We summarise the mean salinity anomalies in Table 3. basin (Fig. 4b) after 2006. In the CORA and ARMOR3D, in- stead, there is a clear increase in the salinity anomaly in the eastern Mediterranean and the entire basin with a less evident positive trend in the western basin. We summarise the mean salinity anomalies in Table 3. A. Aydogdu et al.: The dynamical role of upper layer salinity in the Mediterranean Sea Time series of mean salinity in the upper 300 m in the (a) entire Mediterranean Sea, (b) western Mediterranean basin and (c) eastern Mediterranean basin between the period 1993 and 2021 from MEDREA24, until the end of 2020 for ARMOR3D and CORA, and until the end of 2019 for GREP. The mean of all the products is drawn in black with their standard deviation shaded around the mean. The GREP ensemble members are depicted in thin blue curves. The GREP product covers the period until 2019, while the observational products CORA and ARMOR3D cover the period until 2020. The time series for MEDREA24 is extended until 2021 using the interim products. Panel (d) shows the salinity trend in the ﬁrst 300 m from the MEDREA24 practical salinity unit (psu) per year. Figure 3. The maps of the (a) mean and (b) spread of inte- grated salinity in the period between 1993–2014 in 0–300 m com- puted from the GREP ensemble mean, CORA, ARMOR3D and MEDREA24 products. We refer to the text for the information on the data products used. The analysis is performed only if the water column is deeper than 300 m. Note that in (b), the colour scale is not linear to show the smaller standard deviation. Table 3. The temporal mean salinity anomaly (in psu) in the 0– 300 m between the common period 1993–2019 for separate prod- ucts and their average in Fig. 3. Figure 2. Time series of mean salinity in the upper 300 m in the (a) entire Mediterranean Sea, (b) western Mediterranean basin and (c) eastern Mediterranean basin between the period 1993 and 2021 from MEDREA24, until the end of 2020 for ARMOR3D and CORA, and until the end of 2019 for GREP. The mean of all the products is drawn in black with their standard deviation shaded around the mean. The GREP ensemble members are depicted in thin blue curves. The GREP product covers the period until 2019, while the observational products CORA and ARMOR3D cover the period until 2020. The time series for MEDREA24 is extended until 2021 using the interim products. Panel (d) shows the salinity trend in the ﬁrst 300 m from the MEDREA24 practical salinity unit (psu) per year. https://doi.org/10.5194/sp-1-osr7-6-2023 4 Conclusions In this study, we presented the salinity characteristics of the Mediterranean Sea in the upper 300 m deduced from vari- ous products including reanalysis and gridded observational datasets released by the Copernicus Marine Service. The products with dynamically constructed ocean reanalysis and objectively analysed observations show signiﬁcantly large spread at the beginning of the period of investigation, while In 2021, the anomaly is mostly positive, with some neg- ative anomaly structures on the path of the Atlantic Waters (Fig. 4d), Alboran Sea and upwelling favouring the Balearic Islands. Fedele et al. (2022) reports a positive salinity trend in the modiﬁed Atlantic and Levantine intermediate waters using 18-year-long (2001–2019) Argo proﬁles, which in gen- https://doi.org/10.5194/sp-1-osr7-6-2023 State Planet, 1-osr7, 6, 2023 CHAPTER2.4 CHAPTER2.4 A. Aydogdu et al.: The dynamical role of upper layer salinity in the Mediterranean Sea A. Aydogdu et al.: The dynamical role of upper layer salinity in the Mediterranean Sea 6 Figure 4. Time series of the salinity anomaly from the MEDREA24, GREP, ARMOR3D and CORA products in the (a) western Mediter- ranean Sea, (b) eastern Mediterranean basin and (c) entire Mediterranean basin computed with respect to the monthly reference salinity estimates in the corresponding area in Fig. 1 calculated from the MEDREA24 in the period 1993–2014. The GREP products cover the period until 2019, while the observational products CORA and ARMOR3D cover the period until 2020. The time series for MEDREA24 is extended until 2021 using the interim products. Panel (d) shows the salinity anomaly in 2021 in the Mediterranean Sea against the mean of salinity in Fig. 3a. Figure 4. Time series of the salinity anomaly from the MEDREA24, GREP, ARMOR3D and CORA products in the (a) western Mediter- ranean Sea, (b) eastern Mediterranean basin and (c) entire Mediterranean basin computed with respect to the monthly reference salinity estimates in the corresponding area in Fig. 1 calculated from the MEDREA24 in the period 1993–2014. The GREP products cover the period until 2019, while the observational products CORA and ARMOR3D cover the period until 2020. The time series for MEDREA24 is extended until 2021 using the interim products. Panel (d) shows the salinity anomaly in 2021 in the Mediterranean Sea against the mean of salinity in Fig. 3a. Data availability. All datasets used in this article can be obtained from the Copernicus Marine Service catalogues as described in Ta- ble 1 with their names, temporal coverages and documentation. 4 Conclusions the uncertainty is possibly reduced with the emergence of Argo proﬁlers, which allowed wider spatial sampling and a higher frequency of sampling in the ocean. The mean salin- ity with its anomaly and trend is computed and analysed in the entire basin as well as in the western and eastern basins for all the datasets separately and averaged. The spatial maps of the mean and the spread of the salinity are depicted and discussed. The overall results show a saliniﬁcation of the Mediterranean Sea agreeing with earlier studies (e.g. Skliris et al., 2018). The subbasin-scale investigation shows negative salinity anomalies in the western basin in the upwelling re- gions, which may imply stronger upwelling events, and wa- terway following the North African coast, which may be a consequence of the freshening North Atlantic water masses (Holliday et al., 2020). There is a large spread in the salinity estimates among different products, which is reduced with the introduction of the Argo proﬁlers in the data assimila- tion components of the reanalysis systems. Besides the large spread, considering the reported discrepancies in the salinity measurements after 2016 (Barnoud et al., 2021), it is essen- tial to use all available information sources for a more accu- rate state estimate and uncertainty quantiﬁcation. the uncertainty is possibly reduced with the emergence of Argo proﬁlers, which allowed wider spatial sampling and a higher frequency of sampling in the ocean. The mean salin- ity with its anomaly and trend is computed and analysed in the entire basin as well as in the western and eastern basins for all the datasets separately and averaged. The spatial maps of the mean and the spread of the salinity are depicted and discussed. The overall results show a saliniﬁcation of the Mediterranean Sea agreeing with earlier studies (e.g. Skliris et al., 2018). The subbasin-scale investigation shows negative salinity anomalies in the western basin in the upwelling re- gions, which may imply stronger upwelling events, and wa- terway following the North African coast, which may be a consequence of the freshening North Atlantic water masses (Holliday et al., 2020). There is a large spread in the salinity estimates among different products, which is reduced with the introduction of the Argo proﬁlers in the data assimila- tion components of the reanalysis systems. References Aagaard, K. and Carmack, E. 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Cessi, P., Pinardi, N., and Lyubartsev, V.: Energetics of semien- closed basins with two-layer ﬂows at the strait, J. Phys. Ooceanogr., 44, 967–979, 2014. Cheng, L., Trenberth, K. E., Gruber, N., Abraham, J. A. Aydogdu et al.: The dynamical role of upper layer salinity in the Mediterranean Sea A. Aydogdu et al.: The dynamical role of upper layer salinity in the Mediterranean Sea 7 Financial support. This study has been conducted using EU Copernicus Marine Service Information. This work has been funded through the EU Copernicus Marine Med-MFC Service, lot no. 21002L5-COP-MFC MED-5500. ment for the Global Ocean Ensemble Physics Reanalysis, GLOBAL_REANALYSIS_PHY_001_031, Issue 1.1, Merca- tor Ocean International, https://catalogue.marine.copernicus.eu/ documents/QUID/CMEMS-GLO-QUID-001-031.pdf (last ac- cess: 3 May 2023), 2022. Escudier, R., Clementi, E., Cipollone, A., Pistoia, J., Drudi, M., Grandi, A., Lyubartsev, V., Lecci, R., Aydogdu, A., Delrosso, D., Omar, M., Masina, S., Coppini, G., and Pinardi, N.: A High Res- olution Reanalysis for the Mediterranean Sea, Front. Earth Sci., 9, 702285, https://doi.org/10.3389/feart.2021.702285, 2021. Review statement. This paper was edited by Piero Lionello and reviewed by Katrin Schroeder and one anonymous referee. Review statement. This paper was edited by Piero Lionello and reviewed by Katrin Schroeder and one anonymous referee. Escudier, R., Clementi, E., Nigam, T., Aydogdu, A., Fini, E., Pistoia, J., Grandi, A., and Miraglio, P.: EU Coper- nicus Marine Service Quality Information Document for the Mediterranean Sea Physics Reanalysis, MED- SEA_MULTIYEAR_PHY_006_004, Issue 2.3, Mercator Ocean International, https://catalogue.marine.copernicus.eu/ documents/QUID/CMEMS-MED-QUID-006-004.pdf (last access: 3 May 2023), 2022. 4 Conclusions Besides the large spread, considering the reported discrepancies in the salinity measurements after 2016 (Barnoud et al., 2021), it is essen- tial to use all available information sources for a more accu- rate state estimate and uncertainty quantiﬁcation. Author contributions. AA and PM have done the investigation, developed the methodology and performed the data analysis. AA wrote the initial draft. All the authors reviewed and edited the paper. Competing interests. The contact author has declared that none of the authors has any competing interests. Disclaimer. Publisher’s note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Acknowledgements. We would like to express our thanks to Ka- trin Schroeder and one anonymous referee for their useful com- ments, which helped us to improve the paper. We thank to Piero Lionello for his editorial suggestions. Finally, we would like to thank to Karina von Schuckmann for very useful discussions dur- ing course of the study. State Planet, 1-osr7, 6, 2023 https://doi.org/10.5194/sp-1-osr7-6-2023 CHAPTER2.4 A. Aydogdu et al.: The dynamical role of upper layer salinity in the Mediterranean Sea State Planet, 1-osr7, 6, 2023 References and Losch, M.: “Freshwater” in the ocean is not a use- ful parameter in climate research, J. Phys. Oceanogr., 49, 2309– 2321, 2019. 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Aydogdu et al.: The dynamical role of upper layer salinity in the Mediterranean Sea Baltic Sea freshwater content n of the Copernicus Ocean State Report (OSR7) Urmas Raudsepp, Ilja Maljutenko, Amirhossein Barzandeh, Rivo Uiboupin, and Priidik Lagemaa Department of Marine Systems, Tallinn University of Technology, Tallinn, 12618, Estonia Correspondence: Ilja Maljutenko (ilja.maljutenko@taltech.ee) Received: 1 September 2022 – Discussion started: 29 September 2022 Revised: 8 August 2023 – Accepted: 29 August 2023 – Published: 27 September 2023 Urmas Raudsepp, Ilja Maljutenko, Amirhossein Barzandeh, Rivo Uiboupin, and Priidik Lagemaa Department of Marine Systems, Tallinn University of Technology, Tallinn, 12618, Estonia Correspondence: Ilja Maljutenko (ilja.maljutenko@taltech.ee) Received: 1 September 2022 – Discussion started: 29 September 2022 Revised: 8 August 2023 – Accepted: 29 August 2023 – Published: 27 September 2023 Abstract. The Baltic Sea is a brackish shallow sea, the state of which is determined by the mixing of freshwater from net precipitation and runoff with the salty water from the North Sea inﬂows. The freshwater content (FWC) of the Baltic Sea is calculated from the Copernicus regional reanalysis data covering the period 1993–2021. The FWC in the Baltic Sea has shown a steady decrease over the past 2 decades, with a linear trend of 23.9 km3 yr−1; however, the trend has signiﬁcant spatial variability. The Gulf of Bothnia has a positive FWC tendency, while the Baltic Proper has a negative FWC tendency. Temporal changes of FWC are opposite between the Bothnian Bay in the north and the southern Baltic Proper. In the Bothnian Bay, interannual changes of FWC are positively correlated with river runoff and net precipitation and negatively correlated with salt transport. In the southern Baltic Proper, the variations of FWC and salt transport through the Danish straits are negatively correlated from 1993 until 2010 but positively correlated thereafter. The seasonal freshwater content reﬂects the speciﬁc hydrophysical conditions of each sub-basin, with northern basins being inﬂuenced by seasonal river runoff and ice formation and melting, while the southern basins are more responsive to subsurface salinity changes due to salt transport through the Danish straits. us Ocean State Report (OSR7) The Baltic Sea is one of the marginal seas where water salinity and FWC are strongly inﬂuenced by the water ex- change with the North Sea. The major Baltic inﬂows (MBIs) are the most voluminous event-type sources of saline wa- ter to the Baltic Sea (Mohrholz, 2018). Baltic Sea freshwater content The frequency and intensity of the MBIs and other large volume inﬂows have no long-term trends but do have a multidecadal variabil- ity of about 30 years (Mohrholz, 2018; Lehmann and Post, 2015; Lehmann et al., 2017; Radtke et al., 2020). Smaller barotropic and baroclinically driven inﬂows transport saline water into the halocline or below it, depending on the density of the inﬂow water (Reissmann et al., 2009). The inﬂows of saline water are forced by winds from the west and outﬂows by winds from the east. Published by Copernicus Publications. References E., Santoleri, R., and Buongiorno Nardelli, B.: New Evi- State Planet, 1-osr7, 6, 2023 State Planet, 1-osr7, 6, 2023 https://doi.org/10.5194/sp-1-osr7-6-2023 CHAPTER2.4 CHAPTER2.4 A. Aydogdu et al.: The dynamical role of upper layer salinity in the Mediterranean Sea 9 Zuo, H., Balmaseda, M. A., and Mogensen, K.: The new eddy- permitting ORAP5 ocean reanalysis: description, evaluation and uncertainties in climate signals, Clim. Dynam., 49, 791–811, https://doi.org/10.1007/s00382-015-2675-1, 2017. Tuel, A. and Eltahir, E. A. B.: Why Is the Mediterranean a Climate Change Hot Spot?, J. Climate, 33, 5829–5843, https://journals. ametsoc.org/view/journals/clim/33/14/JCLI-D-19-0910.1.xml (retrieved 25 Jannuary 2022), 2020. Zunino, P., Schroeder, K., Vargas-Yáñez, M., Gasparini, G. P., Cop- pola, L., García-Martínez, M. C., and Moya-Ruiz, F.: Effects of the Western Mediterranean Transition on the resident water masses: Pure warming, pure freshening and pure heaving, J. Mar. Syst., 15, 96–97, https://doi.org/10.1016/j.jmarsys.2012.01.011, 2012. https://doi.org/10.5194/sp-1-osr7-6-2023 State Planet, 1-osr7, 6, 2023 CHAPTER2.4 CHAPTER2.4 Chapter 2.5 – 7th edition of the Copernicus Ocean State Report (OSR7) Chapter 2.5 – 7th edition of the Copernicus Ocean State Report (OSR7) Chapter 2.5 – 7th edition of the Coper State Planet, 1-osr7, 7, 2023 https://doi.org/10.5194/sp-1-osr7-7-2023 © Author(s) 2023. This work is distributed under the Creative Commons Attribution 4.0 License. Chapter 2.5 – 7th edition of the Copernicus Ocean ter 2.5 – 7th edition of the Copernicus Ocean State Report (O U. Raudsepp et al.: Baltic Sea freshwater content 2 Table 1. CMEMS and non-CMEMS products used in this study, including information on data documentation. Product Product ID & type Data access Documentation ref. no. 1 BALTICSEA_REANALYSIS_PHY_003_011; numerical models EU Copernicus Marine Service Product (2023) Quality Information Document (QUID): Liu et al. (2019) Product User Manual (PUM): Axell (2021) 2 ERA5; numerical models Hersbach et al. (2023) Hersbach et al. (2020) 3 IOW-THREDDS-BMIP_bmip_rivers_2019-10-10-1; numerical models and observations IOW THREDDS (2019) Väli et al. (2019) 4 EMODNET_CHEMISTRY_Baltic_Sea_aggregated_ eutrophication_and_acidity_datasets_1902- 2017_v2018; observations SMHI (2023) Buga et al. (2018), Giorgetti et al. (2020) The aim of this study is to analyse the changes of the Baltic Sea FWC during the period of 1993–2021. The MBI in 1993 ended the stagnation period with no MBIs that lasted for about 10 years (1983–1993). During the stagnation period, salinity was below average, stratiﬁcation weakened, and hy- poxic area decreased (Lehmann et al., 2022). The period of 1993–2021 includes the third largest MBI in 2014 (Mohrholz et al., 2015) and several of the other barotropic large vol- ume inﬂows (Mohrholz, 2018). We focus on the changes of the FWC in the whole Baltic Sea as well as its sub-basins. We investigate the trends in FWC and observe its seasonal changes. A qualitative explanation of the physical processes behind the dynamics of FWC is provided. in volume-averaged Baltic Sea salinity (Meier and Kauker, 2003; Lehmann et al., 2022), while the direct dilution of the Baltic Sea water by freshwater accounts for 27 % of the inter- annual variations (Radtke et al., 2020). During 1950–2018, precipitation averaged over the Baltic Sea catchment area had a trend of 1.44 mm yr−1 (Meier et al., 2022). in volume-averaged Baltic Sea salinity (Meier and Kauker, 2003; Lehmann et al., 2022), while the direct dilution of the Baltic Sea water by freshwater accounts for 27 % of the inter- annual variations (Radtke et al., 2020). During 1950–2018, precipitation averaged over the Baltic Sea catchment area had a trend of 1.44 mm yr−1 (Meier et al., 2022). Thus, the long-term salinity of the Baltic Sea is determined by saline water inﬂows from the North Sea (wind forcing) and its dilution with freshwater originating from numerous rivers across the Baltic coast and net precipitation (Winsor et al., 2001; Meier and Kauker, 2003; Gustafsson and Omstedt, 2009; Schimanke and Meier, 2016; Lehmann et al., 2022). U. Raudsepp et al.: Baltic Sea freshwater content There is no clear long-term trend of the mean salinity of the Baltic Sea, but there are multidecadal oscillations of about 30 years (Kniebusch et al., 2019). A 30-year variability has been found for the salinity, river runoff, and saltwater inﬂows (Radtke et al., 2020). The Baltic Sea salinity also has a natu- ral centennial variability (Kniebusch et al., 2019). https://doi.org/10.5194/sp-1-osr7-7-2023 1 Introduction Climate warming has resulted in the intensiﬁcation of the global hydrological cycle but not necessarily on the regional scale (Pratap and Markonis, 2022). The increase in net pre- cipitation over land and sea areas, decrease in ice cover, and increase in river runoff are the main components of the global hydrological cycle that increase freshwater content (FWC) in the ocean (Boyer et al., 2007) and decrease ocean salinity. All the components can be directly estimated but might have sig- niﬁcant uncertainties. Instead, the ocean salinity change can be used as a marker of the water cycle change (Durack et al., 2012). In the case of an open part of the ocean, for example, a regional sea, using salinity as a proxy for FWC includes an additional blurring aspect, which is water transport through the open boundaries between the basin under consideration and its surrounding area. The impact of water exchange on the changes of FWC is signiﬁcant if not dominant. In that case, changes of FWC may not represent the actual changes of freshwater input from the above-mentioned sources. Direct total input of freshwater to the Baltic Sea consists of river runoff and net precipitation. The total river runoff from the Baltic Sea catchment area shows no statistically signif- icant trend but a variability of about 30 years (Meier et al., 2019a, b) and a pronounced decadal variability of accumu- lated anomaly of runoff (Lehmann et al., 2022). The varia- tions in runoff explain about 50 % of the long-term variability CHAPTER2.5 CHAPTER2.5 2 Data and methods The BALMFC CMEMS reanalysis product (data ref. 1, Ta- ble 1) is calculated using the Nemo-Nordic 1.0 ocean model (Hordoir et al., 2019). The horizontal resolution of the model is approximately 2 nmi, and there are 56 vertical levels. Ver- tical resolution varies from 3 m at the surface to 10 m below the 100 m depth. The model without data assimilation has been thoroughly validated (Hordoir et al., 2019). The Coper- nicus model system uses the local singular evolutive inter- polated Kalman ﬁlter data assimilation method (Liu and Fu, 2018). A detailed quality assessment of the reanalysis prod- uct (data ref. 1, Table 1), using the K-means clustering algo- rithm (Raudsepp and Maljutenko, 2022), is provided in Ap- pendix A. A speciﬁc feature of the Baltic Sea is the large difference in sea surface salinity, ranging from about 20 g kg−1 in the Kattegat to 2 g kg−1 in the Bothnian Bay (Leppäranta and Myrberg, 2009). Kniebusch et al. (2019) found a positive trend of centennial changes in the north–south gradient of the surface salinity and river runoff in the northern catchment area. Multidecadal oscillations control the long-term varia- tions of surface salinity and its meridional gradient with a period of about 30 years (Radtke et al., 2020). A common approach is to use salinity to describe the en- ergy and water cycles in the Baltic Sea (Lehmann et al., 2022; Meier et al., 2022). In this study, instead of using spatially mean salinity of the Baltic Sea, we suggest the concept of FWC (Boyer et al., 2007) for the description of the physi- cal state of the Baltic Sea. Previously, a concept of FWC has been used to estimate the freshwater budget of the Baltic Sea (Winsor et al., 2001) and for the geographical spreading of spring-time river runoff (Eilola and Stigebrandt, 1998). The FWC is calculated according to Boyer et al. (2007): FWC = ρ (Sref,Tref,p) ρ (0,Tref,p) Sref −S S , (1) (1) where S(x,y,z,t) and Sref(x,y,z) are actual salinity and ref- erence salinity, respectively, and x, y, z, and t are zonal, meridional, vertical, and temporal coordinates, respectively. The density, ρ, is calculated according to the TEOS10 (IOC State Planet, 1-osr7, 7, 2023 https://doi.org/10.5194/sp-1-osr7-7-2023 CHAPTER2.5 U. Raudsepp et al.: Baltic Sea freshwater content 3 Figure 1. Map of the Baltic Sea depth distribution (data ref. 1, Ta- ble 1). 2 Data and methods Boxes indicate the boundaries used to calculate the fresh- water content for different sub-basins. The transects used to cal- culate salt transport between sub-basins are represented by dashed yellow–black lines. The abbreviations for the sub-basins are as fol- lows: KAT (Kattegat), SBP (southern Baltic Proper), NBP (northern Baltic Proper), BOS (Bothnian Sea), BOB (Bay of Bothnia), GOF (Gulf of Finland), and GOR (Gulf of Riga). et al., 2010). The key issue of FWC calculations lies in how the reference salinity is deﬁned. The climatological range of salinity in the Baltic Sea varies from the freshwater condi- tions in the northern and eastern parts to the oceanic water conditions in the Kattegat. We follow the Boyer et al. (2007) formulation and calculate the climatological FWC from the three-dimensional temperature (Tref) and salinity (Sref) ﬁelds averaged over the period of 1993–2020. The other widely used formulation of FWC is as follows (e.g. Gustafsson and Stigebrandt, 1996): FWC = ρ (Sref,Tref,p) ρ (0,Tref,p) Sref −S Sref . (2) (2) Both formulations are derived from the concept of the mixing of two water masses with different salinities using conserva- tion of salt but have different mechanistic approaches, which are explained in detail in Appendix B. Both formulations are derived from the concept of the mixing of two water masses with different salinities using conserva- tion of salt but have different mechanistic approaches, which are explained in detail in Appendix B. The total volume of freshwater needed to dilute the water with salinity Sref to the salinity S, if S < Sref or should be removed to obtain water with the salinity S, if S > Sref is Figure 1. Map of the Baltic Sea depth distribution (data ref. 1, Ta- ble 1). Boxes indicate the boundaries used to calculate the fresh- water content for different sub-basins. The transects used to cal- culate salt transport between sub-basins are represented by dashed yellow–black lines. The abbreviations for the sub-basins are as fol- lows: KAT (Kattegat), SBP (southern Baltic Proper), NBP (northern Baltic Proper), BOS (Bothnian Sea), BOB (Bay of Bothnia), GOF (Gulf of Finland), and GOR (Gulf of Riga). FWC(t) = V FWC(x,y,z,t)dxdydz (m3 m−3). where dA is the area of each grid cell. where dA is the area of each grid cell. 2 Data and methods A vertical distribution of the FWC is calculated as A vertical distribution of the FWC is calculated as FWC(z,t) = A FWC(x,y,z,t)dxdy (m2 m−3), A vertical distribution of the FWC is calculated as FWC(z,t) = A FWC(x,y,z,t)dxdy (m2 m−3), Baltic Model Intercomparison Project (Gröger et al., 2022). The runoff to each sub-basin was calculated by summing the runoffs from each river discharging to the corresponding sub- basin (Fig. 1). The runoff data covered the period 1993–2018, and the anomalies were calculated relative to the same refer- ence period. and horizontal distribution of the FWC is calculated as FWC(x,y,t) = D FWC(x,y,z,t)dz (mm−3). and horizontal distribution of the FWC is calculated as FWC(x,y,t) = D FWC(x,y,z,t)dz (mm−3). V and A correspond to the volume and area of the Baltic Sea or its sub-region, as shown in Fig. 1. D corresponds to depth from surface to bottom at a speciﬁc location. Salt transport was estimated by calculating the salt ﬂux at the boundaries of each sub-basin (see Fig. 1 for the location of the transects). Daily salt transport through each transect was calculated as a salinity and perpendicular velocity prod- uct. The annual mean salt transports were calculated by av- eraging the transects of daily transports over each year and later integrated over the vertical and corresponding horizon- tal dimension. The positive direction is determined accord- ing to the estuarine transport deﬁnition; i.e. inﬂow is from the ocean and outﬂow is from the head of the estuary. The sea ice volume, Vi, is calculated from the same BALMFC CMEMS reanalysis product (data ref. 1, Table 1), based on the LIM3 model conﬁguration (Pemberton et al., 2017). The Vi is calculated for each model grid cell (x,y) using total ice thickness, Hi, and ice concentration, Ci: Vi (x,y,t) = Hi (x,y,t) × Ci (x,y,t) × dA(x,y), where dA is the area of each grid cell. U. Raudsepp et al.: Baltic Sea freshwater content This suggests that although temporal variability is opposite, the dynamics of FWC are linked over the Baltic Sea. The changes in the FWC of the entire Baltic Sea are in- ﬂuenced by the changes occurring in its sub-basins, resulting in a complex integrated effect. This is illustrated by the si- multaneous positive trend of FWC in the northern Baltic Sea and negative trend in the southern Baltic Sea (Fig. 2) as well as their negative correlation (Table 2). Hence, it is necessary to examine the changes in variables separately for each sub- basin. It is important to note that while the FWC, net precipi- tation, and river runoff for the entire Baltic Sea represent the sum of contributions from each sub-basin, the net salt ﬂux does not represent an integrated value. Horizontal transition of the trends from positive to neg- ative points to the three-dimensional structure of the FWC variability ﬁeld. Therefore, time–depth variations of the FWC in each sub-basin were calculated (Fig. 4). Vertically, in the whole Baltic Sea, FWC is the most variable in the halo- cline layer and beneath it (Fig. 4a). Vertical distribution of the trends shows the absence of the trend in the upper layer of 50 m but a strong negative trend within and below the halo- cline. Thus, the decrease in the FWC in the whole Baltic Sea is mostly contributed by the drop of the FWC below the up- per mixed layer. The variability as well as negative trends is strongest in the southern and the northern Baltic Proper (Fig. 4e, c). Moving further northward and eastward in the Baltic Sea, we can notice that the negative tendencies are only present in the deeper layer (deeper than 50 m) of the Gulf of Finland and the Bothnian Sea (Fig. 4f, d). On the other side, there is a strong positive trend of the FWC in the Bothnian Bay and in the upper 50 m layer of the Bothnian Sea (Fig. 4b, d). It is relevant to note that a positive tendency in the FWC is seen in the layer of 10–50 m in the Gulf of Finland (Fig. 4f). In the northern Baltic Proper, the trend is absent in the upper layer of 30 m but turns negative in the sur- face layer of the southern Baltic Proper. U. Raudsepp et al.: Baltic Sea freshwater content U. Raudsepp et al.: Baltic Sea freshwater content Supplement). Our aim is to examine the co-variability of these factors and the FWC response of both the entire Baltic Sea as well as its sub-basins. To accomplish this, we present a time series of detrended standardized yearly variables in Fig. 5. basins to neutral in the eastern sub-basins and to negative in the central and southern sub-basins (Fig. 2). The decrease in the FWC in the southern Baltic Proper (Fig. 2) contributes the most to the overall decreasing trend in the Baltic Sea. If we look at the continuous distribution of the trends in the Baltic Sea, we see opposite temporal regimes of the FWC in the Bothnian Bay and in the Baltic Proper, with the Bothnian Sea being the transition area (Fig. 3). Although there is no trend in the Gulf of Finland as a whole (Fig. 2), the eastern part has a small negative tendency, while the western part shows a small positive tendency. The shallow Gulf of Riga has a negligible trend. The trends vanish in the southwestern Baltic Sea and in the Kattegat area (Fig. 3). For the entire Baltic Sea, the study period can be divided into two sub-periods. From 1993 to 2003, i.e. the ﬁrst pe- riod, changes in the FWC of the Baltic Sea correlated with freshwater sources, namely net precipitation and river runoff (Fig. 5a). A prominent example is the period from 1997 to 2003. Meanwhile, the salt transport to the Baltic Sea re- mained relatively low. Since 2004, i.e. the second period, we have observed a coherent relationship between salt trans- port and the FWC in the Baltic Sea, which is difﬁcult to explain. We may speculate that an increase in net precipi- tation might trigger an increase in the FWC between 2007 and 2012, while river runoff has no effect. The spatial distribution of the trends indicates a possi- ble coherence of salinity dynamics in different sub-basins, which is checked further on by calculating correlation coefﬁ- cients between the basins. The correlation coefﬁcients calcu- lated pairwise between detrended FWC time series (Table 2) show a high positive value between the southern and northern Baltic Proper (R = 0.8) and between the Bothnian Bay and the Bothnian Sea (R = 0.6), while the correlation between the Bothnian Bay and southern and northern Baltic Proper is negative (R = −0.6). State Planet, 1-osr7, 7, 2023 3 Results Hourly precipitation and evaporation data have been ex- tracted from the ERA5 reanalysis (data ref. 2, Table 1) from the period of 1993–2020. Net precipitation was calculated by subtracting evaporation from precipitation. Thereafter, the net precipitation was interpolated onto a reanalysis model grid, and total net precipitation was estimated for the wet grid cells of each sub-basin (Fig. 1). The net precipitation anomalies were calculated relative to the period 1993–2020. Time series of the FWC and linear trends of the Baltic Sea and its sub-basins are presented in Fig. 2. Even if the calcu- lated trends are not statistically signiﬁcant, they provide in- formation about the tendency of FWC changes. The FWC of the Baltic Sea has a negative trend of −23.9 ± 0.7 km3 yr−1 (p < 10−3) superimposed by irregular decadal variations (Fig. 2a). The trends are variable over the whole Baltic Sea (Fig. 2). It changes sign from positive in the northern sub- The total runoff from the Baltic Sea rivers was estimated from the river discharge database (data ref. 3, Table 1) of the https://doi.org/10.5194/sp-1-osr7-7-2023 State Planet, 1-osr7, 7, 2023 CHAPTER2.5 CHAPTER2.5 4 U. Raudsepp et al.: Baltic Sea freshwater content In the Gulf of Riga, the variability is low, and the trends are negligible and do not show systematic vertical distribution. We speciﬁcally focus on two sub-basins: the southern Baltic Proper, which is characterized by saltwater domi- nance, and the Bothnian Bay, which is characterized by freshwater dominance. The variations in the southern Baltic Proper differ in several features from the variations in the whole Baltic Sea. The southern Baltic Proper exhibits two distinct periods (Fig. 5e), which is qualitatively similar to the entire Baltic Sea, but the periods do not coincide tem- porally. The ﬁrst period spans from 1993 to 2007, while the second period extends from 2008 until the end of recorded data. In the ﬁrst period, FWC, net precipitation, and river runoff show positive covariation, while the ﬂux exhibits a negative covariation pattern. In the second period, there is positive covariation between FWC and salt ﬂux and partially net precipitation, but the pattern is reversed for river runoff. In the Bothnian Bay, there is positive covariation between FWC, net precipitation, and river runoff but negative covari- ation with salt ﬂux (Fig. 5b). In the northern Baltic Proper and the Bothnian Sea, the main pattern is negative covaria- tion between FWC and salt ﬂux (Fig. 5c and d). Changes in net precipitation and river runoff generally support changes in FWC, but the variability pattern remains complex. FWC exhibits noticeable irregular decadal variations over time, as shown in Figs. 2 and 4. To understand the interan- nual variability of FWC, we calculated a time series of river runoff, net precipitation, and salt transport through the cross- sections between the sub-basins of the Baltic Sea (Fig. 1; The Gulf of Finland (Fig. 5f) and the Gulf of Riga (Fig. 5g) do not exhibit a well-deﬁned pattern in the variability of the variables. In the latter basin, the changes in FWC align with the changes in river runoff from 1993 to 2009 but not there- after. https://doi.org/10.5194/sp-1-osr7-7-2023 State Planet, 1-osr7, 7, 2023 CHAPTER2.5 U. Raudsepp et al.: Baltic Sea freshwater content 5 5 Figure 2. Freshwater content time series in the Baltic Sea (a) and in different sub-basins (b–h). The trend of FWC in the corresponding basin is shown in the upper-right corner (km3 yr−1, asterisk p > 0.05) and plotted using the blue line. U. Raudsepp et al.: Baltic Sea freshwater content The trends of FWC are present in our data, but this could be characteristic of the period of our study. An increase in the salinity in the central Baltic Proper since 1993 has been reported by Lehmann et al. (2022), which is consistent with our results about the decline of FWC. Going back further in time, the positive salinity trend becomes weaker (Lehmann et al., 2022) until it vanishes (Radtke et al., 2020). The study by Radtke et al. (2020) was prolonged into the past until 1850 but does not cover the last 15 years. In the southern Baltic Proper, FWC is low in winter and high in summer (Fig. 6e), while in the Gulf of Finland the situation is opposite (Fig. 6f). Similarly, net salt transport to these basins is opposite in time (Fig. 7c, d). The seasonal course of FWC is almost absent in the northern Baltic Proper (Fig. 6c) where the inﬂuence from adjacent sub-basins, the southern Baltic Proper and the Gulf of Finland, which have opposite FWC seasonality, could compensate for each other. In the Gulf of Riga, FWC is at its maximum in spring and decreases monotonically until winter (Fig. 6h). Surprisingly, the seasonal course of the FWC in the Kattegat (Fig. 6g) is like the seasonal course of the FWC in the Gulf of Riga (Fig. 6h). Dynamically, these two areas cannot be interlinked due to their geographical separation. y Many authors have reported dominant 30-year variability in mean, surface, and bottom salinity of the Baltic Sea as well as river runoff into the Baltic Sea and even salt transport across Darss sill (Kniebusch et al., 2019; Radtke et al., 2020; Lehmann et al., 2022). Our time series of 28 years are short to reveal 30-year variability in FWC, river runoff, or net precip- itation with a statistical signiﬁcance. Visual inspection of the time series of FWC does not hint at the presence of a 30-year cycle (Fig. 2). We admit the presence of decadal variability in the time series, which has also been reported by Lehmann et al. (2022). The trends and multi-scale variability of FWC are opposite in the Baltic Proper and in the Bothnian Bay (Fig. 2, Table 1). This raises the following question: if FWC has op- posite changes in the southern and northern Baltic Sea, how could changes in river runoff explain the opposite variabil- ity? U. Raudsepp et al.: Baltic Sea freshwater content Qualitatively, low https://doi.org/10.5194/sp-1-osr7-7-2023 State Planet, 1-osr7, 7, 2023 CHAPTER2.5 CHAPTER2.5 U. Raudsepp et al.: Baltic Sea freshwater content 6 Figure 3. Spatial distribution of depth normalized FWC trends, with the zero isoline highlighted in black. 2018 (Lehmann et al., 2022). The rate of 0.2–0.25 g kg−1 per decade was estimated for the period 1979–2018 (Lehmann et al., 2022). In the Baltic Sea, there was no trend in net precipitation, nor river runoff. Therefore, the decrease in FWC should be explained by accumulated salt ﬂux from the North Sea to the Baltic Sea due to the major Baltic inﬂows (Mohrholz, 2018), large barotropic inﬂows (Lehmann et al., 2017), and smaller inﬂows of barotropic origin (Lehmann et al., 2022). Our estimations of the annual salt transport to the southern Baltic Proper did not show an increase in the salt ﬂux (Supplement). In our reanalysis model data, the salt ﬂux at the Danish straits could have large uncertainty due to rel- atively high salinity errors there (Appendix A). Inside the Baltic Sea area, as reanalysis model data have been calcu- lated using assimilation of the observations, the estimates of FWC have low uncertainty due to low salinity errors (Ap- pendix A). Figure 3. Spatial distribution of depth normalized FWC trends, with the zero isoline highlighted in black. Net precipitation and river runoff increased over the Both- nian Bay (Supplement), which contributed to the increase in the FWC (Fig. 2b). An increase in FWC was fastest at the sur- face of the bay and monotonically slowed down with depth (Fig. 4b). We would like to note that there are no studies that support or refute our ﬁndings on the increase in the FWC or the decrease in salinity in the Gulf of Bothnia. spring and summer. Indeed, net precipitation is low in that period. The Gulf of Bothnia has low FWC in winter and early spring and high FWC in summer and autumn (Fig. 6b, d). The seasonal course is more pronounced in the Bothnian Bay (Fig. 6b) than in the Bothnian Sea (Fig. 6d). In the Gulf of Bothnia, the decrease in FWC in winter could be associ- ated with the freezing of seawater (Fig. 7f). An increase in the FWC in the Bothnian Bay in spring coincides with the melting of sea ice (Fig. 7f), high river runoff (Fig. 7a), and negative salt transport (Fig. 7e). U. Raudsepp et al.: Baltic Sea freshwater content The abbreviations for the sub-basins are as follows: BS (Baltic Sea), NBP (northern Baltic Proper), SBP (southern Baltic Proper), KAT (Kattegat), BOB (Bay of Bothnia), BOS (Bothnian Sea), GOF (Gulf of Finland), and GOR (Gulf of Riga) (data ref. 1, Table 1). Figure 2. Freshwater content time series in the Baltic Sea (a) and in different sub-basins (b–h). The trend of FWC in the corresponding basin is shown in the upper-right corner (km3 yr−1, asterisk p > 0.05) and plotted using the blue line. The abbreviations for the sub-basins are as follows: BS (Baltic Sea), NBP (northern Baltic Proper), SBP (southern Baltic Proper), KAT (Kattegat), BOB (Bay of Bothnia), BOS (Bothnian Sea), GOF (Gulf of Finland), and GOR (Gulf of Riga) (data ref. 1, Table 1). Table 2. Correlation table of the FWC between the sub-basins (Fig. 1) of the Baltic Sea (data ref. 1, Table 1). The abbreviations for the sub-basins are as follows: KAT (Kattegat), SBP (southern Baltic Proper), NBP (northern Baltic Proper), BOS (Bothnian Sea), BOB (Bay of Bothnia), GOF (Gulf of Finland), and GOR (Gulf of Riga). Table 2. Correlation table of the FWC between the sub-basins (Fig. 1) of the Baltic Sea (data ref. 1, Table 1). The abbreviations for the sub-basins are as follows: KAT (Kattegat), SBP (southern Baltic Proper), NBP (northern Baltic Proper), BOS (Bothnian Sea), BOB (Bay of Bothnia), GOF (Gulf of Finland), and GOR (Gulf of Riga). BS BOB BOS GOF GOR KAT NBP SBP BS 1.00 BOB −0.38 1.00 BOS 0.06 0.57 1.00 GOF 0.11 0.01 −0.08 1.00 GOR 0.13 −0.03 −0.07 −0.09 1.00 KAT 0.42 −0.04 −0.03 −0.10 0.30 1.00 NBP 0.78 −0.64 −0.22 0.28 −0.17 0.01 1.00 SBP 0.88 −0.65 −0.25 −0.08 0.13 0.21 0.79 1.00 FWC could be explained by high salt transport in autumn and winter (Fig. 7c) accompanied by low river runoff (Fig. 7a), but this is interfered with by high precipitation in autumn (Fig. 7b). Contrarily, the high FWC could be explained by high river runoff and low or even negative salt transport in The seasonal dynamics of FWC further emphasize the ef- fect of freshwater discharge in the northern basins and salt transport in the southern basins of the Baltic Sea (Fig. 6). In the whole Baltic Sea, FWC is low in autumn and winter but high in spring and summer (Fig. 6a). U. Raudsepp et al.: Baltic Sea freshwater content Our analysis showed that there are multi-year periods when river runoff is in phase or out of phase with the FWC. An example of an in-phase period in the whole Baltic Sea is 1993–2009 and an out-of-phase period 2010–2018 (Fig. 5a). In the Bothnian Bay, we mostly have river runoff and the FWC in phase and out of phase with salt transport. State Planet, 1-osr7, 7, 2023 4 Discussion A distinct feature of the Baltic Sea salinity evolution over the period 1993–2021 was the decreasing trend of the FWC in the southern Baltic Proper and increasing trend in the Both- nian Bay (Fig. 2). Overall, the FWC in the whole Baltic Sea had a statistically signiﬁcant negative trend (Fig. 2). Salt transport to the Baltic Sea, net precipitation, and total river runoff to the sea (Supplement) did not explain the calcu- lated trends. No steady increase in the salt transport to the Baltic Sea has been reported elsewhere, although deepwa- ter salinity has increased in the Gotland Basin from 1993 to https://doi.org/10.5194/sp-1-osr7-7-2023 State Planet, 1-osr7, 7, 2023 CHAPTER2.5 U. Raudsepp et al.: Baltic Sea freshwater content 7 Figure 4. Vertical distribution of the horizontal mean FWC anomaly and corresponding trends for each Baltic Sea sub-basin. Trends with statistical signiﬁcance less than 5 % (p value < 0.05) are shown as grey line segments. The abbreviations for the sub-basins are as follows: BS (Baltic Sea), NBP (northern Baltic Proper), SBP (southern Baltic Proper), KAT (Kattegat), BOB (Bay of Bothnia), BOS (Bothnian Sea), GOF (Gulf of Finland), and GOR (Gulf of Riga) (data ref. 1, Table 1). Figure 4. Vertical distribution of the horizontal mean FWC anomaly and corresponding trends for each Baltic Sea sub-basin. Trends with statistical signiﬁcance less than 5 % (p value < 0.05) are shown as grey line segments. The abbreviations for the sub-basins are as follows: BS (Baltic Sea), NBP (northern Baltic Proper), SBP (southern Baltic Proper), KAT (Kattegat), BOB (Bay of Bothnia), BOS (Bothnian Sea), GOF (Gulf of Finland), and GOR (Gulf of Riga) (data ref. 1, Table 1). Figure 5. Normalized time series of detrended annual mean FWC (black asterisk), runoff, net precipitation, and net salt ﬂuxes. The abbrevi- ations for the sub-basins are as follows: BS (Baltic Sea), NBP (northern Baltic Proper), SBP (southern Baltic Proper), BOB (Bay of Bothnia), BOS (Bothnian Sea), GOF (Gulf of Finland), and GOR (Gulf of Riga) (data ref. 1, 2, 3, 1; Table 1). Figure 5. Normalized time series of detrended annual mean FWC (black asterisk), runoff, net precipitation, and net salt ﬂuxes. The abbrevi- ations for the sub-basins are as follows: BS (Baltic Sea), NBP (northern Baltic Proper), SBP (southern Baltic Proper), BOB (Bay of Bothnia), BOS (Bothnian Sea), GOF (Gulf of Finland), and GOR (Gulf of Riga) (data ref. 1, 2, 3, 1; Table 1). U. Raudsepp et al.: Baltic Sea freshwater content U. Raudsepp et al.: Baltic Sea freshwater content Future climate model scenarios could provide insight into how combinations of different factors affect long-term salin- ity changes in the Baltic Sea, although the uncertainties in the climate projections are high. Three main factors that affect salinity and FWC are the wind ﬁelds over the Baltic Sea re- gion (Lass and Matthäus, 1996), river runoff to the Baltic Sea (Schinke and Matthäus, 1998), and global mean sea level rise (Meier et al., 2017, 2021). With increasing precipitation and river runoff, the salinity in the Baltic Sea decreases (Saraiva et al., 2019). Mean sea level rise, in turn, tends to increase salinity because saltwater imports through the Danish straits are larger (Meier et al., 2017, 2021). An increasing westerly wind could block the freshwater ﬂow out of the Baltic Sea, causing reduced salt transport to the Baltic Sea (Meier and Kauker, 2003), but Schimanke et al. (2014) showed that the intensity and frequency of MBIs were projected to slightly increase due to changes in the wind ﬁelds. In summary, dif- ferent factors affect salinity and FWC differently, so the ob- served effect cannot be explained in a simple way. For in- stance, Meier et al. (2021) stated that no changes in the Baltic Sea salinity were found because river runoff and sea level rise approximately compensated each other. Therefore, also in our study, we could not explain the trends and multidecadal variations, although we have considered the main factors that affect the FWC of the Baltic Sea. through the sections that border the corresponding sub-basin. Seasonal changes of sea ice volume affect the seasonal cycle of the FWC in the Gulf of Bothnia. By taking into consideration the spatial and temporal ten- dencies of the FWC shown in each separate sub-basin, we can characterize the Baltic Sea as a typical estuarine sys- tem with a strengthening exchange ﬂow in time. Geograph- ically, the system spans from the Danish straits in the south to the Bothnian Bay in the north. The southern part corre- sponds to the estuary mouth, where saltwater transport from the ocean prevails and leads to a decrease in FWC. At the other end, the Bothnian Bay is a typical estuary head char- acterized by a signiﬁcant inﬂuence of freshwater discharge, resulting in an increase in FWC over time. 5 Conclusion We utilize a clustering method to assess the accuracy of the hydrodynamic model. This method provides insights into the overall model accuracy by clustering the errors. The cluster- ing process employs the K-means algorithm, which is a form of unsupervised machine learning (Jain, 2010). The original description of this method can be found in the work of Raud- sepp and Maljutenko (2022). In our assessment, all available data within the model domain and simulation period are in- cluded, even if the veriﬁcation data are unevenly distributed or occasionally sparse. This approach allows us to evaluate the model quality at each speciﬁc location and time instance where measurements have been obtained. Temporal variability of the FWC at different timescales has an opposite pattern in the northern and southern sub-basins of the Baltic Sea. The Gulf of Bothnia shows positive tenden- cies of FWC, while in the Baltic Proper, negative tendencies can be witnessed. The total FWC of the Baltic Sea has de- creased steadily with a rate of 23.9 km3 yr−1 over the years 1993–2021. There is no solid explanation for what caused it because different drivers could compensate for each other to a certain extent. Interannual variations of the FWC in the Bothnian Bay are supported by interannual variability of river runoff, net precipitation, and salt transport from the Bothnian Sea, the latter being opposite to the changes of FWC. In the Both- nian Sea and northern Baltic Proper, interannual variations of the FWC and net salt ﬂux to the basins are opposite to each other. The changes in river runoff and net precipitation have a complex contribution to the changes of FWC. In the southern Baltic Proper, the changes of FWC, river runoff, net precipi- tation, and net salt ﬂux have rather complex relationships. A separate study is needed to understand the interplay of these factors, especially seeing as the variations of salt transport and FWC are opposite to each other from 1993 until 2010 but positively correlated thereafter. The ﬁrst step of the method is the formation of a two- dimensional error space of two simultaneously measured pa- rameters. A two-dimensional error space (dS,dT ), where dS = (Smod−Sobs) and dT = (Tmod−Tobs), of simultaneously measured temperature and salinity values was formed as the basis for the clustering. The dataset utilized in this validation study was obtained from the EMODnet dataset compiled by SMHI (SMHI, 2023; data ref. U. Raudsepp et al.: Baltic Sea freshwater content The northern Baltic Proper and the Bothnian Sea converge in the transi- tional zone between the saltwater-dominated region and the freshwater-dominated region. In terms of vertical distribu- tion, the freshwater-inﬂuenced area extends towards the estu- ary mouth in the upper layer, while the saltwater-dominated area extends towards the estuary head in the lower layer. Moving eastward, the Gulf of Finland represents a branch of the main estuarine system and shares general characteris- tics of a transition zone, although it possesses its own unique estuarine structure and dynamics (Maljutenko and Raudsepp, 2019; Westerlund et al., 2019; Liblik et al., 2018). 4 Discussion https://doi.org/10.5194/sp-1-osr7-7-2023 State Planet, 1-osr7, 7, 2023 Figure 5. Normalized time series of detrended annual mean FWC (black asterisk), runoff, net precipitation, and net salt ﬂuxes. The abbrevi- ations for the sub basins are as follows: BS (Baltic Sea) NBP (northern Baltic Proper) SBP (southern Baltic Proper) BOB (Bay of Bothnia) Figure 5. Normalized time series of detrended annual mean FWC (black asterisk), runoff, net precipitation, and net salt ﬂuxes. The abbrevi- ations for the sub-basins are as follows: BS (Baltic Sea), NBP (northern Baltic Proper), SBP (southern Baltic Proper), BOB (Bay of Bothnia), BOS (Bothnian Sea), GOF (Gulf of Finland), and GOR (Gulf of Riga) (data ref. 1, 2, 3, 1; Table 1). State Planet, 1-osr7, 7, 2023 https://doi.org/10.5194/sp-1-osr7-7-2023 CHAPTER2.5 CHAPTER2.5 CHAPTER2.5 8 U. Raudsepp et al.: Baltic Sea freshwater content Figure 6. Seasonality of detrended FWC in the different Baltic Sea sub-basins. The abbreviations for the sub-basins are as follows: BS (Baltic Sea), NBP (northern Baltic Proper), SBP (southern Baltic Proper), KAT (Kattegat), BOB (Bay of Bothnia), BOS (Bothnian Sea), GOF (Gulf of Finland), and GOR (Gulf of Riga) (data ref. 1, Table 1). Figure 6. Seasonality of detrended FWC in the different Baltic Sea sub-basins. The abbreviations for the sub-basins are as follows: BS (Baltic Sea), NBP (northern Baltic Proper), SBP (southern Baltic Proper), KAT (Kattegat), BOB (Bay of Bothnia), BOS (Bothnian Sea), GOF (Gulf of Finland), and GOR (Gulf of Riga) (data ref. 1, Table 1). Figure 7. Seasonality of runoff and net precipitation over the Baltic Sea (BS) (a, b); salt transport through the western boundary of the southern Baltic Proper (SBP) (c), the Gulf of Finland (GOF) (d), and the southern boundary of the Bothnian Bay (BOB) (e); and the sea ice volume in the Bothnian Bay (f). Figure 7. Seasonality of runoff and net precipitation over the Baltic Sea (BS) (a, b); salt transport through the western boundary of the southern Baltic Proper (SBP) (c), the Gulf of Finland (GOF) (d), and the southern boundary of the Bothnian Bay (BOB) (e); and the sea ice volume in the Bothnian Bay (f). https://doi.org/10.5194/sp-1-osr7-7-2023 State Planet, 1-osr7, 7, 2023 CHAPTER2.5 9 https://doi.org/10.5194/sp-1-osr7-7-2023 5 Conclusion 4, Table 1). It comprises a total of 651 565 observations that align with the CMEMS reanaly- sis simulation period, encompassing the years 1993 to 2021. We extracted the nearest model values from the reanalysis dataset for each observation. The seasonal course of FWC in different sub-basins high- lights the local dynamics and explains the FWC dynamics in relation to the local sources of freshwater and salt transport The second step is the selection of the number of clusters. For simplicity, we preselected ﬁve clusters. The third step https://doi.org/10.5194/sp-1-osr7-7-2023 State Planet, 1-osr7, 7, 2023 CHAPTER2.5 CHAPTER2.5 CHAPTER2.5 10 U. Raudsepp et al.: Baltic Sea freshwater content Figure A1. Distribution of normalized error clusters for k = 5 (a). The spatial distribution (b, shaded sub-plots), vertical distribution (c), temporal distribution (d), and seasonal distribution (e) of the share of error points belonging to the ﬁve different clusters. Figure A1. Distribution of normalized error clusters for k = 5 (a). The spatial distribution (b, shaded sub-plots), vertical distribution (c), temporal distribution (d), and seasonal distribution (e) of the share of error points belonging to the ﬁve different clusters. Table A1. The share (%), bias, root-mean-square error (RMSE), standard deviation (SD), and correlation coefﬁcient (Corr) for each of the ﬁve clusters. Shares Bias SD RMSE Corr k % dS dT S T S T S T dSdT (g kg−1) (◦C) (g kg−1) (◦C) (g kg−1) (◦C) 1 2.8 −3.201 −0.169 1.763 1.250 3.654 1.261 0.950 0.721 −0.155 2 5.5 2.379 1.140 1.164 1.508 2.649 1.891 0.983 0.626 0.206 3 8.0 0.137 2.567 0.621 1.465 0.636 2.955 0.994 0.637 0.030 4 6.7 −0.026 −2.859 0.587 1.630 0.587 3.291 0.985 0.693 −0.006 5 77.0 −0.029 0.006 0.412 0.549 0.413 0.549 0.994 0.907 0.113 tistical metrics of non-normalized clustered errors. Common statistics, like SD, RMSE, and correlation coefﬁcient, can be calculated for the parameters belonging to each cluster. is to perform a K-means clustering of the 2-dimensional er- rors. The clustering is applied to the normalized errors. Nor- malization was done for temperature and salinity errors sepa- rately using corresponding standard deviations of the errors. The K-means algorithm ﬁnds the location of the centroids of a predeﬁned number of clusters in the error space. The location of the centroids represents the bias of the set of er- rors for each cluster. 5 Conclusion The fourth step is the calculation of sta- The ﬁfth step is the analysis of spatio-temporal distribu- tions of the errors belonging to different clusters. In the for- mation of the error space, we retained the coordinates of each error point (dS,dT )(x,y), which enables us to map the errors belonging to each cluster back to the location where the mea- CHAPTER2.5 State Planet, 1-osr7, 7, 2023 https://doi.org/10.5194/sp-1-osr7-7-2023 State Planet, 1-osr7, 7, 2023 https://doi.org/10.5194/sp-1-osr7-7-2023 CHAPTER2.5 U. Raudsepp et al.: Baltic Sea freshwater content 11 surements were performed. In order to do that, the model do- main is divided into horizontal grid cells (i,j) of 1 × 1 km2 in size. Subsequently, the number of error points belonging to different clusters at each grid cell (i,j) is counted. The total number of error points belonging to the grid cell (i,j) is the sum of the points of each cluster. The share of error points in each grid cell belonging to cluster k is the ratio of the number of error points of cluster k and the total number of error points in each grid cell. S will be (Vref + Vf). If S > Sref, then Eq. (B3) shows how much freshwater should be removed from the volume of wa- ter (Vref) with the salinity Sref to obtain water with the salinity S. In this case, the volume of resulting water with S will be (Vref −Vf). We would like to note that the relationship (B3) is not a lin- ear relationship between salinity and Vf. There are straight- forward conclusions that can be drawn from Eq. (B3). First, to obtain water with a salinity close to zero, an inﬁnite amount of freshwater is needed, independent of the Sref, Figure A1 displays the results of the K-means clustering for non-normalized errors. Table A1 presents the correspond- ing metrics. Within cluster k = 5, the salinity and tempera- ture values closely align with the observations, with a bias of dS = −0.03 g kg−1 and dT = 0.006 ◦C, respectively. This cluster encompasses 77 % of all data points. The points are distributed throughout the entire Baltic Sea, with a dominant share exceeding 0.5 (Fig. A1b). Clusters k = 3 and k = 4 ex- hibit relatively even spatial distributions over the Baltic Sea, accounting for 8 % and 7 % of the points, respectively. Appendix B The mixing of two water masses (Mref, Mf) with different salinities (Sref, Sf) results in the mixture (Mref + Mf) with unknown salinity S: In the natural system, water volume and salinity are con- served when volume and salt exchange with the outside sys- tem is allowed. First, we assume that the natural system is initially ﬁlled with water Vref with salinity Sref. Then, if at some time instance we observe that water salinity has de- creased to S, so that S < Sref, then Vf was added to the sys- tem either by river runoff or net precipitation. (Without losing generality, we neglect the presence of ice in the system and variations of the water volume of the system.) At the same time, to ﬁx the volume of the natural system, the amount of water Vf with salinity S must be removed from the system due to outﬂow through the open boundary, for instance. If water salinity has increased to S, so that S > Sref, then the amount of freshwater Vf has been removed from the system either by evaporation or outﬂow through the open boundary. Simultaneously, to conserve the volume of water in the nat- ural system, the amount of water Vf with salinity S must be transported to the system due to inﬂow from the open bound- ary. We would like to note that in the current argumentation, the actual inﬂow/outﬂow volume and salt transport through the open boundary cannot be estimated from knowing the Vf. MrefSref + MfSf = (Mref + Mf)S. (B1) (B1) If one of the water masses, Mf, is freshwater with Sf = 0, and we assume that the densities of the water masses of different salinities have negligible difference, then Eq. (B1) simpliﬁes to If one of the water masses, Mf, is freshwater with Sf = 0, and we assume that the densities of the water masses of different salinities have negligible difference, then Eq. (B1) simpliﬁes to VrefSref = VrefS + VfS, (B2) (B2) where Vref and Vf are the volumes of reference water and freshwater, respectively. The formulation of Boyer et al. (2007) for freshwater content (FWC) follows directly from Eq. (B2), where the volume of freshwater in the total volume of the mixture is Vf = Vref Sref −S S . (B3) (B3) In the derivation of Eq. 5 Conclusion These clusters are particularly noteworthy due to their small salin- ity biases and variability, which are crucial for estimating the freshwater content (FWC) directly impacted by salinity. Collectively, approximately 92 % of all validation points ex- hibit relatively low salinity bias, standard deviation (SD), and root-mean-square error (RMSE) (Table A1). Consequently, we anticipate that the model reanalysis data provide sufﬁ- ciently accurate information for calculating the FWC of the Baltic Sea. lim S→0Vf = ∞. Second, no freshwater is needed to add to the water Vref if the mixture has a salinity equal to Sref. Third, if Second, no freshwater is needed to add to the water Vref if the mixture has a salinity equal to Sref. Third, if lim S→∞Vf = −Vref, then all water should be removed from the mixture. In its practical application, the formulation (Eq. B3) means that a ﬁxed volume equal to Vref was initially ﬁlled with the water of salinity Sref. Then, Vf is the volume of freshwater that was needed to dilute the water to the observed salinity S. In this case, the volume of the mixture (Vref+Vf) with salinity S is larger than the initial volume Vref. This means that the amount of water in the mixture, Vf, with salinity S should be removed from the system after the mixing is complete. If S > Sref, then Vf < 0, and this is the volume of freshwater that was removed from the system to obtain water with the salinity S. In such a case, the volume of the mixture (Vref − Vf) with salinity S is smaller than the initial volume Vref. This means that the amount of water in the mixture, Vf with salinity S, should be added to the system. https://doi.org/10.5194/sp-1-osr7-7-2023 (B5) Competing interests. The contact author has declared that none of the authors has any competing interests. The relationship between salinity and Vf is linear in Eq. (B5). Now some conclusions will be drawn from Eq. (B5). First, if the salinity is S = 0, then Vf = V ; i.e. all water in the mixture is freshwater. Second, if S = Sref, then Vf = 0; i.e. there is no freshwater in the mixture. If S > Sref, then we obtain that Vf < 0, which means that a fraction of the water in the mix- ture becomes negative. Therefore, in its initial applications (Gustafsson and Stigebrandt, 1996; Eilola and Stigebrandt, 1998; Winsor et al., 2001), the relationship (B5) is bounded to zero when S > Sref. The argumentation about the dynam- ics of the freshwater content is not applicable to the formu- lation by Gustafsson and Stigebrandt (1996). In the applica- bility range S ≤Sref, Eq. (B5) can be interpreted as follows: initially, a ﬁxed volume, V , is ﬁlled in with the water with salinity Sref, and, then, the amount of water Vf with salin- ity Sref is removed from the ﬁxed volume and replaced with freshwater with volume Vf to obtain the mixture with salinity S. Disclaimer. The Copernicus Marine Service offering is regularly updated to ensure it remains at the forefront of user requirements. In this process, some products may undergo replacement or renaming, leading to the removal of certain product IDs from our catalogue. If you have any questions or require assistance regarding these modiﬁcations, please feel free to reach out to our user support team for further guidance. They will be able to provide you with the necessary information to address your concerns and ﬁnd suitable alternatives, maintaining our commitment to delivering top-quality services. Publisher’s note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Review statement. This paper was edited by Pierre Brasseur and reviewed by three anonymous referees. Technically, Eq. (B5) can also be used for the calculation of Vf if S > Sref. We have calculated FWC for the whole Baltic Sea using Eqs. (B3) and (B5) (Fig. B1). The difference of the calculated FWC of the Baltic Sea is around 100 km3 and rarely exceeds 200 km3, while the FWC anomaly of the Baltic Sea varies in the range of ±1000 km3. (B5) The esti- mates used by either formulation are between 10 %–20 %. We would like to point out that FWC calculated by Eq. (B3) (Boyer et al., 2007) is always larger than FWC calculated by Eq. (B5) (Gustafsson and Stigebrandt, 1996): Appendix B (B2), we can express FWC as Vf = V (Sref −S) Sref . (B5) (B5) Appendix B (B3), the volume of the mixture of two water masses is not limited. The formulation (B3) answers the question of how much freshwater (Vf) is needed to dilute the water (Vref) with salin- ity Sref to the salinity S if S < Sref. The volume of water with The derivation of the formulation by Gustafsson and Stige- brandt (1996) is based on the conservation of salt in mixing State Planet, 1-osr7, 7, 2023 State Planet, 1-osr7, 7, 2023 https://doi.org/10.5194/sp-1-osr7-7-2023 CHAPTER2.5 CHAPTER2.5 12 U. Raudsepp et al.: Baltic Sea freshwater content U. Raudsepp et al.: Baltic Sea freshwater content U. Raudsepp et al.: Baltic Sea freshwater content Figure B1. Time series of FWC in the Baltic Sea as calculated according to Eq. (B3) (black line, 1) and Eq. (B5) (brown line, 2) and difference between Eqs. (B5) and (B3) (blue). From Eq. (B6) we can see that the difference in FWC of two formulations increases with the increase in the difference be- tween reference water salinity and the salinity of the mixture. Data availability. This study is based on public databases and the references are listed in Table 1. Supplement. The supplement related to this article is available online at: https://doi.org/10.5194/sp-1-osr7-7-2023-supplement. Figure B1. Time series of FWC in the Baltic Sea as calculated according to Eq. (B3) (black line, 1) and Eq. (B5) (brown line, 2) and difference between Eqs. (B5) and (B3) (blue). Author contributions. In the study, UR was responsible for con- ceptualizing the research, developing the methodology, conducting the investigation, performing formal analysis, and writing the orig- inal draft of the paper. IM carried out formal analyses, conducted data curation, and handled data visualization, while also participat- ing in the initial drafting of the manuscript. AB was involved in formal analysis, investigation, and data visualization. RU and PL obtained funding and managed project administration tasks. All au- thors collaborated in the review and editing of the manuscript. of two water masses; i.e. Eqs. (B1) and (B2) are valid. Addi- tionally, the assumption behind their formulation is that the water volume of the mixture, V = Vf + Vref, is known, while Vref is an unknown volume. Thus, in addition to Eq. (B2), the unknown volume can be expressed as Vref = V −Vf. (B4) Vref = V −Vf. (B4) Substituting Eq. (B4) into Eq. (B2), we can express FWC as Substituting Eq. (B4) into Eq. Axell, L.: EU Copernicus Marine Service Product User Man- ual for Baltic Sea Physical Reanalysis Product, BALTIC- SEA_REANALYSIS_PHY_003_011, Issue 2.1, Mercator Ocean International, https://doi.org/10.5281/zenodo.7935113, 2021. Boyer, T., Levitus, S., Antonov, J., Locarnini, R., Mishonov, A., Garcia, H., and Josey, S. A.: Changes in freshwater content in References Axell, L.: EU Copernicus Marine Service Product User Man- ual for Baltic Sea Physical Reanalysis Product, BALTIC- SEA_REANALYSIS_PHY_003_011, Issue 2.1, Mercator Ocean International, https://doi.org/10.5281/zenodo.7935113, 2021. 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Sci., 9, 30, https://doi.org/10.1186/s40645- 022-00489-0, 2022. https://doi.org/10.5194/sp-1-osr7-7-2023 State Planet, 1-osr7, 7, 2023 High-frequency radar-derived coastal upwelling index ition of the Copernicus Ocean State Report (OSR7) Pablo Lorente1, Anna Rubio2, Emma Reyes3, Lohitzune Solabarrieta2, Silvia Piedracoba4, Joaquín Tintoré3,5, and Julien Mader2 1Puertos del Estado, Madrid, 28042, Spain 2AZTI Marine Research, Basque Research and Technology Alliance (BRTA), Pasaia, 20110, Spain 3Balearic Islands Coastal Ocean Observing and Forecasting System (SOCIB), Palma, 07122, Spain 4CETMAR (Centro Tecnológico del Mar), Vigo, 36208, Spain 5Mediterranean Institute for Advanced Studies (IMEDEA), Esporles, 07190, Spain Pablo Lorente1, Anna Rubio2, Emma Reyes3, Lohitzune Solabarrieta2, Silvia Piedracoba4, Joaquín Tintoré3,5, and Julien Mader2 1Puertos del Estado, Madrid, 28042, Spain 2AZTI Marine Research, Basque Research and Technology Alliance (BRTA), Pasaia, 20110, Spain 3Balearic Islands Coastal Ocean Observing and Forecasting System (SOCIB), Palma, 07122, Spain 4CETMAR (Centro Tecnológico del Mar), Vigo, 36208, Spain 5Mediterranean Institute for Advanced Studies (IMEDEA), Esporles, 07190, Spain rnicus Ocean State Report (OSR7) Correspondence: Pablo Lorente (plorente@puertos.es) Received: 30 July 2022 – Discussion started: 30 September 2022 Revised: 24 March 2023 – Accepted: 30 May 2023 – Published: 27 September 2023 Received: 30 July 2022 – Discussion started: 30 September 2022 Revised: 24 March 2023 – Accepted: 30 May 2023 – Published: 27 September 2023 an State Report (OSR7) Abstract. Coastal upwelling has been extensively studied since it plays a critical role in the connectivity be- tween offshore waters and coastal ecosystems, which has impacts on water quality, ﬁsheries, and aquaculture production. Signiﬁcant efforts have been devoted to the quantiﬁcation of the intensity, duration, and variability of this phenomenon by means of coastal upwelling indexes (CUIs), derived from wind, sea level pressure, or sea surface temperature data. Although valuable, ﬁrst-order descriptors, such classical indexes have been reported to present some limitations. As one of the major shortcomings is the omission of the direct inﬂuence of ocean cir- culation, this work introduces a novel CUI, generated from remotely sensed hourly surface current observations provided by a high-frequency radar (HFR). The consistency of the proposed index (CUI-HFR) is assessed in two different oceanographic areas during two distinct time periods: in the north-western Iberian (NWI) peninsula for 2021 and in the Bay of Biscay (BOB) for 2014. To this aim, CUI-HFR is compared against a traditional CUI based on hourly wind observations (CUI-WIND) provided by two buoys. Likewise, the skill of CUI-HFR to identify upwelling and downwelling processes is also qualitatively evaluated. High-frequency radar-derived coastal upwelling index Complementarily, the prognostic capabilities of the GLOBAL analysis and forecasting system to accurately reproduce upwelling and downwelling events in the NWI area are also analysed (CUI-GLOBAL). Results obtained in these two pilot areas revealed the following: (i) noticeable agreement between CUI-HFR and CUI-WIND, with correlation coefﬁcients above 0.67; and (ii) a proven ability of CUI-HFR and CUI-GLOBAL to categorize a variety of upwelling and downwelling episodes, which highlights their potential applicability for direct upwelling monitoring over any coastal area of the global ocean. Published by Copernicus Publications. CHAPTER2.5 CHAPTER2.5 Chapter 2.6 – 7th edition of the Copernicus Ocean State Report (OSR7) Chapter 2.6 – 7th edition of the Copernicus Ocean State Planet, 1-osr7, 8, 2023 https://doi.org/10.5194/sp-1-osr7-8-2023 © Author(s) 2023. This work is distributed under the Creative Commons Attribution 4.0 License. ter 2.6 – 7th edition of the Copernicus Ocean State Report (O 1 Introduction sensing instrument is high-frequency radar – HFR (Roarty et al., 2019). HFR networks have become an essential com- ponent of ocean observatories as they collect, in near-real time, ﬁne-resolution maps of the upper-layer ﬂow over broad coastal areas, providing a dynamical framework for other tra- ditional in situ observation platforms (Lorente et al., 2022; Rubio et al., 2017). HFR-derived surface circulation is a reliable source of information for search-and-rescue oper- ations and oil spill tracking, among other practical appli- cations (Reyes et al., 2022; Roarty et al., 2019; Rubio et al., 2017). Equally, it can be used for a detailed investiga- Since coastal waters encompass many unique habitats and support a wide range of anthropogenic activities (tourism, transportation, ﬁsheries, etc.), accurate monitoring is re- quired for efﬁcient marine resources management, the preservation of vulnerable ecosystems, and the sustained development of the so-called blue economy (Trincardi et al., 2020). Cutting-edge technology that has been steadily gaining worldwide recognition as an effective shore-based remote P. Lorente et al.: High-frequency radar-derived coastal upwelling index De- spite being valuable ﬁrst-order descriptors, such classical in- dexes have been reported to provide an incomplete picture of coastal UPW due to a number of limitations, encompass- ing uncertainties related to the estimation of wind stress or derived from the coarse spatio-temporal resolution of the at- mospheric pressure ﬁelds used (Jacox et al., 2018). As one of the major shortcomings is the omission of the direct inﬂuence of ocean circulation, the present work in- tends to ﬁll this gap by introducing a novel CUI, gener- ated from HFR-derived hourly surface current observations (CUI-HFR). Although such an ocean-based index was ﬁrstly presented in Lorente et al. (2020) as a proof-of-concept in- vestigation, here we gain further insight into the credibil- ity of the proposed approach by analysing two different re- gions (Fig. 1a), the north-western Iberian (NWI) peninsula upwelling system and the Bay of Biscay (BOB), for distinct time periods (2021 and 2014, respectively). Nowadays, there is an emerging question about the im- pact of anthropogenic pressures on future changes in coastal UPW ecosystems and the potential implications for biodi- versity conservation (IPCC, 2022; Abrahams et al., 2021a; Xiu et al., 2018; Bakun et al., 2015; Cropper et al., 2014; Di Lorenzo, 2015). It has been hypothesized that climate change could promote greater land–sea temperature gradients to- gether with stronger alongshore winds, eventually resulting in intensiﬁed wind-driven coastal UPW (Di Lorenzo, 2015; Varela et al., 2015; Barton et al., 2013). In Wang et al. (2015), results from an ensemble of climate models showed relevant changes in the timing, duration, intensity, and spatial hetero- geneity of coastal UPW in response to future warming in most eastern boundary upwelling systems. p p y In the NWI region, which extends from 40 to 44◦N (Fig. 1a), the ocean dynamics are regulated by the relative changes in the strength and position of the Azores high- pressure system with respect to the Iceland low-pressure sys- tem, deﬁning two largely wind-driven oceanographic sea- sons (Lorente et al., 2020). The UPW season is predominant from March to October when northerly winds prevail and in- duce a south-westward surface ﬂow. The upward ﬂux of cold deep waters supplies nutrients to the euphotic zone, result- ing in an elongated alongshore strip of maximum concentra- tion of chlorophyll (Fig. 1b). P. Lorente et al.: High-frequency radar-derived coastal upwelling index 2 Table 1. Products from the Copernicus Marine Service used in this study, including the Product User Manual (PUM) and QUality Information Document (QUID). Last access for all web pages cited in this table: 2 June 2023. Product Product ID and type Data access Documentation ref. no. 1 INSITU_GLO_PHY_UV_DISCRETE_NRT_013_048, EU Copernicus Marine PUM: Verbrugge et al. (2022a); in situ observations Service Product (2022a) QUID: Verbrugge et al. (2022b) 2 INSITU_GLO_PHY_UV_DISCRETE_MY_013_044, EU Copernicus Marine PUM: Etienne et al. (2022); in situ observations Service Product (2023a) QUID: Etienne et al. (2023) 3 INSITU_IBI_PHYBGCWAV_DISCRETE_MYNRT_013_033, EU Copernicus Marine PUM: Copernicus Marine In Situ Tac in situ observations Service Product (2022b) Data Management Team (2021); QUID: Wehde et al. (2022) 4 GLOBAL_ANALYSISFORECAST_PHY_001_024, EU Copernicus Marine PUM: Le Galloudec et al. (2022); numerical models Service Product (2022c) QUID: Lellouche et al. (2022) 5 SST_GLO_SST_L4_REP_OBSERVATIONS_010_011, EU Copernicus Marine PUM: Worsfold et al. (2022); satellite observations Service Product (2023b) QUID: Worsfold et al. (2023) 6 OCEANCOLOUR_GLO_BGC_L4_MY_009_104, EU Copernicus Marine PUM: Colella et al. (2022); satellite observations Service Product (2022d) QUID: Garnesson et al. (2022) Table 1. Products from the Copernicus Marine Service used in this study, including the Product User Manual (PUM) and QUality Information Document (QUID). Last access for all web pages cited in this table: 2 June 2023. tion of upwelling (UPW) and downwelling (DOW) processes that modulate the connectivity between offshore waters and coastal ecosystems (Lorente et al., 2020; Paduan et al., 2018; Kaplan and Largier, 2006). It occurs when alongshore winds and the Coriolis effect (due to Earth’s rotation) combine to drive a near-surface layer of water offshore, a process re- ferred to as Ekman transport (Ekman, 1905). Such cross- shelf transport is compensated for by the vertical uplift of cold and enriched waters that fertilize the uppermost layer. Conversely, during DOW events winds induce net onshore displacement and subduction of surface coastal waters that foster the retention of organic matter and pollutants onto the shoreline with a subsequent impact on residence times and water renewal mechanisms. hams et al., 2021a, b; González-Nuevo et al., 2014; Benaz- zouz et al., 2014; Wooster et al., 1976; Bakun, 1973). State Planet, 1-osr7, 8, 2023 CHAPTER2.6 CHAPTER2.6 CHAPTER2.6 P. Lorente et al.: High-frequency radar-derived coastal upwelling index 3 Figure 1. (a) Study areas are marked with black squares: the north-western Iberian (NWI) peninsula upwelling system and the Bay of Biscay (BOB), where two high-frequency radars (HFRs) are deployed – product ref. nos. 1 and 2 (Table 1). Green dots denote HFR sites, whil orange dots represent the Silleiro buoy and Bilbao buoy locations (product ref. no. 3) (Table 1), moored within the NWI and BOB areas respectively. Red lines indicate selected transects: HFR-derived surface circulation and satellite-derived chlorophyll distribution – produc ref. no. 6 (Table 1) – during a 2 d (b) summer upwelling event and (c) winter downwelling event in the NWI area, when intense northerly and southerly winds were predominant, respectively. Figure 1. (a) Study areas are marked with black squares: the north-western Iberian (NWI) peninsula upwelling system and the Bay of Biscay (BOB), where two high-frequency radars (HFRs) are deployed – product ref. nos. 1 and 2 (Table 1). Green dots denote HFR sites, while orange dots represent the Silleiro buoy and Bilbao buoy locations (product ref. no. 3) (Table 1), moored within the NWI and BOB areas, respectively. Red lines indicate selected transects: HFR-derived surface circulation and satellite-derived chlorophyll distribution – product ref. no. 6 (Table 1) – during a 2 d (b) summer upwelling event and (c) winter downwelling event in the NWI area, when intense northerly and southerly winds were predominant, respectively. The south-eastern BOB (Fig. 1a) is characterized by com- plex topography wherein a combination of different temporal and spatial ocean processes (i.e. slope currents, eddies) gov- erns the local hydrodynamics (Llope et al., 2006). Coastal UPW and DOW processes depict a marked seasonal vari- ability, linked to seasonal changes in the wind regime (Prego et al., 2012; Botas et al., 1990). During autumn and win- ter, south-westerly winds are prevalent and induce DOW events. In spring and summer, winds are less intense and more variable, with the dominance of easterlies and north- easterlies driving weak but frequent UPW episodes (Fontán et al., 2008). Although UPW (whenever present) can be con- sidered weak in comparison with typical UPW areas world- wide (Borja et al., 2008), this phenomenon has been investi- gated in the BOB region as they are signiﬁcantly correlated with the recruitment of commercial ﬁsh species (Caballero et al., 2018). P. Lorente et al.: High-frequency radar-derived coastal upwelling index During the rest of the year, the predominant DOW season is characterized by (i) prominent southerly winds that drive the surface circulation northwards and (ii) low biological productivity, with nutrient concentra- tions conﬁned in shallower coastal areas (Fig. 1c). Since there is no available ground-truth observation of ver- tical currents, diverse coastal upwelling indexes (CUIs) have been derived from historical estimations of other met-ocean parameters (e.g. wind, sea level pressure, or sea surface tem- perature) to indirectly infer seasonal trends of UPW along the shoreline (Gunduz et al., 2022; Mourre et al., 2023; Abra- https://doi.org/10.5194/sp-1-osr7-8-2023 State Planet, 1-osr7, 8, 2023 CHAPTER2.6 CHAPTER2.6 State Planet, 1-osr7, 8, 2023 P. Lorente et al.: High-frequency radar-derived coastal upwelling index In particular, 55 % of the recruitment variabil- ity of anchovy ﬁsheries can be explained by upwelling over the spawning area (Borja et al., 2008). Therefore, the HFR deployed in this region might be very useful to monitor the surface circulation and characterize the UPW variability by means of a CUI (Caballero et al., 2018). The main goal of this contribution is twofold: ﬁrstly, to assess the validity of the proposed ocean-based CUI-HFR through its comparison against a traditional CUI based on hourly wind observations (CUI-WIND) provided by adjacent in situ buoys (Fig. 1a). Likewise, the skill of CUI-HFR to cat- egorize UPW and DOW events was also qualitatively evalu- ated. The second part of the goal is to infer the prognostic capabilities of the GLOBAL analysis and forecasting system State Planet, 1-osr7, 8, 2023 https://doi.org/10.5194/sp-1-osr7-8-2023 CHAPTER2.6 CHAPTER2.6 P. Lorente et al.: High-frequency radar-derived coastal upwelling index 4 Table 1), is a two-site CODAR SeaSonde network (Fig. 1a) that operates at a central frequency of 4.46 MHz, providing hourly current maps (representative of the ﬁrst 1.5 m of the water column) with a spatial resolution of 5 km in an area up to 150 km from the coast (Solabarrieta et al., 2014). The con- sistent performance of this system and its potential for the study of ocean processes and transport patterns have already been demonstrated by previous works (Manso-Narvarte et al., 2018; Solabarrieta et al., 2014, 2015, 2016; Rubio et al., 2011, 2018). (Lellouche et al., 2018) to accurately reproduce UPW and DOW events previously detected in the NWI region. In this context, hydrodynamic models can serve as ancillary tools for gappy HFR surface current maps by offering a seamless predictive picture of the ocean state. This work is structured as follows: Sect. 2 outlines the ob- servational and modelled data sources. Section 3 describes the methodology adopted. Results are presented and dis- cussed in Sect. 4. Finally, principal conclusions are drawn in Sect. 5. Data are collected, processed, and quality-controlled fol- lowing standard recommendations (Mantovani et al., 2020). They are later distributed in near-real time through the Eu- ropean HFR Node as part of the Copernicus Marine Ser- vice products. Again, a cut-off ﬁlter of 3 was imposed for the GDOP parameter to mitigate the impact of geometrical uncertainties on total current vectors accuracy (Fig. A1b). 2 Data All the observational and modelled products used in this study are gathered in Table 1 and thoroughly described be- low. 2.1 The HFR system deployed in NWI A ﬁve-site CODAR SeaSonde HFR network, deployed along the Galician–Portuguese coast (Fig. 1b) since 2004, was used in this work (product ref. nos. 1 and 2 in Table 1). The network is jointly operated by Puertos del Estado, INTECMAR–Xunta de Galicia, and the Portuguese Hydro- graphic Institute. While the southernmost HFR site operates at 13.5 MHz, the other four sites operate at a central fre- quency of 4.86 MHz, providing hourly radial vectors that are representative of the currents moving toward or away from the site. All radial current vectors (from two or several sites) within a predeﬁned search radius of 25 km are geometri- cally combined to estimate hourly total current vectors on a Cartesian regular mesh of 6×6 km horizontal resolution and 200 km range (Lorente et al., 2015, 2020). Data are quality- controlled and delivered freely through the European HFR Node, which oversees the harvesting, harmonization, format- ting, and distribution of HFR data (Corgnati et al., 2021). 2.4 GLOBAL analysis and forecasting system The operational Mercator global ocean analysis and forecast system provides 10 d of 3D global ocean forecasts updated daily. This product (product ref. no. 4 in Table 1), named GLOBAL_ANALYSIS_FORECAST_PHY_001_024 and freely available through the Copernicus Marine Service catalogue, includes daily and monthly mean ﬁles of temper- ature, salinity, currents, sea level, mixed layer depth, and ice parameters from the surface to seaﬂoor (5500 m depth) over the global ocean. It also includes hourly mean surface ﬁelds for sea level height, temperature, and currents. The global ocean output ﬁles are displayed on 50 vertical levels with a 1/12◦horizontal resolution over a regular latitude and longitude equirectangular projection. This product presents temporal coverage from November 2020 to the present. HFR-derived data used in this study were collected from August to December 2021, once the southernmost site (LEÇA) was calibrated and integrated into the pre-existing network. During this 5-month period, the ﬁve sites were si- multaneously operational, and the spatial coverage was at its maximum extent. The speciﬁc geometry of the HFR domain and, hence, the intersection angles of radial vectors handicap the accuracy of the total current vectors resolved at each grid point. Such a source of uncertainty is quantiﬁed by a dimen- sionless parameter called geometrical dilution of precision – GDOP (Chapman et al., 1997; Barrick, 2006), which typi- cally increases with the distance from the HFR sites. In this work, a cut-off ﬁlter of 3 was imposed for the GDOP to get rid of estimations affected by higher uncertainties (Fig. A1a). The system is based on the Nucleus for European Mod- elling of the Ocean (NEMO) v3.1 ocean model (Madec and the NEMO team, 2008) and is forced with 3-hourly atmo- spheric ﬁelds provided by the European Centre for Medium- Range Weather Forecasts. The GLOBAL system does not in- clude tides or pressure forcing. Altimeter data, in situ tem- P. Lorente et al.: High-frequency radar-derived coastal upwelling index Finally, HFR-derived current estimations used in this work were collected from April to August 2014, when the system operated consistently and provided the longest data series (Solabarrieta et al., 2015). 2.3 In situ buoys Basic features of two deep-ocean buoys (product ref. no. 3 in Table 1), deployed within each HFR footprint (Fig. 1a) and operated by Puertos del Estado, are gathered in Table 2. Both in situ devices collect quality-controlled estimations of sea surface temperature, salinity, and currents, among other physical parameters. Furthermore, both buoys are equipped with a wind sensor, providing hourly averaged wind vectors at 3 m height. State Planet, 1-osr7, 8, 2023 P. Lorente et al.: High-frequency radar-derived coastal upwelling index Lorente et al.: High-frequency radar-derived coastal upwelling index 5 Table 2. Description of the buoys (product ref. no. 3 in Table 1) deployed within the coverage of each HFR system (product ref. no. 1 and no. 2 in Table 1), as shown in Fig. 1. More information on products can be found in the text and in the product table. Name (area) Type Deployment Longitude Latitude Depth Sampling Coriolis parameter Silleiro (NWI) Seawatch 1998 9.44◦W 42.12◦N 600 m 1 h 9.75 × 10−5 s−1 Bilbao (BOB) Seawatch 1990 3.04◦W 43.64◦N 580 m 1 h 10 × 10−5 s−1 parameter (whose values are shown in Table 2). In this case, u and v denote the hourly time series of zonal and meridional wind at 3 m height, respectively, as measured by each buoy. The sign is changed to deﬁne positive (negative) magnitudes of CUI-WIND as a response to the predominant equatorward (poleward) wind over NWI and the predominant westward (eastward) wind over BOB. perature and salinity vertical proﬁles, and satellite sea sur- face temperature data are jointly assimilated to estimate the initial conditions for numerical ocean forecasting. For fur- ther details, the reader is referred to the GLOBAL Product User Manual (Le Galloudec et al., 2022) and to Lellouche et al. (2018). Assuming the prompt and direct reaction of the upper ocean layer to intense and prolonged wind forcing in NWI (Herrera et al., 2005) and BOB (Solabarrieta et al., 2015), it seems reasonable to develop an ocean-based indicator for UPW and DOW conditions. Analogously, the CUI-HFR is deﬁned as follows. 2.5 Satellite-derived data Surface ﬁelds of temperature and chlorophyll (product ref. nos. 5 and 6 in Table 1, respectively) provided by satellite missions were downloaded from the Copernicus Marine Ser- vice catalogue and used to analyse the impact of UPW and DOW events on their spatial distribution. For the NWI region: For the NWI region: UEkman = CUI −HFR m3 skm UEkman = CUI −HFR m3 skm 2.2 The HFR system deployed in BOB This HFR system, deployed in the south-eastern BOB since 2009 and owned by Euskalmet (product ref. nos. 1 and 2 in State Planet, 1-osr7, 8, 2023 https://doi.org/10.5194/sp-1-osr7-8-2023 CHAPTER2.6 CHAPTER2.6 https://doi.org/10.5194/sp-1-osr7-8-2023 3 Methodology 2f) at the northernmost sector of the transect, in the vicinity of Cape Finisterre (Fig. 1a), which has been largely documented to act as the locus of frequent UPW (Lorente et al., 2020; Tor- res et al., 2003). Finally, the intense DOW event identiﬁed by CUI-HFR and CUI-GLOBAL for 21 December 2021 (de- noted as DOW-1 in Fig. 2b) was coincident with a general minimum of Chl concentration (Fig. 2f), resulting in reduced primary production. These ﬁndings conﬁrm the strong con- nection between the wind-induced circulation, coastal UPW, and the modulation of SST and Chl ﬁelds at the uppermost layer, in line with previous works. For instance, Alvarez et al. (2012) postulated that the high seasonal variability of Chl in NWI was mainly related to UPW episodes during spring and summer, while Chl variations depended on other addi- tional factors (such as the input of nutrients from land run- off) during autumn and winter. To gain further insight into the three speciﬁc events pre p y g With regard to the 5-month period (August–December 2021) of concurrent CUI estimations (Fig. 2b), the degree of agreement was rather satisfactory. Both ocean-based CUIs were able to reproduce the temporal variability, capturing the most prominent events fairly well (marked and denoted in black). Hovmöller diagrams of CUI were computed at a selected transect of constant longitude (9.43◦W, shown in Fig. 1a) to infer its spatio-temporal evolution (Fig. 2c–d). There were clear similarities (in terms of intensity and tim- ing) between CUI-HFR and CUI-GLOBAL during the en- tire 5-month period, with a predominance of UPW (DOW) events during the summer and autumn (early winter) of 2021. Representative UPW and DOW episodes were selected for each study area to analyse the prevailing met-ocean con- ditions (wind and surface circulation, among others) along with the spatial distribution of CUI. In the case of the NWI area, a CUI was also derived from the surface currents pre- dicted by the GLOBAL analysis and forecast system (Lel- louche et al., 2018) following Eq. (3). CUI-GLOBAL and CUI-HFR were intercompared to infer strengths and weak- nesses of each index and infer the potential of the GLOBAL model as a predictive tool for UPW conditions in this speciﬁc region. 3 Methodology To this aim, a variety of outcomes were computed, ranging from 2D maps, time series, and the best linear ﬁt of scatter plots to Hovmöller diagrams, which are the common way of plotting met-ocean data to depict changes over time of scalar quantities such as the CUI (Benazzouz et al., 2014). g y The Hovmöller diagrams of sea surface temperature (SST) and chlorophyll a (Chl) concentration at the same tran- sect corroborated the consistency of the proposed approach (Fig. 2e–f). An abrupt cooling (with SST below 14 ◦C) and a relevant peak of Chl (with values above 2 mg m−3) were observed by mid-August 2021, coincident with the UPW-1 event previously categorized (Fig. 2b–d). Equally, the mod- erate UPW episode that took place on 20 September 2021 (denoted as UPW-2 in Fig. 2b) could also be related to a local drop in SST (Fig. 2e) and a maximum of Chl (Fig. 2f) at the northernmost sector of the transect, in the vicinity of Cape Finisterre (Fig. 1a), which has been largely documented to act as the locus of frequent UPW (Lorente et al., 2020; Tor- res et al., 2003). Finally, the intense DOW event identiﬁed by CUI-HFR and CUI-GLOBAL for 21 December 2021 (de- noted as DOW-1 in Fig. 2b) was coincident with a general minimum of Chl concentration (Fig. 2f), resulting in reduced primary production. These ﬁndings conﬁrm the strong con- nection between the wind-induced circulation, coastal UPW, and the modulation of SST and Chl ﬁelds at the uppermost layer, in line with previous works. For instance, Alvarez et al. (2012) postulated that the high seasonal variability of Chl in NWI was mainly related to UPW episodes during spring and summer, while Chl variations depended on other addi- tional factors (such as the input of nutrients from land run- off) during autumn and winter. 3 Methodology Bakun (1973, 1975) proposed a CUI (the so-called Bakun index) based on Ekman theory (Ekman, 1905) and available estimates of atmospheric conditions to derive estimates of cross-shore Ekman transport as a proxy for coastal UPW. Following the same approach, in the present contribution two CUIs were computed (as benchmarks to compare CUI-HFR against) from hourly wind estimations provided by in situ buoys moored within each HFR coverage (Fig. 1a). Then, the CUI-WIND is deﬁned as follows. = −ρa · Cd · √ u2 + v2 · v · 1000 f · ρw · C (3) (3) For the BOB region: g VEkman = CUI −HFR m3 skm = −ρa · Cd · √ u2 + v2 · u · 1000 f · ρw · C (4) For the NWI region: For the NWI region: (4) f · ρw UEkman = CUI −WIND m3 skm Here, u and v represent the ﬁltered hourly time series of zonal and meridional surface current velocities (m s−1) pro- vided by the HFR, respectively. A 25 h running-mean ﬁl- ter was used to smooth time series data by suppressing the main diurnal and semidiurnal tidal constituents (Shirahata et al., 2016), particularly the M2 signal, which is the largest harmonic constituent in the study area. Furthermore, C rep- resents a dimensionless parameter that acts as a proportional- ity constant between wind and surface current observations, assuming a direct relationship between the two parameters (Lorente et al., 2020; Solabarrieta et al., 2015). The value of this parameter, which depends on the selected study area, can be easily derived from the best linear ﬁt against CUI-WIND. For instance, C is equal to 2300 (3500) for the NWI (BOB) region. = −ρa · Cd · √ u2 + v2 · v · 1000 f · ρw (1) (1) For the BOB region: For the BOB region: VEkman = CUI −WIND m3 skm = −ρa · Cd · √ u2 + v2 · u · 1000 f · ρw (2) (2) Here, ρa is the air density at standard temperature and pres- sure conditions (1.22 kg m−3), ρw is the seawater density (1025 kg m−3), Cd is a dimensionless empirical drag coef- ﬁcient (1.4×10−3), and f is the latitude-dependent Coriolis In order to obtain a single time series of CUI-HFR, which is representative of the entire study area, hourly 2D maps State Planet, 1-osr7, 8, 2023 https://doi.org/10.5194/sp-1-osr7-8-2023 CHAPTER2.6 P. State Planet, 1-osr7, 8, 2023 3 Methodology Lorente et al.: High-frequency radar-derived coastal upwelling index 6 were spatially averaged over a delimited HFR spatial sub- domain (see Fig. A1). In particular, two prerequisites were imposed to select such a subdomain: (i) it should be between 60 and 1200 m bathymetric depths to represent coastal waters while avoiding shallow water effects and the potential impact of impulsive-type freshwater discharges on the coastal circu- lation, and (ii) a predeﬁned threshold of 3 was established for the GDOP parameter to minimize geometrical uncertain- ties in the remotely sensed currents. Finally, as a rough ap- proximation, we also assumed that the local coastline was perfectly aligned in the south–north axis for the NWI region and in the east–west axis for the BOB region, which implies that v and u surface current components were parallel to the coastline. of the most relevant UPW (positive values) and DOW (nega- tive values) episodes such as those observed during February 2021, whose values were higher than 3000 m3 s−1 km−1 and lower than −5000 m3 s−1 km−1, respectively (Fig. 2a). lower than −5000 m3 s 1 km 1, respectively (Fig. 2a). With regard to the 5-month period (August–December 2021) of concurrent CUI estimations (Fig. 2b), the degree of agreement was rather satisfactory. Both ocean-based CUIs were able to reproduce the temporal variability, capturing the most prominent events fairly well (marked and denoted in black). Hovmöller diagrams of CUI were computed at a selected transect of constant longitude (9.43◦W, shown in Fig. 1a) to infer its spatio-temporal evolution (Fig. 2c–d). There were clear similarities (in terms of intensity and tim- ing) between CUI-HFR and CUI-GLOBAL during the en- tire 5-month period, with a predominance of UPW (DOW) events during the summer and autumn (early winter) of 2021. The Hovmöller diagrams of sea surface temperature (SST) and chlorophyll a (Chl) concentration at the same tran- sect corroborated the consistency of the proposed approach (Fig. 2e–f). An abrupt cooling (with SST below 14 ◦C) and a relevant peak of Chl (with values above 2 mg m−3) were observed by mid-August 2021, coincident with the UPW-1 event previously categorized (Fig. 2b–d). Equally, the mod- erate UPW episode that took place on 20 September 2021 (denoted as UPW-2 in Fig. 2b) could also be related to a local drop in SST (Fig. 2e) and a maximum of Chl (Fig. https://doi.org/10.5194/sp-1-osr7-8-2023 P. Lorente et al.: High-frequency radar-derived coastal upwelling index 7 Figure 2. Time series of diverse hourly coastal upwelling index (CUI) in the north-western Iberia (NWI) region (a) for the entirety of 2021 and (b) for August–December 2021, as derived from wind observations from the Silleiro buoy (CUI-WIND) (product ref. no. 3; Table 1), HFR surface currents (CUI-HFR) (product ref. nos. 1 and 2; Table 1), and modelled surface currents (CUI-GLOBAL) (product ref. no. 4; Table 1). CUI-WIND raw (grey dots) was ﬁltered by applying a 24 h moving mean (blue line). Hovmöller diagrams of (c) CUI-HFR, (d) CUI- GLOBAL, (e) sea surface temperature (SST) (product ref. no. 5; Table 1), and (f) chlorophyll (Chl) (product ref. no. 6; Table 1) concentration during the period August–December 2021 for a selected transect of constant longitude (9.43◦W), depicted in Fig. 1a. Figure 2. Time series of diverse hourly coastal upwelling index (CUI) in the north-western Iberia (NWI) region (a) for the entirety of 2021 and (b) for August–December 2021, as derived from wind observations from the Silleiro buoy (CUI-WIND) (product ref. no. 3; Table 1), HFR surface currents (CUI-HFR) (product ref. nos. 1 and 2; Table 1), and modelled surface currents (CUI-GLOBAL) (product ref. no. 4; Table 1). CUI-WIND raw (grey dots) was ﬁltered by applying a 24 h moving mean (blue line). Hovmöller diagrams of (c) CUI-HFR, (d) CUI- GLOBAL, (e) sea surface temperature (SST) (product ref. no. 5; Table 1), and (f) chlorophyll (Chl) (product ref. no. 6; Table 1) concentration during the period August–December 2021 for a selected transect of constant longitude (9.43◦W), depicted in Fig. 1a. tial distribution of CUI was quite similar for HFR (Fig. 3c) and GLOBAL (Fig. 3d), with a core of maximum UPW ob- served to the south of Rías Baixas (several coastal embay- ments characterized by high biodiversity, denoted in Fig. 1a). The main discrepancy between the two patterns was that in the case of GLOBAL, the peak appeared in the form of an elongated belt of positive values of CUI (conﬁned landward close to the shoreline), whereas the HFR-derived pattern re- vealed a wider dipole-like structure, with two main cores of maximum CUI-HFR (close to 4000 m3 s−1 km−1). The drop in CUI-HFR is consistent (in timing and location) with the drop in Chl concentration shown in Fig. 1b, supporting this HFR pattern, which was already documented in Lorente et al. (2020). Finally, during the DOW-1 event (Fig. 4 Results To quantify the consistency of both CUI-HFR and CUI- GLOBAL in the NWI area, hourly time series were com- pared against CUI-WIND, derived from wind estimations provided by the Silleiro buoy (Fig. 2a–b). The visual re- semblance between the three different CUIs was notice- able, with signiﬁcantly high correlation coefﬁcients: (i) 0.72 and 0.74 between CUI-GLOBAL and CUI-WIND for the entirety of 2021 and for August–December 2021, respec- tively, as reﬂected by their best linear ﬁt of scatter plots (Fig. A2a–b); (ii) 0.80 between CUI-HFR and CUI-WIND for August–December 2021 (Fig. A2c); (iii) 0.91 between CUI-GLOBAL and CUI-HFR for August–December 2021 (Fig. A2d). According to the statistical results in Fig. A2a–c, we can state that (i) the slope and intercept values were close to 1 and moderately low, respectively, and (ii) ocean-based CUI and CUI-WIND are strongly correlated, likely due to the role of alongshore wind stress as a primary driver of UPW conditions in the NWI area. Hourly alongshore winds (from Silleiro buoy) and HFR-derived alongshore currents (at the grid point closest to the buoy) are highly correlated (0.80) for August–December 2021 (not shown). It is also worth high- lighting the ability of the GLOBAL model to capture many To gain further insight into the three speciﬁc events pre- viously identiﬁed (Fig. 2b), daily averaged maps of CUI and circulation patterns were computed from current esti- mations provided by both HFR and the GLOBAL model (Fig. 3). During the UPW-1 (Fig. 3a–b) and UPW-2 (Fig. 3c– d) events, intense northerlies (with gusts above 10 m s−1) were predominant over the study area according to the wind roses derived from Silleiro buoy observations. Despite the observed discrepancies in magnitude and direction, maps of wind-induced surface currents shared some common fea- tures: (i) the prevailing S-SW surface circulation (as a re- sponse to northerly winds) along with the typical offshore deﬂection of the ﬂow, associated with UPW-favourable con- https://doi.org/10.5194/sp-1-osr7-8-2023 State Planet, 1-osr7, 8, 2023 CHAPTER2.6 State Planet, 1-osr7, 8, 2023 5 Conclusions In this previous work, it was sug- gested that positive divergence, localized at the tip of Cape Finisterre, induced topographic UPW and then up- welled waters were advected southwards away from the promontory. Similar initiatives with HFR current obser- vations were effectively addressed on the west coast of the USA (Roughan et al., 2005), proposing that conﬁned areas of semi-persistent UPW were not due to local or remote wind forcing but rather to the divergence of the prevailing southerly ﬂow as it passed the Point Loma headland. ii. It provides high-resolution two-dimensional maps that can aid in elucidating the spatial distribution and magni- tude of the coastal, together with the potential existence of recurrent patterns and/or ﬁlaments in intricate regions with complex geometry conﬁgurations. The small-scale belt of UPW, conﬁned in shallower coastal areas and ev- idenced in Fig. 3a and c, is consistent with HFR-derived maps of horizontal divergence previously published in Lorente et al. (2020). In this previous work, it was sug- gested that positive divergence, localized at the tip of Cape Finisterre, induced topographic UPW and then up- welled waters were advected southwards away from the promontory. Similar initiatives with HFR current obser- vations were effectively addressed on the west coast of the USA (Roughan et al., 2005), proposing that conﬁned areas of semi-persistent UPW were not due to local or remote wind forcing but rather to the divergence of the prevailing southerly ﬂow as it passed the Point Loma headland. A Hovmöller diagram of CUI-HFR was computed at a selected transect of constant latitude (43.54◦N, denoted in Fig. 1a) to deduce its spatio-temporal variability (Fig. 4b). The six events previously categorized were clearly observed and marked in the diagram, where the UPW and DOW fea- tures were evidenced throughout the entire transect. A num- ber of secondary UPW events could be observed: although relevant in strength and duration, they were spatially con- ﬁned to the westernmost sector of the transect (coincident with Cape Matxitxako, denoted in Fig. 1a) during the be- ginning of April, late July, and early August 2014. This fact highlighted the importance of coastal promontories as mod- ulators of UPW processes by inducing important wind stress variations and zones of retention (Pitcher et al., 2010). During DOW-1 event (Fig. P. Lorente et al.: High-frequency radar-derived coastal upwelling index 2b), both HFR and GLOBAL captured the so-called Iberian poleward current fairly well, a narrow surface poleward ﬂow along the ditions; (ii) the rather uniform circulation westwards (south- westwards) to the north (south) of Cape Finisterre (indicated in Fig. 1a); (iii) the absence of submesoscale structures (i.e. eddies, small meanders) due to the strong wind-induced ho- mogenization. It is also worth mentioning that the GLOBAL model predicted a more pronounced alongshore circulation (i.e. a more intense meridional – v – velocity component) to the south of this coastal promontory, which probably gave rise to higher CUI values (Fig. 3b) than those derived from HFR current estimations (Fig. 3a), according to Eq. (3). In- deed, this could be also observed in Fig. 2b where the peak of CUI-GLOBAL (green line) was higher than the concomi- tant peak of CUI-HFR (red line) during the UPW-1 episode. By contrast, local maximums of CUI-GLOBAL and CUI- HFR time series during the UPW-2 event were rather alike in terms of timing and strength (Fig. 2b). Analogously, the spa- State Planet, 1-osr7, 8, 2023 State Planet, 1-osr7, 8, 2023 https://doi.org/10.5194/sp-1-osr7-8-2023 CHAPTER2.6 CHAPTER2.6 P. Lorente et al.: High-frequency radar-derived coastal upwelling index P. Lorente et al.: High-frequency radar-derived coastal upwelling index 8 NWI shelf edge (Torres and Barton, 2006). As reﬂected in Fig. 3e–f, DOW-favourable southerly winds induced a net ﬂow to the north that settled in the continental shelf and cir- cuited the western and northern Iberia margins. Again, the main difference resided in the existence of both a longitu- dinal gradient and a narrow strip of very negative CUI val- ues close to the coastline in the case of GLOBAL (Fig. 3f), while the HFR-derived map of CUI exhibited a dipole-like distribution wherein the values were not so negative (Fig. 3e). This was also evidenced in the spatially averaged hourly time series of CUI shown in Fig. 2b, where the drop in CUI- GLOBAL was sharper than observed in CUI-HFR. events denoted in Fig. 4a (not shown) were very similar to the DOW-1 case shown here (Fig. 4c), sharing common circu- lation features and comparable spatial distributions of CUI. Most of the surface current patterns were clearly related to speciﬁc wind patterns that are recurrent in the study area, in line with Borja et al. (2008) and Solabarrieta et al. (2015). State Planet, 1-osr7, 8, 2023 5 Conclusions Over the last decades, relevant efforts have been dedicated to the indirect quantiﬁcation of the intensity, duration, and variability of UPW since it seriously impacts biogeochemi- cal cycles and ecosystem productivity (Lachkar and Gruber, 2011). Diverse CUIs have been traditionally derived from wind or sea level pressure estimations, but they present some limitations such as the omission of the direct inﬂuence of ocean circulation (Jacox et al., 2018). In the present contri- bution, attention was placed on the development of a purely ocean-based CUI for the NWI and BOB regions (Fig. 1a), constructed from reliable hourly surface current maps pro- vided by two different HFR remote sensing systems. To further verify the validity of the proposed methodology, a similar CUI was generated for a 5-month period (April– August 2014) from an hourly surface current estimation pro- vided by another HFR system deployed in the BOB since 2009 (Solabarrieta et al., 2014). This CUI-HFR was vali- dated against a CUI-WIND based on hourly estimations pro- vided by the Bilbao buoy (located within the HFR footprint and depicted in Fig. 1a), used here as a reference benchmark (Fig. 4a). The visual resemblance between the two time se- ries was signiﬁcantly high, with a correlation coefﬁcient and a slope of 0.68 and 1, respectively, for a set of 3672 hourly data (Fig. A2e). During the analysed period, DOW events (5) were predominant with respect to UPW episodes (1). It seemed that CUI-HFR tended to slightly underestimate the intensity of some DOW events (i.e. DOW-2, DOW-3, and DOW-4), while it clearly overestimated the strength of the UPW-1 episode (Fig. 4a). In this context, the proposed CUI-HFR presents additional advantages with respect to previous traditional CUIs, namely the following. i. It takes into consideration the direct inﬂuence of coastal waters dynamics, thereby providing a more complete portrait of this phenomenon. ii. It provides high-resolution two-dimensional maps that can aid in elucidating the spatial distribution and magni- tude of the coastal, together with the potential existence of recurrent patterns and/or ﬁlaments in intricate regions with complex geometry conﬁgurations. The small-scale belt of UPW, conﬁned in shallower coastal areas and ev- idenced in Fig. 3a and c, is consistent with HFR-derived maps of horizontal divergence previously published in Lorente et al. (2020). 5 Conclusions 4c), intense westerly winds (up to 10–12 m s−1) were dominant and a counterclockwise re- circulation pattern prevailed, revealing the associated mech- anisms of convergence and subduction (and the subsequent vertical mixing), especially in the vicinity of Cape Matx- itxako (denoted in Fig. 1a) where a local minimum of CUI (down to −3000 m3 s−1 km−1) was detected. By contrast, weak north-easterly winds induced a general surface ﬂow to the west during UPW-1 (Fig. 4d). Two local cores of UPW (ranging from 1000 to 1500 m3 s−1 km−1) could be observed: one on the south-western French coast and a sec- ondary one near Cape Matxitxako. The rest of the DOW iii. It is generated from consistent remotely sensed hourly surface current observations, not from coarse-resolution atmospheric forecasts, which are probably affected by higher uncertainties. This interpretation is supported by the fact that operational atmospheric and ocean mod- els include assimilation schemes in which remote ob- servations are routinely ingested to improve their pre- https://doi.org/10.5194/sp-1-osr7-8-2023 State Planet, 1-osr7, 8, 2023 CHAPTER2.6 P. Lorente et al.: High-frequency radar-derived coastal upwelling index 9 y averaged maps of circulation and coastal upwelling index (CUI) as derived from surface current estimations p cy radar (HFR, left column, product ref. no. 1 and no. 2 in Table 1) and the GLOBAL model (right column, pro ﬁc upwelling (UPW) and downwelling (DOW) episode categorized in Fig. 2b. Daily wind roses derived from h m the Silleiro buoy (represented by an orange dot, product ref. no. 3 in Table 1) are also provided, indicating inco peed. coastal upwelling index (CUI) as derived from surface current estimations provided by ref. no. 1 and no. 2 in Table 1) and the GLOBAL model (right column, product ref. 4) ng (DOW) episode categorized in Fig. 2b. Daily wind roses derived from hourly wind an orange dot, product ref. no. 3 in Table 1) are also provided, indicating incoming wind Figure 3. Daily averaged maps of circulation and coastal upwelling index (CUI) as derived from surface current estimations provided by a high-frequency radar (HFR, left column, product ref. no. 1 and no. 2 in Table 1) and the GLOBAL model (right column, product ref. 4) for each speciﬁc upwelling (UPW) and downwelling (DOW) episode categorized in Fig. 2b. Daily wind roses derived from hourly wind estimations from the Silleiro buoy (represented by an orange dot, product ref. no. 3 in Table 1) are also provided, indicating incoming wind direction and speed. CHAPTER2.6 https://doi.org/10.5194/sp-1-osr7-8-2023 State Planet, 1-osr7, 8, 2023 State Planet, 1-osr7, 8, 2023 https://doi.org/10.5194/sp-1-osr7-8-2023 CHAPTER2.6 P. Lorente et al.: High-frequency radar-derived coastal upwelling index 10 P. Lorente et al.: High frequency radar derived coastal upwelling index 4. (a) Coastal upwelling indexes (CUIs) in the Bay of Biscay (April–August 2014) based on hourly wind observations (from the buoy, product ref. no. 3 in Table 1) and hourly surface currents (from an HFR system, product ref. no. 1 and no. 2 in Table 1) IND raw (grey dots) was ﬁltered by applying a 24 h moving mean (blue line). Identiﬁcation of six UPW–DOW events. (b) Hovmölle m of CUI-HFR for a transect of constant latitude, where UPW (DOW) events are marked with black dotted squares. (c–d) Daily d maps of surface circulation and CUI-HFR for two events. Daily wind roses from the Bilbao buoy (represented by an orange dot ref. no. 3 in Table 1) are also provided, indicating incoming wind direction and speed. Figure 4. P. Lorente et al.: High-frequency radar-derived coastal upwelling index (a) Coastal upwelling indexes (CUIs) in the Bay of Biscay (April–August 2014) based on hourly wind observations (from the Bilbao buoy, product ref. no. 3 in Table 1) and hourly surface currents (from an HFR system, product ref. no. 1 and no. 2 in Table 1). CUI-WIND raw (grey dots) was ﬁltered by applying a 24 h moving mean (blue line). Identiﬁcation of six UPW–DOW events. (b) Hovmöller diagram of CUI-HFR for a transect of constant latitude, where UPW (DOW) events are marked with black dotted squares. (c–d) Daily averaged maps of surface circulation and CUI-HFR for two events. Daily wind roses from the Bilbao buoy (represented by an orange dot, product ref. no. 3 in Table 1) are also provided, indicating incoming wind direction and speed. Figure 4. (a) Coastal upwelling indexes (CUIs) in the Bay of Biscay (April–August 2014) based on hourly wind observations (from the Bilbao buoy, product ref. no. 3 in Table 1) and hourly surface currents (from an HFR system, product ref. no. 1 and no. 2 in Table 1). CUI-WIND raw (grey dots) was ﬁltered by applying a 24 h moving mean (blue line). Identiﬁcation of six UPW–DOW events. (b) Hovmöller diagram of CUI-HFR for a transect of constant latitude, where UPW (DOW) events are marked with black dotted squares. (c–d) Daily averaged maps of surface circulation and CUI-HFR for two events. Daily wind roses from the Bilbao buoy (represented by an orange dot, product ref. no. 3 in Table 1) are also provided, indicating incoming wind direction and speed. CHAPTER2.6 State Planet, 1-osr7, 8, 2023 https://doi.org/10.5194/sp-1-osr7-8-2023 https://doi.org/10.5194/sp-1-osr7-8-2023 State Planet, 1-osr7, 8, 2023 State Planet, 1-osr7, 8, 2023 CHAPTER2.6 P. Lorente et al.: High-frequency radar-derived coastal upwelling index dictive skills (Wilczak et al., 2019; Hernandez-Lasheras et al., 2021). favourab between 11 favourable conditions (Fig. 3e–f). The discrepancies detected between CUI-HFR and CUI-GLOBAL maps in this speciﬁc subregion could be attributed to the fact that coastal and shelf phenomena are still poorly replicated or even misrep- resented as the model grid mesh is too coarse (e.g. nominal 1/12◦). This is especially true for complex geometry regions like semi-enclosed coastal embayments where the coastline, seamounts, and bottom topography are not well resolved. In this context, mixing schemes, river inﬂows, and atmospheric forcings have been traditionally identiﬁed as areas of fur- ther research in global ocean modelling (Holt et al., 2017). P. Lorente et al.: High-frequency radar-derived coastal upwelling index Nevertheless, this comparison potentially opens future path- ways for the design of ﬁt-for-purpose metrics that quantify the model skill in reproducing speciﬁc ocean processes and their seasonal variability. dictive skills (Wilczak et al., 2019; Hernandez-Lasheras et al., 2021). To assess the credibility of the proposed CUI-HFR in- dexes, they were spatially averaged over speciﬁc subdomains (Fig. A1) and later validated against CUI-WIND time series, based on hourly wind observations collected by two in situ buoys moored within each HFR footprint, during two distinct time periods: in NWI for August–December 2021 (Fig. 2) and in BOB for April–August 2014 (Fig. 4), respectively. The results obtained evidenced the signiﬁcant agreement be- tween CUI-HFR and CUI-WIND, with correlation coefﬁ- cients of 0.80 (Fig. A2c) and 0.68 (Fig. A2e), respectively, for these two pilot areas. Furthermore, CUI-HFRs were able to adequately identify a variety of UPW and DOW episodes, regardless of their length and intensity, corroborating the re- current relationship between the surface circulation and pre- dominant wind patterns in NWI (Herrera et al., 2005) and BOB (Solabarrieta et al., 2015). Thanks to the computation of Hovmöller diagrams at selected transects, coastal promon- tories such as Cape Finisterre (NWI) and Cape Matxitxako (BOB) were revealed as important modulators of UPW pro- cesses, in line with previous works that acknowledged that capes and bays may induce signiﬁcant variations in wind stress (Torres and Barton, 2006; Enriquez and Friehe, 1995). Therefore, the obtained outcomes support the potential im- plementation of a predictive application to infer UPW–DOW conditions even 10 d ahead, which is the maximum forecast horizon provided by the GLOBAL ocean model. Comple- mentarily, the methodology proposed in this work could also be applied to the GLOBAL ocean physics reanalysis prod- uct (which covers the period 1 January 1993–31 December 2020) to analyse long-term seasonal trends in both study areas, as extremely active and persistent UPW and DOW episodes might negatively impact coastal ecosystems. Dur- ing periods of increased offshore advection, some ﬁsh and in- vertebrate populations are exported from coastal habitats and exhibit reduced recruitment success (Bjorkstedt and Rough- garden, 1997). Moreover, an excessive enrichment of surface waters inshore may support the proliferation of harmful algal blooms (Pitcher et al., 2010). By contrast, the opposite-phase circulation patterns during DOW-favourable wind conditions might be related to the transport and retention of pollutants onto the shoreline, with subsequent biological and socioeco- nomic consequences. P. Lorente et al.: High-frequency radar-derived coastal upwelling index The strong link between HFR-derived wind-induced circu- lation and the modulation of surface ﬁelds of temperature and chlorophyll (provided by two independent satellite missions) was also shown in the NWI area, coherent with prior liter- ature (Picado et al., 2013). Intense northerly winds and the general ﬂow induced to the south-west are closely associated with the cooling and fertilization of coastal waters with nu- trients, fuelling high primary production that ultimately sup- ports the notable biological diversity in this region. Within this framework, future investigations should explore the con- nection between ocean dynamics and marine communities as well as how the latter respond to persistent changes in the former. This could be achieved by analysing the relation- ship between the proposed CUI-HFRs and food availability, which is often represented by recruitment indexes (Borja et al., 1996, 2008). In summary, results seem to suggest that the HFR perfor- mances were sound and credible for the two distinct peri- ods and areas analysed, providing reliable surface current estimations that could be effectively used for coastal mon- itoring and the characterization of recurring UPW and DOW episodes. Future research endeavours should include the syn- ergetic combination of HFRs and GLOBAL model products to implement a valuable long-term ocean monitoring indi- cator that might be used over any coastal area of the global ocean for wise decision-making to mitigate adverse effects of climate change. As a proof-of-concept investigation, the prognostic capa- bilities of the GLOBAL analysis and forecasting system to accurately reproduce UPW–DOW events in the NWI area were also assessed (Figs. 2 and 3). Following the same approach, CUI-GLOBAL demonstrated signiﬁcant consis- tency, with annual correlation coefﬁcients of 0.74 and 0.72 for 2020 (Fig. A3) and 2021 (Fig. 2a). From a qualitative perspective, the daily maps of surface circulation and spatial patterns of CUI-GLOBAL presented similarities with those derived from HFR remotely sensed observations, namely (i) the rather uniform S-SW surface circulation as well as the related offshore deﬂection of the main ﬂow during UPW- favourable conditions (Fig. 3a–d) and (ii) the narrow sur- face poleward ﬂow along the NWI shelf edge during DOW- State Planet, 1-osr7, 8, 2023 https://doi.org/10.5194/sp-1-osr7-8-2023 CHAPTER2.6 CHAPTER2.6 P. Lorente et al.: High-frequency radar-derived coastal upwelling index The blue area represents the entire HFR spatial grid, while the green area denotes the subdomain where the value of the dimensionless GDOP parameter is below 3. The yellow area is the selected HFR subdomain to compute the coastal upwelling index (CUI) and must fulﬁl two requirements: (i) GDOP must be below 3, and (ii) it must be between bathymetric contours (red lines) of 60 and 1200 m depth. Green dots represent the HFR sites that compose each network deployed in the NWI (a) Figure A2. Best linear ﬁt of scatter plots between the following: (a–b) CUI-GLOBAL (product ref. no. 4 in Table 1) and CUI-WIND (ﬁltered), product ref. no. 3 (Table 1), in the NWI area for the en- tirety of 2021 and August–December 2021; (c) CUI-HFR, prod- uct ref. nos. 1 and 2 (Table 1), and CUI-WIND (ﬁltered), product ref. no. 2 (Table 1), in the NWI area for August–December 2021; (d) CUI-GLOBAL, product ref. no. 4 (Table 1), and CUI-HFR, product ref. nos. 1 and 2 (Table 1) in the NWI area for August– December 2021; (e) CUI-HFR, product ref. nos. 1 and 2 (Table 1), and CUI-WIND (ﬁltered), product ref. no. 3 (Table 1), in the BOB area for April–August 2014. Statistical metrics are gathered in white boxes. Figure A1. Selection of subdomains within each HFR coverage – product ref. nos. 1 and 2 (Table 1). The blue area represents the entire HFR spatial grid, while the green area denotes the subdomain where the value of the dimensionless GDOP parameter is below 3. The yellow area is the selected HFR subdomain to compute the coastal upwelling index (CUI) and must fulﬁl two requirements: (i) GDOP must be below 3, and (ii) it must be between bathymetric contours (red lines) of 60 and 1200 m depth. Green dots represent the HFR sites that compose each network deployed in the NWI (a) and BOB (b) regions. https://doi.org/10.5194/sp-1-osr7-8-2023 State Planet, 1-osr7, 8, 2023 P. Lorente et al.: High-frequency radar-derived coastal upwelling index P. Lorente et al.: High-frequency radar-derived coastal upwelling index 12 Appendix A Figure A1. Selection of subdomains within each HFR coverage – product ref. nos. 1 and 2 (Table 1). The blue area represents the entire HFR spatial grid, while the green area denotes the subdomain where the value of the dimensionless GDOP parameter is below 3. The yellow area is the selected HFR subdomain to compute the coastal upwelling index (CUI) and must fulﬁl two requirements: (i) GDOP must be below 3, and (ii) it must be between bathymetric contours (red lines) of 60 and 1200 m depth Green dots represent Figure A2. Best linear ﬁt of scatter plots between the following: (a–b) CUI-GLOBAL (product ref. no. 4 in Table 1) and CUI-WIND (ﬁltered), product ref. no. 3 (Table 1), in the NWI area for the en- tirety of 2021 and August–December 2021; (c) CUI-HFR, prod- uct ref. nos. 1 and 2 (Table 1), and CUI-WIND (ﬁltered), product ref. no. 2 (Table 1), in the NWI area for August–December 2021; (d) CUI-GLOBAL, product ref. no. 4 (Table 1), and CUI-HFR, product ref. nos. 1 and 2 (Table 1) in the NWI area for August– December 2021; (e) CUI-HFR, product ref. nos. 1 and 2 (Table 1), and CUI-WIND (ﬁltered), product ref. no. 3 (Table 1), in the BOB area for April–August 2014. Statistical metrics are gathered in white boxes. Appendix A Figure A2. Best linear ﬁt of scatter plots between the following: (a–b) CUI-GLOBAL (product ref. no. 4 in Table 1) and CUI-WIND (ﬁltered), product ref. no. 3 (Table 1), in the NWI area for the en- tirety of 2021 and August–December 2021; (c) CUI-HFR, prod- uct ref. nos. 1 and 2 (Table 1), and CUI-WIND (ﬁltered), product ref. no. 2 (Table 1), in the NWI area for August–December 2021; (d) CUI-GLOBAL, product ref. no. 4 (Table 1), and CUI-HFR, product ref. nos. 1 and 2 (Table 1) in the NWI area for August– December 2021; (e) CUI-HFR, product ref. nos. 1 and 2 (Table 1), and CUI-WIND (ﬁltered), product ref. no. 3 (Table 1), in the BOB area for April–August 2014. Statistical metrics are gathered in white boxes. Figure A1. Selection of subdomains within each HFR coverage – product ref. nos. 1 and 2 (Table 1). P. Lorente et al.: High-frequency radar-derived coastal upwelling index 13 Figure A3. (a) Time series of hourly coastal upwelling index (CUI) in the north-western Iberia (NWI) region for the entirety of 2020 as derived from wind observations from Silleiro Buoy (CUI-WIND), product ref. no. 3 (Table 1), and from modelled surface currents (CUI-GLOBAL), product ref. no. 4 (Table 1). CUI-WIND raw (grey dots) was ﬁltered by applying a 25 h moving mean (blue line); (b) best linear ﬁt of the scatter plot between CUI-GLOBAL and CUI-WIND (ﬁltered). Statistical metrics are gathered in the white box. Competing interests. The contact author has declared that none of the authors has any competing interests. Competing interests. The contact author has declared that none of the authors has any competing interests. Disclaimer. Publisher’s note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Acknowledgements. This study has been developed in the framework of the Work Package WP3 of EuroSea project (https: //eurosea.eu/, last access: 29 June 2023), which is aimed at improv- ing and integrating the European ocean observing and forecasting system. The authors acknowledge the ﬁnancial support of the EU H2020 Programme’s JERICO-S3 project. It is also worth mention- ing that this work builds upon a previous proof-of-concept inves- tigation conducted within the context of the RADAR_ON_RAIA project (Interreg V, a Spain–Portugal programme). Finally, the au- thors are grateful to INTECMAR–Consellería do Mar–Xunta de Galicia and the Hydrographic Institute of Portugal for the strong co- operation to jointly operate the Galician HFR network. Equally, the authors thank the Emergencies and Meteorology Directorate (Secu- rity Department) of the Basque Government for public data provi- sion from the Basque Operational Oceanography System EuskOOS. This study has been conducted using EU Copernicus Marine Ser- vice Information. Financial support. This research has been partially funded through the EU H2020 Programme’s JERICO-S3 project (grant agreement no. 871153). Financial support. This research has been partially funded through the EU H2020 Programme’s JERICO-S3 project (grant agreement no. 871153). Figure A3. (a) Time series of hourly coastal upwelling index (CUI) in the north-western Iberia (NWI) region for the entirety of 2020 as derived from wind observations from Silleiro Buoy (CUI-WIND), product ref. no. 3 (Table 1), and from modelled surface currents (CUI-GLOBAL), product ref. no. 4 (Table 1). P. Lorente et al.: High-frequency radar-derived coastal upwelling index CUI-WIND raw (grey dots) was ﬁltered by applying a 25 h moving mean (blue line); (b) best linear ﬁt of the scatter plot between CUI-GLOBAL and CUI-WIND (ﬁltered). Statistical metrics are gathered in the white box. Review statement. 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Lorente et al.: High-frequency radar-derived coastal upwelling index Verbrugge, N., Etienne, H., Corgnati, L., Mantovani, C., Reyes, E., Solabarrieta, L., Rubio, A., Carval, T., Mader, J., and Drouineau, L.: EU Copernicus Marine Service Quality Information Document for the Global Ocean- in-situ Near real time observations of ocean currents, Rubio, A., Mader, J., Corgnati, L., Mantovani, C., Griffa, A., Novellino, A., Quentin, C., Wyatt, L., Schulz-Stellenﬂeth, J., Horstmann, J., Lorente, P., Zambianchi, E., Hartnett, M., Fer- nandes, C., Zervakis, V., Gorringe, P., Melet, A., and Puillat, I.: State Planet, 1-osr7, 8, 2023 https://doi.org/10.5194/sp-1-osr7-8-2023 CHAPTER2.6 Ocean heat content in the Iberian–Biscay–Ireland regional seas n of the Copernicus Ocean State Report (OSR7) Álvaro de Pascual-Collar1, Roland Aznar1, Bruno Levier2, and Marcos García-Sotillo1 1Nologin Consulting, Avda. de Ranillas 1D, 50018 Zaragoza, Spain 2Mercator Ocean, 8-10, rue Hermès, 31520, Ramonville Saint-Agne, France Álvaro de Pascual-Collar1, Roland Aznar1, Bruno Levier2, and Marcos García-Sotillo1 1Nologin Consulting, Avda. de Ranillas 1D, 50018 Zaragoza, Spain 2Mercator Ocean, 8-10, rue Hermès, 31520, Ramonville Saint-Agne, France Correspondence: Álvaro de Pascual-Collar (alvaro.depascual@nologin.es) Received: 28 July 2022 – Discussion started: 23 September 2022 Revised: 12 April 2023 – Accepted: 23 April 2023 – Published: 27 September 2023 o de Pascual-Collar1, Roland Aznar1, Bruno Levier2, and Marcos García-Sotillo1 1Nologin Consulting, Avda. de Ranillas 1D, 50018 Zaragoza, Spain 2Mercator Ocean, 8-10, rue Hermès, 31520, Ramonville Saint-Agne, France rnicus Ocean State Report (OSR7) Received: 28 July 2022 – Discussion started: 23 September 2022 Revised: 12 April 2023 – Accepted: 23 April 2023 – Published: 27 September 2023 Abstract. The ocean has the largest heat capacity of any single component of the climate system and plays a key dominant role in global heat content changes. To analyse ocean heat content (OHC) variability in the Iberia–Biscay–Ireland (IBI) region, several Copernicus Marine reanalysis and observational products are used together to provide multi-product estimations of OHC anomalies over the water column (at upper layers 150, 700, and 2000 m). This work provides a study of spatial and temporal variability of OHC in the northeastern Atlantic region through the analysis of area-averaged time series, trend maps, and trends linked with the main water masses found in the IBI region. an State Report (OSR7) The work states the key role that subsurface water masses play in the OHC trends in the IBI region. The study shows that, although no signiﬁcant trends are detected for the surface water layers, the intermediate and deep water layers show signiﬁcant trends (with rates of +0.40 W m−2). However, the high interannual variability of the subsurface water masses masks these trends. Two of the main water masses described in the region (i.e. Sub- Arctic Intermediate Water and Mediterranean Outﬂow Water) are found to explain more than 50 % of the OHC variability. Since the variability of both water masses is linked to the North Atlantic Oscillation, the work shows the mechanisms by which atmospheric forcing is able to affect the subsurface water layers without showing a signal in the surface. P. Lorente et al.: High-frequency radar-derived coastal upwelling index 17 Wooster, W. S., Bakun, A., and McLain, D. R.: Seasonal upwelling cycle along eastern boundary of North-Atlantic, J. Mar. Res., 34, 131–141, 1976. INSITU_GLO_PHY_UV_DISCRETE_NRT_013_048, Is- sue: 2.2, Mercator Ocean International, https://catalogue.marine. copernicus.eu/documents/QUID/CMEMS-INS-QUID-013-048. pdf (last access: 2 June 2023), 2022b. 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S.: Future changes in coastal upwelling ecosystems with global warming: The case of the California Current System, Scientiﬁc Reports, 8, 2866, https://doi.org/10.1038/s41598-018-21247-7, 2018. Wilczak, J. M., Olson, J. B., Djalalova, I., Bianco, L., Berg, L. K., Shaw, W. J., Coulter, R. L., Eckman, R. M., Freedman, J., Finley, C., and Cline, J.: Data assimilation impact of in situ and remote sensing meteorological observations on wind power forecasts during the ﬁrst Wind Forecast Improvement Project (WFIP), Wind Energy, 22, 932–944, https://doi.org/10.1002/we.2332, 2019. https://doi.org/10.5194/sp-1-osr7-8-2023 State Planet, 1-osr7, 8, 2023 CHAPTER2.6 Chapter 2.7 – 7th edition of the Copernicus Ocean State Report (OSR7) Chapter 2.7 – 7th edition of the Copernicus Ocean State Planet, 1-osr7, 9, 2023 https://doi.org/10.5194/sp-1-osr7-9-2023 © Author(s) 2023. This work is distributed under the Creative Commons Attribution 4.0 License. ter 2.7 – 7th edition of the Copernicus Ocean State Report (O Published by Copernicus Publications. 1 Introduction (2022b) 3 INSITU_GLO_PHY_TS_OA_MY_013_052 (CORA) In situ observations EU Copernicus Marine Service Product (2022c) QUID: Szekely (2022a) PUM: Szekely (2022b) 4 MULTIOBS_GLO_PHY_TSUV_3D_MYNRT_015_012 (ARMOR) In situ observations and Satellite observations EU Copernicus Marine Service Product (2021a) QUID: Greiner et al. (2021) PUM: Guinehut (2021) 5 GLOBAL_OMI_OHC_trend (GLO-OMI-trend) Numerical models, in situ observations, and satellite observations EU Copernicus Marine Service Product (2021b) QUID: von Schuckmann et al. (2021) PUM: Monier et al. (2021) and the white ocean, e.g. the sea ice extent OMI (ARC- TIC_OMI_SI_extent). Among the multiple OMIs provided by Copernicus Marine, an important family includes the indicators focused on evaluating OHC trends and variability. Particularly, the indicator GLOBAL_OMI_OHC_trend (see Tables 1 and 2 for more details on the product; hereafter this product will be referred to as GLO-OMI-trend) provides a global map of trends of OHC integrated in the upper 2000 m. Attending to the spatial variability of trends in the IBI region, this global OMI shows spatial variability of trends, ranging from negative trends of −5 W m−2 northward of the parallel 40◦N up to positive trends of 2.5 W m−2 observed westward of the Gulf of Cádiz. However, this global information should be treated cautiously when looking at regions close to coastal areas and marked by the combination of on-shelf and open waters, such as occurs in the IBI domain. in the IBI region we could mention the northern limit of the Canary Current, the upwelling system that inﬂuences the Iberian Peninsula and African coasts, the spreading Mediter- ranean waters that inﬂuence the intermediate hydrographic properties and promote the formation of long-lasting ed- dies, and the inﬂuence of the eastern closure of the sub- tropical gyre that brings warm waters from the Gulf Stream, inﬂuencing the climate of western Europe. In this region, the literature describes a number of water masses but three groups of salinity extrema mainly determine the intermedi- ate hydrographic structure: a subsurface salinity minima con- nected with Sub-Arctic Intermediate Water (SAIW) at po- tential density anomalies 27.3 < σθ kg m−3, salinity maxima connected with Mediterranean Outﬂow Water (MOW) at the neutral density 27.25 < σθ < 27.45 kg m−3, and salinity min- ima connected with Labrador Sea Water (LSW) underneath the isopycnal 27.8 > σθ kg m−3 (Talley and McCarney, 1982; Iorga and Lozier, 1999a, b; van Aken, 2000; Prieto et al., 2013; de Pascual-Collar et al., 2019). 1 Introduction system, this is typically done from the ocean’s surface down to a reduced depth (such as 700 or 2000 m). The choice of these levels is due to the fact before the year 2000, tempera- ture measurements were most often taken in the upper 700 m of the water column, but by about 2005 the Argo array had sufﬁcient space–time sampling to yield an improved mea- surement of OHC down to a depth of 2000 m (von Schuck- mann et al., 2016; Abraham et al., 2013; Cheng et al., 2022, 2017). It is well established that the main storage (> 90 %) of ex- tra heat from increasing greenhouse gases is absorbed by the oceans; consequently, oceans are the dominant source of changes in global warming (Levitus et al., 2005, 2012; Tren- berth et al., 2014; Cheng et al., 2017; von Schuckmann et al., 2020; Gulev et al., 2021). Additionally, the heat stored in oceans has increased during the last decades at basin and global scales (Gulev et al., 2021; Abraham et al., 2013). Therefore, ocean heat content (OHC) is one of the six global climate indicators recommended by the World Meteorolog- ical Organization for Sustainable Development Goal 13 im- plementation (WMO, 2017). The Copernicus Marine service provides marine data cov- ering the global ocean and European seas, splitting them into six different regions. One of those, the Iberian–Biscay– Ireland (IBI) region, covers the northeastern Atlantic from the Canary Islands (26◦N) up to Ireland (55◦N), being limited by the 20◦W meridian and the continental margin. Among the variety of hydrographic processes taking place Ideally, the estimations of OHC are integrated over the full depth of the ocean, but for limitations related to the observing CHAPTER2.7 CHAPTER2.7 Á. de Pascual-Collar et al.: Ocean heat content in the Iberian–Biscay–Ireland regional seas 2 Table 1. List of Copernicus Marine products used for the computation of ocean heat content (OHC) in the Iberia–Biscay–Ireland region (IBI). Product ref. no. Product ID/acronym/type Data access Documentation QUID: Quality Information Document PUM: Product User Manual 1 IBI_MULTIYEAR_PHY_005_002 (IBI-REA) Numerical models EU Copernicus Marine Service Product (2022a) QUID: Levier et al. (2022) PUM: Amo-Baladrón et al. (2022) 2 GLOBAL_MULTIYEAR_PHY_001_030 (GLO-REA) Numerical models EU Copernicus Marine Service Product (2022b) QUID: Drévillon et al. (2022a) PUM: Drévillon et al. Á. de Pascual-Collar et al.: Ocean heat content in the Iberian–Biscay–Ireland regional seas 3 Table 2. Spatial and temporal coverage of products. Product reference Resolution Temporal Vertical coverage coverage 1. IBI-REA 0.083◦× 0.083◦ 1993–2020 0–5500 m (50 levels) 2. GLO-REA 0.083◦× 0.083◦ 1993–2020 0–5500 m (50 levels) 3. CORA ∼0.36◦× 0.5◦ 1993–2021 0–2000 m (152 levels) 4. ARMOR 0.25◦× 0.25◦ 1993–2021 0–5500 m (50 levels) 5. GLO-OMI-trend 0.25◦× 0.25◦ 2005–2009 Integrated 0–2000 (1 level) Table 2. Spatial and temporal coverage of products. Earth’s positive energy imbalance dominates the observed re- gional changes around Europe, but the year-to-year variabil- ity in the region potentially masks the long-term warming trend. Earth’s positive energy imbalance dominates the observed re- gional changes around Europe, but the year-to-year variabil- ity in the region potentially masks the long-term warming trend. a coupling between the spreading areas of both water masses and the NAO index. The present work pursues two main objectives: on one hand, the availability of higher-resolution speciﬁc regional products allows us to reﬁne the study of OHC trends in the IBI region; thus, this work explores the spatial and temporal variability of OHC trends in the region as well as explain- ing its causes in the period 1993–2021, providing a better understanding of the processes controlling such trends. Addi- tionally, since OMIs are simpliﬁed indicators that statistically summarise ocean information, a study of the oceanographic processes behind each indicator is needed to have a proper understanding of the changes represented by the OMI. Thus, the work analyses the use and interpretation of OHC OMIs on regional scales and its sources of variability and uncer- tainty. Since the distribution of various water masses is one of the main sources of variability (spatial and temporal) in the IBI region, analysis of the heat stored by them can provide infor- mation on OHC trends at regional and local scales. For exam- ple, Potter and Lozzier (2004), based on 50 years of hydro- graphic data, studied the temperature and salinity trends of the MOW, ﬁnding positive trends that lead to a heat content gain in the MOW reservoir that surpasses the average gain of the North Atlantic basin. Nonetheless, the literature on OHC variability of speciﬁc water masses in the IBI region is rather limited. Alternatively, since several studies have con- centrated efforts on assessing the thermohaline variability of water masses, they may provide some clues as to what can be expected with respect to OHC. Á. de Pascual-Collar et al.: Ocean heat content in the Iberian–Biscay–Ireland regional seas This paper is organised as follows: Sect. 2 presents the datasets used, as well as the methodology applied, to com- pute the OHC in the IBI region. In Sect. 3 the time series of OHC averaged over the whole IBI region are discussed to provide a general view of the regional trends estimated. Sec- tion 4 is devoted to showing and discussing maps of trends computed in the same way as the ones provided in the GLO- OMI-trend product. Section 5 analyses the vertical proﬁles of OHC trends, studying the variability associated with dif- ferent water masses. Section 6 summarises the availability of the data used in this article. Finally, main conclusions are summed up in Sect. 7. Regarding the SAIW, Leadbetter et al. (2007) studied the temperature variability of the water column in the period 1981–2005 in a section at 36◦N, concluding that the vari- ability in the SAIW is consistent with the displacement of neutral density surfaces driven by changes in surface wind forcing and linked with North Atlantic Oscillation (NAO). Several studies have focused on the temporal variability of MOW. The analysis of hydrographic properties in the MOW core concluded that changes in MOW properties are not dom- inated by changes in Mediterranean Sea Water properties (Lozier and Sindlinger, 2009; Bozec et al., 2011). However, the variability of MOW is strongly inﬂuenced by year-to- year processes. Some studies described the MOW interan- nual variability as an oscillation of the water tongue shifting the dominant spreading pathway and interacting with the un- derlying water masses such as North Atlantic Deep Water and LSW (Bozec et al., 2011; de Pascual-Collar et al., 2019); this variability is also correlated with NAO. 1 Introduction The density ranges for these water masses show considerable overlap, which allows both diapycnal and isopycnal mixing. In the last decades, the upper North Atlantic Ocean experi- enced a reversal of climatic trends for temperature and salin- ity. While the period 1990–2004 is characterised by decadal- scale ocean warming, the period 2005–2014 shows a substan- tial cooling and freshening (González-Pola et al., 2020; Hol- liday et al., 2020; Somavilla et al., 2009). Such changes are discussed to be linked to ocean internal dynamics and air–sea interactions (Fox-Kemper et al., 2021; Collins et al., 2019; Robson et al., 2016), together with changes linked to the con- nectivity between the North Atlantic Ocean and the Mediter- ranean Sea (Masina et al., 2022). Previous works show a con- sistency of regional OHC with this decadal-scale variabil- ity in the IBI region (von Schuckmann et al., 2016, 2018); however, in spite of the year-to-year variability, a long-term warming trend of 0.9 ± 0.4 W m−2 in the upper 700 m of the IBI region is also detected. These studies concluded that the Among the Copernicus Marine Service products are the Ocean Monitoring Indicators (OMIs), which are free downloadable trends and datasets covering the past quarter of a century. These are indicators that allow us to track the vital health signs of the ocean and changes in line with climate change. These OMIs are proposed to cover the blue ocean, e.g. the mean volume transport across sections from Reanalysis OMI (GLOBAL_OMI_WMHE_voltrp); the green ocean, e.g. the chlorophyll a time se- ries and trends from Observations Reprocessing OMI (OMI_HEALTH_CHL_GLOBAL_OCEANCOLOUR_trend); https://doi.org/10.5194/sp-1-osr7-9-2023 State Planet, 1-osr7, 9, 2023 CHAPTER2.7 CHAPTER2.7 3 Analysis of OHC time series Figure 1 shows combined results of products IBI-REA, GLO-REA, CORA, and ARMOR (listed in Tables 1 and 2), with panels displaying the ensemble mean and the ensem- ble standard deviation of OHC for the period 1993–2021. Corresponding to the ensemble spread, time series reveal an increase in uncertainties when deeper layers are included (0–700 and 0–2000 m); this result can be explained by the decrease in observational data available in deeper levels. It is worth noting the decrease in uncertainties observed after 2003 in the time series integrated over the upper 2000 m. These differences in uncertainties are explained considering the remarkable improvement in the global ocean observing system achieved with the implementation of the global Argo array in 2005 (von Schuckmann et al., 2016; Abraham et al., 2013; Cheng et al., 2022, 2017). The period 1999–2001 shows larger uncertainties observed in the record. This in- dicates larger discrepancies between ensemble members in representing the minimum of OHC observed in this period. These discrepancies affect both the year when the minimum is reached (while GLO-PHY-REA estimates the minimum in 2000, the other products reach the minimum in 2001) and the magnitude of the minimum values (with all products agree- ing on observed negative anomalies during this year). Each product used is supplied for speciﬁc vertical levels with speciﬁc thickness; all vertical integrations were com- puted taking into consideration the thickness of each product level. Similarly, since most of the products used are provided with regular lat–long grids, the surface of each grid cell de- pends on the latitude, and the spatial averages (when used in this study) were computed considering the speciﬁc surface of each product grid cell. This work shows different results of OHC, mixing differ- ent products with speciﬁc spatial resolutions (see Table 2). The OHC has been computed for all products using Eq. (1) on the grid of the distributed products and integrating results (from the surface down to 150, 700, and 2000 m) using the product layer thicknesses. The presentation of the results as time series or as maps implies a difference in the way the re- sults are averaged to compute the ensemble mean and spread. Regarding the time series of OHC, anomalies were calcu- lated referenced to the climatic mean of the period 1993– 2019. Then the vertical integrations and spatial average were obtained, preserving the service grid of each product. 2 Data and methods Following the same methodology as in previous Copernicus Marine Ocean State Report contributions (Lima et al., 2020; Mayer et al., 2021), the estimates of OHC anomalies were computed in the IBI region according to the follow equation: OHC = z2 z1 ρ0Cp (Tm −Tclim)dz, (1) The interannual variability of LSW has been described in previous works, concluding that some of the variability in the water mass can be explained by diapycnal mixing with the overlying MOW (van Aken, 2000) as well as changes in the source regions over the North Atlantic basin (Leadbetter et al., 2007). Additionally, Bozec et al. (2011) studied the distribution of LSW and MOW from 1950 to 2006, observing (1) where ρ0 is the density at a reference depth (ρ0 = 1020 kg m−3), Cp is the speciﬁc heat capacity (Cp = 4181.3 J kg−1 ◦C−1), z1 and z2 are the range of depths to compute the total OHC, Tm is the monthly average https://doi.org/10.5194/sp-1-osr7-9-2023 State Planet, 1-osr7, 9, 2023 State Planet, 1-osr7, 9, 2023 CHAPTER2.7 CHAPTER2.7 CHAPTER2.7 Á. de Pascual-Collar et al.: Ocean heat content in the Iberian–Biscay–Ireland regional seas 4 potential temperature at a speciﬁc depth, and Tclim is the climatological potential temperature of the corresponding month and depth. grid, it was considered to be the reference grid; thus, CORA and ARMOR products were spatially interpolated. This in- terpolation was made over the OHC integrated on the respec- tive layer before averaging the ensemble. The comparison of interpolated and non-interpolated results of each ensemble member did not show relevant changes in the ﬁeld structure or mathematical artefacts. As can be seen in Eq. (1), the OHC anomalies are obtained from integrated differences between the monthly tempera- ture and the climatological one along a vertical proﬁle in the ocean. In the present work the anomalies have been refer- enced to the monthly climatology computed between 1993 and 2019. Additionally, the OHC is presented for the typical depths of 700 and 2000 m, but also for the upper 150 m to analyse the OHC variability in the upper layer. This paper focuses on the analysis of OHC trends in the IBI regions. For this purpose, OHC trends are analysed on different time periods within the registry. 2 Data and methods The selection of time periods corresponds to different criteria such as the de- tection of trend changes, comparison of results with other works, or selection of periods representative of large-scale patterns. In the case of the analysis of vertical trend proﬁles (Sect. 5), the time record 1993–2021 has been divided into two periods characterised by a differentiated behaviour of the NAO (NOAA, 2022), considering that (i) the NAO in- dex in 1994 was moderately positive and (ii) it has a min- imum in 2010 and (iii) a maximum in 2018; the periods 1993–2010 and 2010–2019 were selected as representative of NAO changes in phase from positive to negative (period 1993–2010) and vice versa, from negative to positive (period 2010–2019; see Fig. S1 in the Supplement). In order to allow assessing uncertainties of results, differ- ent Copernicus products were used to provide multi-product estimations of OHC; therefore, all results were previously computed for a collection of Copernicus products and com- bined to give an ensemble mean and the standard deviation of the ensemble. Since the objective of this work is the anal- ysis of OHC in the IBI region integrating results from the surface down to a maximum of 2000 m depth, this study has included all Copernicus Marine products that deliver gridded data on potential temperature with vertical coverage from the surface down to at least 2000 m. As shown in Tables 1 and 2, four different products meet these stated conditions: two model reanalyses (the global and regional ones: GLO-REA and IBI-REA, respectively) and two observation-based prod- ucts (CORA and ARMOR). It is worth mentioning that when OHC was computed, the GLO-REA product did not cover the year 2020, so that year was not considered in the men- tioned product. 4 Analysis of regional trends of OHC Figure 2 shows trend maps of OHC computed at 0–2000 m for the IBI region. The ﬁgure includes the computation of trends considering two different periods. The period 2005– 2019 was selected to allow comparison of results obtained in this work (Fig. 2a) with those obtained with the prod- uct GLO-OMI-trend (Fig. 2b), computed for the same pe- riod and cropped in the ﬁgure to only show the study region. On the other hand, the period 1993–2021 was selected to study the spatial distribution of long-term trends in the IBI region (Fig. 2c). The comparison of results obtained in this work with the GLO-OMI-trend (Fig. 2a and b) shows a high level of agreement, with differences mostly being related to the different resolution. Thus, both ﬁgures estimate a neg- ative trend that mainly affects the offshore ocean north of 38◦N and a tongue between 31 and 38◦N that shows warm- ing trends. Figure 1. OHC averaged in the IBI domain integrated from the ocean’s surface down to 150 m (a), 700 m (b), and 2000 m (c). Time series computed from four Copernicus Marine products (i.e. IBI- REA, GLO-REA, CORA, and ARMOR), providing a multi-product approach. The blue line represents the ensemble mean, and shaded grey areas represent the standard deviation of the ensemble. The analysis of trends (at 95 % conﬁdence interval) computed in the pe- riod 1993–2020 is included (bottom right box). Trend lines (dashed line) are only included in the ﬁgures when a signiﬁcant trend is ob- tained. cant. Considering that the trend integrated at 0–2000 m is at the limit of signiﬁcance, we can consider this to be a mathe- matical result; thus, results suggest a dominance of the vari- ability at intermediate levels over the OHC trend. It is worth mentioning that, despite the positive trends observed in the whole period (1993–2020) of time series integrated down to 700 and 2000 m, a change in trends of these time series after the year 2005 can be appreciated. Table 3 shows the trend analysis by dividing the time record into two halves: the pe- riod 1993–2005 and the period 2005–2021. While the results show a signiﬁcant warming of integration depths 0–700 and It worth mentioning that the higher resolutions of the prod- ucts used in this work allow computing OHC trends along the northwestern European shelf, showing a signiﬁcant warming of the region. 3 Analysis of OHC time series Additionally, this reversal of the trend explains the discrepancies observed between the long- term trends estimated in this work and the ones observed at 0–700 m in von Schuckmann et al. (2016) (0.8 ± 0.3 W m−2) and von Schuckmann et al. (2018) (0.9 ± 0.4 W m−2) for the periods 1993–2015 and 1993–2016, respectively. The cited works exclude the last years, which have a large inﬂuence on the overall trend. 3 Analysis of OHC time series There- fore, the ensemble time series are averaged from time series computed over the service grid of each product. However, to compute ensemble results that comprise spatial informa- tion (i.e. results presented as maps), each ensemble member must be interpolated to obtain all estimations on the same grid. Since IBI-REA and GLO-REA products share the same Trends of time series obtained in Fig. 1 show a barely sig- niﬁcant warming of the IBI region in the upper (0–700 m; 0.39 ± 0.27 W m−2) and intermediate to deep layers (0– 2000 m; 0.40 ± 0.39 W m−2); conversely, such a trend does not affect the surface layer, where the trend is not signiﬁ- State Planet, 1-osr7, 9, 2023 State Planet, 1-osr7, 9, 2023 https://doi.org/10.5194/sp-1-osr7-9-2023 CHAPTER2.7 Á. de Pascual-Collar et al.: Ocean heat content in the Iberian–Biscay–Ireland regional seas Á. de Pascual-Collar et al.: Ocean heat content in the Iberian–Biscay–Ireland regional seas 5 Table 3. Mean OHC trends (in W m−2) averaged in the IBI do- main and integrated from the ocean’s surface down to 150, 700, and 2000 m. The conﬁdence interval of trends is computed with 95 % conﬁdence. In bold are the values for which the absolute trend ex- ceeds the conﬁdence interval. Figure 1. OHC averaged in the IBI domain integrated from the ocean’s surface down to 150 m (a), 700 m (b), and 2000 m (c). Time series computed from four Copernicus Marine products (i.e. IBI- REA, GLO-REA, CORA, and ARMOR), providing a multi-product approach. The blue line represents the ensemble mean, and shaded grey areas represent the standard deviation of the ensemble. The analysis of trends (at 95 % conﬁdence interval) computed in the pe- riod 1993–2020 is included (bottom right box). Trend lines (dashed line) are only included in the ﬁgures when a signiﬁcant trend is ob- tained. Integration OHC trend (W m−2) computed depth (m) in period 1993–2005 2005–2021 1993–2021 0–150 0.36 ± 0.46 0.03 ± 0.25 0.10 ± 0.12 0–700 1.6 ± 0.57 –0.56 ± 0.35 0.39 ± 0.27 0–2000 1.86 ± 1.09 –1.02 ± 0.48 0.40 ± 0.39 0–2000 in the period 1993–2005, the period 2005–2021 is characterised by a cooling of both integration layers. This re- sult is consistent with the decadal-scale warming and fresh- ening observed in the North Atlantic Ocean in the period 1990–2020 (González-Pola et al., 2020; Holliday et al., 2020; Somavilla et al., 2009). 4 Analysis of regional trends of OHC This warming may seem low when compared with the values observed in open ocean, but it should be con- sidered that this warming is affected by the shallower depths in the region. For the sake of brevity, Fig. 2 only shows re- sults for the 0–2000 m layer; however, as occurred in the State Planet, 1-osr7, 9, 2023 State Planet, 1-osr7, 9, 2023 https://doi.org/10.5194/sp-1-osr7-9-2023 CHAPTER2.7 CHAPTER2.7 Á. de Pascual-Collar et al.: Ocean heat content in the Iberian–Biscay–Ireland regional seas Á. de Pascual-Collar et al.: Ocean heat content in the Iberian–Biscay–Ireland regional seas 6 Figure 2. Regional maps of OHC trend computed at 0–2000 m (a) over the periods 2005–2019 and (c) 1993–2021. Trends computed using the four Copernicus Marine products (IBI-REA, GLO-REA, CORA, and ARMOR), providing a multi-product approach. Shaded colours represent mean trends (using all products), while shaded grey indicates areas with less robust signatures (where the noise – ensemble standard deviation of trends – exceeds the signal – ensemble mean). (b) Same as panels (a) and (c) but obtained from the product GLO-OMI-trend over the period 2005–2019. Figure 2. Regional maps of OHC trend computed at 0–2000 m (a) over the periods 2005–2019 and (c) 1993–2021. Trends computed using the four Copernicus Marine products (IBI-REA, GLO-REA, CORA, and ARMOR), providing a multi-product approach. Shaded colours represent mean trends (using all products), while shaded grey indicates areas with less robust signatures (where the noise – ensemble standard deviation of trends – exceeds the signal – ensemble mean). (b) Same as panels (a) and (c) but obtained from the product GLO-OMI-trend over the period 2005–2019. tical proﬁle of trends in the region. However, this analysis cannot be carried out for all products and for all points of the grid. Therefore, it has been decided to study the trend proﬁle obtained from the IBI-REA product for two charac- teristic subregions centred at 35 and 48◦N (Fig. 3a and b). Hereafter these subregions will be referred to as 35N and 48N, respectively. time series analysed in the previous section, the observed trends (positive and negative) are subsurface-intensiﬁed, so the larger the integration depth, the larger the observed trend (results not shown). Such intensiﬁcation of trends suggests that both signals are stronger underneath the upper layer, sug- gesting that they are more related to the evolution of interme- diate and deep water masses than to the year-to-year interac- tion with the atmosphere. 4 Analysis of regional trends of OHC The IBI-REA product was selected for this purpose be- cause it assimilates observational (in situ and satellite) data and uses the GLO-REA product as initial and boundary con- ditions, therefore indirectly incorporating from the parent products information from the other products. On the other hand, the subregions chosen were selected to provide a de- tailed analysis of the main features described in the previous section. A ﬁrst analysis has shown that, as seen with verti- cally integrated data, the resulting trends show a dependency on the selected period. Hence, as explained in the “Data and methods” section (Sect. 2), in order to provide information on the temporal variability of the water bodies in the IBI area, the trend analysis has been performed by dividing the time period into two periods representative of the positive and negative trend of the NAO index: the period 1993–2010 when the NAO evolves from positive to negative and the pe- riod 2010–2019 representing the NAO transition from nega- tive to positive. Examining trends computed for the whole time record (Fig. 2c) we conclude that, as estimated by the averaged time series in the previous section, the trend calculated for the whole time record indicates a generalised warming of the IBI area. However, the signiﬁcance of such warming is smaller, existing in regions with no evidence of OHC trend. As occurred with the analysis of time series, trend maps show a dependence of the results on the time period selected for the analysis (comparing Fig. 2a and c). This suggests a strong inﬂuence of interannual processes on the observed trends. However, it is worth mentioning that the warming trend of the region around 34◦N latitude is consistent for both analyses. State Planet, 1-osr7, 9, 2023 5 Analysis of OHC trends across different water masses 2b and 3b are not directly comparable, since temporal coverage differs. Table 4. Mean thermic trends (in W m−2) of SAIW, MOW, and 0–2000 m in the IBI subregions (35N and 48N) computed for the period 1993–2021 with the product IBI-REA. Signiﬁcance of trends computed with 95 % conﬁdence. Table 4. Mean thermic trends (in W m−2) of SAIW, MOW, and 0–2000 m in the IBI subregions (35N and 48N) computed for the period 1993–2021 with the product IBI-REA. Signiﬁcance of trends computed with 95 % conﬁdence. Water type Averaged trend (W m−2) in IBI subregions (1993–2021) 35N 48N Sub-Arctic Intermediate Water (SAIW) +0.52 (44 %) +0.40 (27 %) Mediterranean Outﬂow Water (MOW) +0.52 (44 %) +0.36 (24 %) Total (0–2000 m) +1.18 +1.46 Water type Averaged trend (W m−2) in IBI subregions (1993–2021) 35N 48N Sub-Arctic Intermediate Water (SAIW) +0.52 (44 %) +0.40 (27 %) Mediterranean Outﬂow Water (MOW) +0.52 (44 %) +0.36 (24 %) Total (0–2000 m) +1.18 +1.46 Water type The trends of OHC averaged for the subregions deﬁned at 35N and 48N have been computed at each level to ob- tain a vertical proﬁle of trends. These trend proﬁles are pre- sented in Fig. 3c, d, e, and f combined with the temperature and salinity data on a yearly basis. Each proﬁle shown in the θ/S diagrams corresponds to the annual mean temperature and salinity observed in the selected period (1993–2010 or 2010–2021) and averaged over the corresponding study re- gion (35N or 48N). The different markers used correspond to the different OHC trends observed for each depth. There- fore, the shown θ/S diagrams allow discussing the trends of OHC at each layer, linking these results with the different water masses observed in the region. Apart from the estimation of density levels deﬁning each water mass (shown in Fig. 3), an estimation of the heat trends for each water mass was done for the entire study period of 1993–2021 (see results in Table 4). It is observed that, in line with the results shown in Fig. 2, the overall OHC trends in both regions are positive. 5 Analysis of OHC trends across different water masses The analysis of OHC trend maps for these two periods shows that during the transition to negative NAO phases (Fig. 3a), the water masses in the region undergo signiﬁ- Since it has been observed in the data that OHC trends are accentuated in depth, the following section analyses the ver- https://doi.org/10.5194/sp-1-osr7-9-2023 State Planet, 1-osr7, 9, 2023 CHAPTER2.7 Á. de Pascual-Collar et al.: Ocean heat content in the Iberian–Biscay–Ireland regional seas Á. de Pascual-Collar et al.: Ocean heat content in the Iberian–Biscay–Ireland regional seas 7 . (a, b) Maps of regional trends of OHC (0–2000 m) over the periods 1993–2010 (a) and 2010–2019 (b) derived from the Grey shaded areas represent regions where the trend is not signiﬁcant (95 % conﬁdence). Dashed rectangles denote the subre N where θ/S diagrams are averaged in other panels. (c–f) θ/S diagrams averaged in subregions (35 and 48◦N) and over th 10 and 2010–2019. (c) Region 48◦N period 1993–2010, (d) region 48◦N period 2010–2019, (e) region 35◦N period 19 gion 35◦N period 2010–2019. Figure 3. (a, b) Maps of regional trends of OHC (0–2000 m) over the periods 1993–2010 (a) and 2010–2019 (b) derived from the IBI-REA product. Grey shaded areas represent regions where the trend is not signiﬁcant (95 % conﬁdence). Dashed rectangles denote the subregions 35 and 48◦N where θ/S diagrams are averaged in other panels. (c–f) θ/S diagrams averaged in subregions (35 and 48◦N) and over the periods 1993–2010 and 2010–2019. (c) Region 48◦N period 1993–2010, (d) region 48◦N period 2010–2019, (e) region 35◦N period 1993–2010, and (f) region 35◦N period 2010–2019. CHAPTER2.7 https://doi.org/10.5194/sp-1-osr7-9-2023 State Planet, 1-osr7, 9, 2023 State Planet, 1-osr7, 9, 2023 https://doi.org/10.5194/sp-1-osr7-9-2023 CHAPTER2.7 CHAPTER2.7 Á. de Pascual-Collar et al.: Ocean heat content in the Iberian–Biscay–Ireland regional seas Á. de Pascual-Collar et al.: Ocean heat content in the Iberian–Biscay–Ireland regional seas 8 Á. de Pascual-Collar et al.: Ocean heat content in the Iberian–Biscay–Ireland regional sea cant warming over almost the entire IBI area. However, this warming is not signiﬁcant in part of the southern half of the domain, in the vicinity of the Gulf of Cádiz and the Seine and Horseshoe Abyssal Plains. In contrast, the trend map associated with the NAO transition period towards positive values (Fig. 3b) reveals a generalised cooling pattern with a warming trend around the Horseshoe region similar to that observed in Fig. 2b. Take into account, however, the fact that Figs. 5 Analysis of OHC trends across different water masses Considering that each wa- ter mass has different thickness (SAIW approximately 300 m and MOW approximately 400–500 m), we must conclude that although the total trend shown in Table 4 is of the same magnitude for both water masses, the heat gain per cubic me- tre is higher for SAIW than for MOW. Additionally, the rel- ative inﬂuence should be noted that both water masses have on the water column trends down to 2000 m, explaining the sum of the two water masses (a layer approximately 700– 800 m thick): 88 % (51 %) of the OHC trend in 35N (48N), respectively. The θ/S proﬁles shown in Fig. 3c, d, e, and f are consis- tent with the water masses that the literature has described in the northeastern Atlantic: the SAIW characterised by a salin- ity minima at potential density σθ = 27.2 kg m−3, the salin- ity maxima connected with MOW at the neutral density sur- face σθ = 27.6 kg m−3, and a deeper salinity minima corre- sponding to LSW underneath the isopycnal σθ = 27.8 kg m−3 (Talley and McCarney, 1982; Iorga and Lozier, 1999a, b; van Aken, 2000; Prieto et al., 2013; de Pascual-Collar et al., 2019); however, the core of LSW is only clearly visible in subregion 48N where this water mass has a greater presence. 6 Conclusions The present work uses several Copernicus Marine products to generate a multi-product OMI of ocean heat content from the surface down to a set of depths over the period 1993– 2021. These indicators are able to detect decadal trends of OHC. Speciﬁcally, the IBI region presents a warming of +0.39 W m−2 in the upper 700 m. However, the study of both time series trends and spatial distribution of trends shows a high sensitivity to the time period selected, and therefore the high inter-decadal variability detected makes these trends not very signiﬁcant. On the other hand, the OMIs integrated up to 150 and 2000 m does not allow detecting clear signiﬁcant trends of results, which suggest that OHC variability in the upper 2000 m may be mainly controlled by intermediate lev- els. Regarding the OHC variability of SAIW, results in Fig. 3 show a similar behaviour of the water mass at 48N and 35N. Thus, at both latitudes, the SAIW shows a warming (cool- ing) associated with the negative (positive) NAO transition. On the contrary, the OHC trends of MOW shows an inverse behaviour between the subregions 35N and 48N. Thus, while in subregion 35N, the MOW experiences a cooling (warm- ing) associated with the negative (positive) NAO transition (periods 1993–2010 and 2010–2021, respectively), in subre- gion 48N the opposite occurs, and a warming (cooling) of the MOW associated with the negative (positive) NAO tran- sition is observed. These are consistent with the processes described in the literature. Thus, the SAIW trends are coher- ent with the changes in wind forcing associated with NAO as described by Hurrel (1995) and Leadbetter et al. (2007), and the MOW results are consistent with studies that observed a signiﬁcant anticorrelation between the westward and north- ward transport of MOW (Bozec et al., 2011) as well as works that describe the east–west shift of water mass boundaries in the Horseshoe basin (de Pascual-Collar et al., 2019). Although the regional analysis of OHC trends integrated in different depths may provide some clues about the ori- gin of such trends, a ﬁner analysis focusing on the differ- ent water masses involved concludes that the vertically in- tegrated trend is the result of different trends (positive and negative) contributing at different layers. 6 Conclusions The analysis of wa- ter masses in the region shows the existence of three water masses (well referenced in the literature): SAIW, MOW, and State Planet, 1-osr7, 9, 2023 https://doi.org/10.5194/sp-1-osr7-9-2023 CHAPTER2.7 Á. de Pascual-Collar et al.: Ocean heat content in the Iberian–Biscay–Ireland regional seas Á. de Pascual-Collar et al.: Ocean heat content in the Iberian–Biscay–Ireland regional seas 9 Á. de Pascual-Collar et al.: Ocean heat content in the Iberian–Biscay–Ireland regional seas LSW. Since the LSW is found in the depth limit established here (2000 m), we put the focus on studying the temporal variability of the heat stored by the other two involved water masses: the SAIW and MOW. The study states that the IBI- REA can simulate the OHC behaviour of SAIW and MOW in a consistent way, with the OHC variability being associ- ated with the NAO, consistent with the variability described in the literature. Supplement. The supplement related to this article is available online at: https://doi.org/10.5194/sp-1-osr7-9-2023-supplement. Author contributions. This work was conceptualised through the participation of all authors. Data analysis and drafting of the ﬁrst version were carried out by AdPC. Additionally, RA, BL, and MGS contributed to the ﬁnal writing and subsequent revisions of the manuscript. Despite the results showing a strong relationship between the variability of the NAO and the OHC of SAIW and MOW, both water masses show a differentiated behaviour. While SAIW shows a homogeneous behaviour over the whole IBI area (OHC trends associated with NAO have the same sign in subregions 35N and 48N), MOW shows an anticorrelated behaviour between subregions 35N and 48N (when the trend is positive in 35N, in 48N it is negative and vice versa). Competing interests. The contact author has declared that none of the authors has any competing interests. Disclaimer. Publisher’s note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. p g The work states the key role that subsurface water masses play in the OHC trends in the IBI region by comput- ing the OHC trends for SAIW and MOW over the period 1993–2021. The results show that, despite high interannual variability, both water masses have experienced signiﬁcant warming in the study period. Additionally, the relative inﬂu- ence that these two water masses have on the integrated OHC trends down to 2000 m is remarkable. 6 Conclusions Thus, it is found that the sum of both water masses accounts for up to 88 % of the integrated OHC variability from the surface down to 2000 m. However, at more northerly latitudes, this relative inﬂuence decreases to 50 %. The greater presence of LSW in the north- ern regions of the IBI domain suggests that this water mass may share relevance with respect to OHC trends with SAIW and MOW; however, this hypothesis has not been demon- strated in the present work. Acknowledgements. The authors thank the Copernicus Marine Environment Monitoring Service for providing the data for the arti- cle. Additionally, the helpful comments of Karina von Schuckmann and the other referees are gratefully acknowledged. Review statement. This paper was edited by Marta Marcos and reviewed by Carolina M. L. Camargo and Cesar Gonzalez-Pola. 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Valeria Di Biagio, Riccardo Martellucci, Milena Menna, Anna Teruzzi, Carolina Amadio, Elena Mauri, and Gianpiero Cossarini National Institute of Oceanography and Applied Geophysics – OGS, Trieste, Italy Correspondence: Valeria Di Biagio (vdibiagio@ogs.it) Received: 22 July 2022 – Discussion started: 30 September 2022 Revised: 13 March 2023 – Accepted: 31 March 2023 – Published: 27 September 2023 rnicus Ocean State Report (OSR7) ean State Report (OSR7) Abstract. Oxygen is essential to all aerobic organisms, and its dynamics in the ocean involve interconnected physical and biological processes that form the basis of the functioning of the marine ecosystem. The study of dissolved oxygen (DO) variations under multiple drivers is currently one of the main goals of climate and marine-ecological scientiﬁc communities, and the quantiﬁcation of DO levels is essential for the assessment of the environmental status, especially in coastal areas. We investigate the 1999–2021 interannual variability in DO in the southern Adriatic Sea, a marginal area of the Mediterranean Sea, where deep-water formation processes occur, contributing signiﬁcantly to the ventilation of the eastern Mediterranean Basin. Following the Marine Strategy Framework Directive, which promotes the integration of different observational platforms, we use DO modelled by the Copernicus Marine Mediterranean Sea biogeochemical reanalysis, which assimilates satellite chlorophyll concentrations and to which we apply a bias correction using DO Argo ﬂoat measurements in 2014–2020. A correlation analysis of the time series of the ﬁrst three modes of variability (86 % of the total variance) of the DO proﬁles extracted from the bias-corrected reanalysis with key meteo-marine indicators shows a link with (i) net heat ﬂuxes related to oxygen solubility, (ii) vertical mixing, (iii) biological production at the surface and in subsurface layers, and (iv) circulation asso- ciated with the entrance of northern Adriatic waters. The alternating entrance of Levantine and Atlantic waters through the North Ionian Gyre (NIG) appears to be the driver of the fourth mode of variability, which explains 8 % of the total variance. Moreover, we ﬁnd that the ﬁrst temporal mode of variability is the main driver of the negative anomaly of DO in the 0–600 m layer in 2021 with respect to the 1999–2020 climatology. We ascribe the lower content of DO in 2021 to a negative anomaly of the subsurface biological production in the same year, in agreement with the previous correlation analysis, but not to heat ﬂuxes. Indeed, in agreement with previous studies, we observe a sharp increase in salinity favoured by the cyclonic circulation of NIG from 2019 onwards. Á. de Pascual-Collar et al.: Ocean heat content in the Iberian–Biscay–Ireland regional seas Cossarini, G., Dagneaux, Q., Desportes, C., Dye, S., Fratianni, C., Good, S., Greiner, E., Gourrion, J., Hamon, M., Holt, J., Hy- der, P., Kennedy, J., Manzano-Muñoz, F., Melet, A, Meyssignac, von Schuckmann, K., Storto, A., Simoncelli, S., Raj, R. P., Samuelsen, A., Pascual-Collar, A., Garcia-Sotillo, M., and Sz- erkely, T.: Ocean Heat Content, in: Copernicus Marine Service State Planet, 1-osr7, 9, 2023 https://doi.org/10.5194/sp-1-osr7-9-2023 CHAPTER2.7 Chapter 3.1 – 7th edition of the Copernicus Ocean State Report (OSR7) State Planet, 1-osr7, 10, 2023 https://doi.org/10.5194/sp-1-osr7-10-2023 © Author(s) 2023. This work is distributed under the Creative Commons Attribution 4.0 License. Chapter 3.1 – 7th edition of the Copernicus Ocean ter 3.1 – 7th edition of the Copernicus Ocean State Report (O Dissolved oxygen as an indicator of multiple drivers of the marine ecosystem: the southern Adriatic Sea case study ition of the Copernicus Ocean State Report (OSR7) Valeria Di Biagio, Riccardo Martellucci, Milena Menna, Anna Teruzzi, Carolina Amadio, Elena Mauri, and Gianpiero Cossarini National Institute of Oceanography and Applied Geophysics – OGS, Trieste, Italy Correspondence: Valeria Di Biagio (vdibiagio@ogs.it) Received: 22 July 2022 – Discussion started: 30 September 2022 Revised: 13 March 2023 – Accepted: 31 March 2023 – Published: 27 September 2023 Valeria Di Biagio, Riccardo Martellucci, Milena Menna, Anna Teruzzi, Carolina Amadio, Elena Mauri, and Gianpiero Cossarini National Institute of Oceanography and Applied Geophysics – OGS, Trieste, Italy Correspondence: Valeria Di Biagio (vdibiagio@ogs.it) Received: 22 July 2022 – Discussion started: 30 September 2022 Revised: 13 March 2023 – Accepted: 31 March 2023 – Published: 27 September 2023 Valeria Di Biagio, Riccardo Martellucci, Milena Menna, Anna Teruzzi, Carolina Amadio, Elena Mauri, and Gianpiero Cossarini National Institute of Oceanography and Applied Geophysics – OGS, Trieste, Italy 1 Introduction elongated, semi-enclosed, and roughly north–south-oriented basin characterized by a shallow northern shelf (shallower than 80 m) and a deep pit in its southern part (maximum depth of approximately 1200 m) which is connected to the Io- nian Sea (central Mediterranean Basin) through the Otranto Strait (with a maximum depth of 800 m). The Adriatic Sea is characterized by a cyclonic circulation governed by several drivers: river runoff, wind stress, surface buoyancy ﬂuxes, and mass exchanges through the Otranto Strait (Cushman- Roisin et al., 2013). Dissolved oxygen (DO) is a key indicator for monitoring the marine ecosystem functioning because it is the result of several atmospheric, hydrodynamic, and biogeochemical driving processes (such as air–sea ﬂuxes, vertical convec- tion and mixing, horizontal transport, and biological produc- tion and consumption; Keeling and Garcia, 2002; Oschlies et al., 2018; Pitcher et al., 2021). Indeed, DO is currently being studied under the global warming scenarios by climate and marine-ecological scientiﬁc communities (e.g. IPCC, 2019; Kwiatkowski et al., 2020; Garcia-Soto et al., 2021), as oxy- gen depletion has been observed in the global ocean as well as at the local scale (Breitburg et al., 2018). Climate models predict a reduction in global-ocean dissolved oxygen con- tent (Matear et al., 2000; Oschlies et al., 2008; Stramma et al., 2010; Reale et al., 2022), so this parameter is of primary interest, especially in those areas where oceanic processes connect surface and deep layers. The SAdr is strongly inﬂuenced by the inﬂow of water masses from the northern Adriatic Sea (i.e. North Adriatic Dense Water; Querin et al., 2016) and the Ionian Sea. In par- ticular, the inﬂow of southern water masses is triggered by the periodic reversal of North Ionian Gyre circulation (Gaˇci´c et al., 2002; Civitarese et al., 2010; Menna et al., 2019; Gaˇci´c et al., 2021). This oscillating system, called the Adriatic– Ionian Bimodal Oscillating System (BiOS), changes the cir- culation of the North Ionian Gyre from cyclonic to anticy- clonic and vice versa, modulating the advection of water masses in the Adriatic Sea (Gaˇci´c et al., 2010; Rubino et al., 2020). The cyclonic circulation of the North Ionian Gyre causes the advection of saline water masses of Levantine ori- gin (i.e. Levantine Intermediate Water, LIW; Cretan Inter- mediate Water; Ionian Surface Water; and Levantine Surface The southern Adriatic Sea (SAdr; Fig. Valeria Di Biagio, Riccardo Martellucci, Milena Menna, Anna Teruzzi, Carolina Amadio, Elena Mauri, and Gianpiero Cossarini We interpret this as a possible regime shift that is not captured by the time series analysis and whose possible consequences for Ionian–Adriatic system ventilation and for marine organisms should be carefully monitored in the near future. Published by Copernicus Publications. CHAPTER3.1 V. Di Biagio et al.: Dissolved oxygen in the southern Adriatic Sea 2 Table 1. Products used in the present work. Prod. ref. nos. 4 and 5 are a forcing for prod. ref. nos. 1 and 2, and prod. ref. no. 8 is a forcing for prod. ref. nos. 4 and 5. Prod. Product ID and type Data access Documentation ref. no. 1 MEDSEA_MULTIYEAR_BGC_006_008 EU Copernicus Marine Service Quality Information Document (multi-year product), numerical models Product (2022a) (QUID): Teruzzi et al. (2022); Product User Manual (PUM): Lecci et al. (2022a) 2 MEDSEA_MULTIYEAR_BGC_006_008 EU Copernicus Marine Service QUID: Teruzzi et al. (2022); (INTERIM product), numerical models Product (2022b) PUM: Lecci et al. (2022a) 3 INSITU_MED_PHYBGCWAV_DISCRETE EU Copernicus Marine Service QUID: Wehde et al. (2022); _MYNRT_013_035, in situ observations Product (2022c) PUM: Pouliquen et al. (2022) 4 MEDSEA_MULTIYEAR_PHY_006_004 EU Copernicus Marine Service QUID: Escudier et al. (2022); (multi-year product), numerical models Product (2022d) PUM: Lecci et al. (2022b) 5 MEDSEA_MULTIYEAR_PHY_006_004 EU Copernicus Marine Service QUID: Escudier et al. (2022); (INTERIM product), numerical models Product (2022e) PUM: Lecci et al. (2022b) 6 SEALEVEL_EUR_PHY_L4_MY_008_068, EU Copernicus Marine Service QUID: Pujol et al. (2023); satellite observations Product (2023b) PUM: Pujol (2022) 7 SEALEVEL_EUR_PHY_L4_NRT EU Copernicus Marine Service QUID: Pujol et al. (2023) _OBSERVATIONS_008_060, Product (2023a) PUM: Pujol (2022) satellite observations 8 ERA5 hourly data on single levels from https://doi.org/10.24381/cds.adbb2d47, Hersbach et al. (2023) 1940 to present, global climate EU Copernicus Climate Change and weather reanalysis Service Product (2023) State Planet, 1-osr7, 10, 2023 V. Di Biagio et al.: Dissolved oxygen in the southern Adriatic Sea V. Di Biagio et al.: Dissolved oxygen in the southern Adriatic Sea 3 V. Di Biagio et al.: Dissolved oxygen in the southern Adriatic Sea 3 Figure 1. (a) Southern Adriatic area (blue square) within the Mediterranean Sea. (b) Cross-correlation map of surface oxygen, nitrate, and chlorophyll concentration provided by Copernicus biogeochemical reanalysis (prod. ref. nos. 1 and 2, Table 1) in the southern Adriatic area with respect to the central point of the pit indicated by the red star; the black contour line delimits the area with cross-correlation equal to or higher than 0.9; dashed lines indicate the trajectories of BGC-Argo ﬂoats (in situ TAC data, prod. ref. no. 3) passing the area. (c) Hovmöller diagrams of the dissolved oxygen concentration from in situ TAC data (prod. ref. no. 3) within the 0.9 cross-correlation area (b). Data have been interpolated for readability of the plot. (d) Hovmöller diagrams of dissolved oxygen concentration from Copernicus biogeochemical reanalysis (prod. ref. nos. 1 and 2), spatially averaged within the area of cross-correlation equal to 0.9 (a) in the time period of 1999–2021, after the bias correction procedure based on in situ TAC data (prod. ref. no. 3). Figure 1. (a) Southern Adriatic area (blue square) within the Mediterranean Sea. (b) Cross-correlation map of surface oxygen, nitrate, and chlorophyll concentration provided by Copernicus biogeochemical reanalysis (prod. ref. nos. 1 and 2, Table 1) in the southern Adriatic area with respect to the central point of the pit indicated by the red star; the black contour line delimits the area with cross-correlation equal to or higher than 0.9; dashed lines indicate the trajectories of BGC-Argo ﬂoats (in situ TAC data, prod. ref. no. 3) passing the area. (c) Hovmöller Figure 1. (a) Southern Adriatic area (blue square) within the Mediterranean Sea. (b) Cross-correlation map of surface oxygen, nitrate, and chlorophyll concentration provided by Copernicus biogeochemical reanalysis (prod. ref. nos. 1 and 2, Table 1) in the southern Adriatic area with respect to the central point of the pit indicated by the red star; the black contour line delimits the area with cross-correlation equal to or higher than 0.9; dashed lines indicate the trajectories of BGC-Argo ﬂoats (in situ TAC data, prod. ref. no. 3) passing the area. (c) Hovmöller diagrams of the dissolved oxygen concentration from in situ TAC data (prod. ref. no. 3) within the 0.9 cross-correlation area (b). V. Di Biagio et al.: Dissolved oxygen in the southern Adriatic Sea Data have been interpolated for readability of the plot. (d) Hovmöller diagrams of dissolved oxygen concentration from Copernicus biogeochemical reanalysis (prod. ref. nos. 1 and 2), spatially averaged within the area of cross-correlation equal to 0.9 (a) in the time period of 1999–2021, after the bias correction procedure based on in situ TAC data (prod. ref. no. 3). Oesterwind et al., 2016) and for understanding anthro- pogenic impacts on the marine environment (IPCC, 2022). The emerging ecosystem-based management method pro- posed by the MSFD (2008/56/EC) promotes the use of dif- ferent observational platforms, allowing information on the space-time distribution of important parameters related to water quality to be synoptically collected (Martellucci et al., 2021). Water; Manca et al., 2006), while the anticyclonic circula- tion favours the inﬂow of Atlantic Water and a relative de- crease in salinity in the SAdr (Gaˇci´c et al., 2011; Menna et al., 2022a). This feature has a strong inﬂuence on the biogeo- chemical properties of the SAdr, affecting nutrient availabil- ity (Civitarese et al., 2010), phytoplankton blooms (Gaˇci´c et al., 2002; Civitarese et al., 2010), and species composition (Batisti´c et al., 2014; Mauri et al., 2021). Water; Manca et al., 2006), while the anticyclonic circula- tion favours the inﬂow of Atlantic Water and a relative de- crease in salinity in the SAdr (Gaˇci´c et al., 2011; Menna et al., 2022a). This feature has a strong inﬂuence on the biogeo- chemical properties of the SAdr, affecting nutrient availabil- ity (Civitarese et al., 2010), phytoplankton blooms (Gaˇci´c et al., 2002; Civitarese et al., 2010), and species composition (Batisti´c et al., 2014; Mauri et al., 2021). In this context, the present work integrates the state-of- the-art approach of in situ measurements (in 2014–2020, distributed by Copernicus In Situ Thematic Centre, INS TAC) with the Copernicus biogeochemical reanalysis in the Mediterranean Sea at high resolution (Cossarini et al., 2021) with the aim of characterizing the DO dynamics in the SAdr in the time period of 1999–2021. In particular, we aim to as- sess DO interannual variability in an area (SAdr) sensitive to multiple drivers (e.g. atmospheric forcing, Mediterranean circulation, and biological processes) and to evaluate the rel- ative importance of the different drivers in this area. While hydrodynamic and biogeochemical properties of the SAdr have been widely described in several studies (e.g. 1 Introduction 1a) is one of these areas, as it is an area of deep-water formation (Gaˇci´c et al., 2002; Pirro et al., 2022) and represents one of the deep engines of the eastern Mediterranean thermohaline circula- tion (Malanotte-Rizzoli et al., 1999), which is crucial for the eastern basin ventilation. The Adriatic Sea (Fig. 1a) is an State Planet, 1-osr7, 10, 2023 https://doi.org/10.5194/sp-1-osr7-10-2023 CHAPTER3.1 2 Data and methods In the present study the DO concentration in the SAdr area (Fig. 1a) was assessed by combining data from the Coper- nicus biogeochemical reanalysis in the Mediterranean Sea (product reference numbers – prod. ref. nos. – 1 and 2 in Table 1; Cossarini et al., 2021) in 1999–2021 and the Coper- nicus in situ dataset (prod. ref. no. 3), available for the period 2014-2020 (Figs. 1b-c). The temporal evolution of the com- bined model–in situ DO concentration proﬁle in the time pe- riod of 1999–2021 is shown in Fig. 1d. – heat ﬂuxes in the SAdr as a proxy for thermal and mix- ing and stratiﬁcation cycles (from prod. ref. no. 8 in Ta- ble 1; Fig. 2a); – mixed-layer depth in the SAdr as a proxy for both lo- cal vertical mixing and water residence times in the pit (prod. ref. nos. 4 and 5; Escudier et al., 2021; Fig. 2b); – chlorophyll concentration at the surface and in the sub- surface in the SAdr as a proxy for biological production in spring and late spring–summer, respectively (prod. ref. nos. 1 and 2; Fig. 2c–d); In particular, we used the Biogeochemical Argo (BGC- Argo) ﬂoat measurements of in situ DO to compute a bias correction of the daily DO concentrations simulated by the biogeochemical reanalysis at 1/24◦horizontal resolution. In fact, the biogeochemical reanalysis does not include BGC- Argo ﬂoat DO assimilation and displays an average RMSD (root mean square difference) of 15 mmol m−3 for DO in the 0–600 m depth layer with respect to the observations in the area (Cossarini et al., 2021; Teruzzi et al., 2022). Quantile mapping, a technique largely used for climate sim- ulations (e.g. Hopson and Webster, 2010; Jakob Themeßl et al., 2011; Gudmundsson et al., 2012), was adopted to perform the reanalysis bias correction. The quantile map- ping technique adjusts the cumulative distribution of the data simulated for the past or future period by applying a transformation between the quantiles of the simulated and observed data in the present. In our application, we adapted the code publicly provided by Beyer et al. (2020) at https://doi.org/10.17605/OSF.IO/8AXW9 (Beyer, 2023) and included available in situ data of daily DO (Fig. 1c) within a representative area (Fig. 1b) of the southern Adriatic in the period 2014–2020 and DO reanalysis data for the same days of measurements. 2 Data and methods The representative area was identi- ﬁed by applying a spatial cross-correlation analysis (Martel- lucci et al., 2021) to the biogeochemical reanalysis centred on the SAdr pit and selecting the correlation threshold of 0.9 (Fig. 1b). Speciﬁcally, we considered the cross-correlation between the surface data of DO, nitrate, and chlorophyll con- centrations in the central point of the pit and those at each spatial grid point in the domain to identify the area that dis- played the same dynamics at the surface from a phenomeno- logical perspective. Further details of the quantile mapping bias correction are included in Appendix A. – heat ﬂuxes in the northern Adriatic Sea (NAdr), as a proxy for oxygen-rich dense-water formation in the NAdr and its transport into the pit (prod. ref. no. 8; Fig. 2e); – North Ionian Gyre (NIG) vorticity derived from satel- lite altimetry, as a proxy for the inﬂow of Levantine wa- ters and Atlantic Water (AW) (prod. ref. nos. 6 and 7; Fig. 2f). In particular, the temporal phases of the NIG are deﬁned as cyclonic and anticyclonic, respectively, when the vortic- ity ﬁeld is positive and negative, as highlighted by the de- seasonalized time series in Fig. 2f. Mixed-layer depth (computed in prod. ref. nos. 4 and 5 considering the 0.03 kg m−3 density difference with respect to the near-surface value at 10 m depth) and the chlorophyll at the surface and in the subsurface (30–80 m, where the deep chlorophyll maximum is located) were spatially averaged in the SAdr area (41.6–42.1◦N, 17.6–18.1◦E; to consider the whole volume of the pit); heat ﬂuxes were calculated in both the SAdr area and in the NAdr area (44.5–45.5◦N, 13– 13.5◦E), while current vorticity was computed in the north- ern Ionian Sea (37–39◦N, 17–19.5◦E). In the correlation analysis, the time series of the heat ﬂuxes in the NAdr (Fig. 2e) has been temporally lagged by 2 months as an estimated mean time for the entrance in the SAdr pit of waters originating in the northern Adriatic area (Vilibi´c and Mihanovi´c, 2013; Querin et al., 2016; Mihanovi´c et al., 2021). Moreover, we tested the signiﬁcance of the cor- relation coefﬁcients between EOF and driver time series us- ing a parametric t test (with a reference signiﬁcance level of 0.05). We then applied the empirical orthogonal function (EOF) analysis (e.g. Thomson and Emery, 2014) to the vertical pro- ﬁles in Fig. V. Di Biagio et al.: Dissolved oxygen in the southern Adriatic Sea 4 dence of the mechanisms driving oxygen concentration and dynamics in the area: 2 Data and methods 1d to describe DO variability in the SAdr area in the period 1999–2021. The EOF analysis allows us to iden- tify the spatial patterns of variability (i.e. EOF vertical pat- terns), describe how they change in time by means of time series (i.e. EOF time series), and associate the explained vari- ance with each mode. 3 Results 3.1 Temporal scales of variability in connection with drivers V. Di Biagio et al.: Dissolved oxygen in the southern Adriatic Sea Civitarese et al., 2010; Cushman-Roisin et al., 2013; Lip- izer et al., 2014; Kokkini et al., 2018, 2020; Mavropoulou et al., 2020; Mihanovi´c et al., 2021; Menna et al., 2022b), to the best of our knowledge DO dynamics in the area in connec- tion with relevant driving processes over decadal timescales have not been addressed yet. Investigating the DO multidecadal variability is crucial for quantifying the state of the marine environment (Ma- rine Strategy Framework Directive, MSFD: EU-COM, 2008; State Planet, 1-osr7, 10, 2023 State Planet, 1-osr7, 10, 2023 https://doi.org/10.5194/sp-1-osr7-10-2023 CHAPTER3.1 CHAPTER3.1 CHAPTER3.1 State Planet, 1-osr7, 10, 2023 3.1 Temporal scales of variability in connection with drivers Dissolved oxygen in the southern Adriatic area (Fig. 1a) shows in the subsurface layers an alternation between peri- ods of enrichment (in 2004–2006, 2010–2013, 2016–2017) Finally, we performed a Pearson correlation analysis be- tween the EOF time series in 1999–2021 and the following series of forcing indexes (reported in Fig. 2), providing evi- State Planet, 1-osr7, 10, 2023 https://doi.org/10.5194/sp-1-osr7-10-2023 https://doi.org/10.5194/sp-1-osr7-10-2023 CHAPTER3.1 CHAPTER3.1 V. Di Biagio et al.: Dissolved oxygen in the southern Adriatic Sea Figure 2. Time series of the main forcing in the time period of 1999–2021: (a) net heat ﬂuxes in SAdr (prod. ref. no. 8 in Table 1 layer depth (prod. ref. nos. 4 and 5), (c) surface chlorophyll concentration (prod. ref. nos. 1 and 2), (d) subsurface chlorophyll c (30–80 m layer in which deep chlorophyll maximum, DCM, is located; prod. ref. nos. 1 and 2), (e) net heat ﬂuxes in the NA no. 8), and (f) NIG current vorticity (grey line) and de-seasonalized time series as obtained by applying a low-pass ﬁlter of 13 m black line) (prod. ref. nos. 6 and 7). V. Di Biagio et al.: Dissolved oxygen in the southern Adriatic Sea V. Di Biagio et al.: Dissolved oxygen in the southern Adriatic Sea 5 Figure 2. Time series of the main forcing in the time period of 1999–2021: (a) net heat ﬂuxes in SAdr (prod. ref. no. 8 in Table 1), (b) mixed- layer depth (prod. ref. nos. 4 and 5), (c) surface chlorophyll concentration (prod. ref. nos. 1 and 2), (d) subsurface chlorophyll concentration (30–80 m layer in which deep chlorophyll maximum, DCM, is located; prod. ref. nos. 1 and 2), (e) net heat ﬂuxes in the NAdr (prod. ref. no. 8), and (f) NIG current vorticity (grey line) and de-seasonalized time series as obtained by applying a low-pass ﬁlter of 13 months (thick black line) (prod. ref. nos. 6 and 7). and sharp declines that impacted the oxygen minimum layer (OML), located between 100 and 300 m. Low concentration values are also observed in the years between 1999 and 2003. The ﬁrst mode (Fig. 3a–b), accounting for 48.9 % of the explained variance, can be associated with the seasonal cycle of oxygen concentration in the upper layers: its vertical pat- tern mainly affects the ﬁrst levels (Fig. 3b), the correspond- ing time series shows relative maximum values in spring (Fig. https://doi.org/10.5194/sp-1-osr7-10-2023 State Planet, 1-osr7, 10, 2023 V. Di Biagio et al.: Dissolved oxygen in the southern Adriatic Sea 6 Figure 3. EOF time series (a, c, e, g) and vertical patterns (b, d, f, h) of the ﬁrst four modes computed on the bias-corrected dissolved oxygen concentration in the southern Adriatic area shown in Fig. 1d. The explained variances of the four modes are 48.9 %, 19.7 %, 17.7 %, and 8.4 %. The fourth mode (Fig. 3g–h), which describes 8 % of the variance, can be ascribed mainly to the vorticity of the NIG (r = −0.37, last column in Table 2), which affects the oxygen concentration in the intermediate layer (100–500 m depth), ﬁlled by LIW, and acts in the opposite direction in the upper and deeper layers (Fig. 3h). Analysing the four modes in order of decreasing explained variance, we ascribe the seasonal variability connected with solubility mainly to the ﬁrst mode, whereas we associate the biological contribution to oxygen dynamics to multiple in- teracting modes. In fact, the ﬁrst mode explains the onset of the subsurface oxygen maximum (SOM) in spring, while the summer dynamics of the SOM are partially related to the third mode. The second mode, whose time series is correlated with surface chlorophyll evolution among other factors, can explain that part of the oxygen variability that is related to winter surface productivity. The SOM, evident in summer oxygen proﬁles in Fig. 1c–d at about 40 m depth, is a feature that has already been ob- served in a great part of oligotrophic oceans (Riser and John- son, 2008; Yasunaka et al., 2022) and of the Mediterranean Sea (e.g. Kress and Herut, 2001; Copin-Montégut and Bé- govic, 2002; Manca et al., 2004; Cossarini et al., 2021; Di Biagio et al., 2022), and it represents an emerging property resulting from multiple interacting ecosystem processes (i.e. air–sea interactions, transport, mixing, and biological pro- duction and consumption) and is, indeed, captured by mul- tiple modes. Figure 3. EOF time series (a, c, e, g) and vertical patterns (b, d, f, h) of the ﬁrst four modes computed on the bias-corrected dissolved oxygen concentration in the southern Adriatic area shown in Fig. 1d. The explained variances of the four modes are 48.9 %, 19.7 %, 17.7 %, and 8.4 %. Table 2. Correlations between the ﬁrst four temporal modes of EOFs of DO (Fig. 3a, c, e, g) and the forcing ﬁelds (with heat ﬂuxes in the northern Adriatic Sea time-lagged by 2 months; Fig. 2). V. Di Biagio et al.: Dissolved oxygen in the southern Adriatic Sea Non- statistically signiﬁcant correlations are identiﬁed by a signiﬁcance level higher than 0.05 and indicated by “n.s.” in the table. The third mode, which also describes the high concentra- tion values in the deep layers in the period 2005–2006 and 2012–2014, is also moderately associated with a multiannual signal of the inﬂow of deep, denser, and oxygenated water from the northern Adriatic Sea (r = 0.68, third column in Ta- ble 2; Querin et al., 2016). Finally, it is worth noting that an EOF analysis of detrended DO time series (not shown) yields fairly similar results but with the third mode only weakly cor- related with the forcing indexes (r < 0.4). Indeed, we can conclude that the third mode captures a signal of long-term evolution of oxygen concentration associated with changes in heat ﬂuxes and chlorophyll concentration. Mode 1 Mode 2 Mode 3 Mode 4 Heat ﬂux (SAdr) 0.56 0.15 0.51 0.32 Mixed-layer depth n.s. −0.28 −0.41 −0.25 (SAdr) Surface chlorophyll n.s. −0.41 −0.61 n.s. (SAdr) Subsurface chlorophyll 0.43 0.13 0.48 0.34 (SAdr) Heat ﬂux (NAdr, n.s. 0.48 0.68 0.16 2-month lag) NIG vorticity n.s. −0.40 n.s. −0.37 (North Ionian) 3.1 Temporal scales of variability in connection with drivers 3a), and it shows a statistically signiﬁcant but moderate correlation (r = 0.56) with heat ﬂux and a lower correlation with the subsurface chlorophyll concentration (r = 0.43) in the SAdr area (ﬁrst column in Table 2). The EOF analysis was performed on the vertical proﬁles of the oxygen anomaly, derived by removing the mean proﬁle in the period 1999–2021 and then normalized dividing by their standard deviation. The time series of the ﬁrst four EOF modes, which explain up to 95 % of the oxygen variability in the water column, are shown along with the corresponding vertical patterns in Fig. 3a, c, e, g and Fig. 3b, d, f, h, respectively. The second and third modes (Fig. 3c–d and Fig. 3e–f, re- spectively), describing 19.7 % and 17.7 % of the variance, re- spectively, affected both the upper and deeper layers. Both modes display relative maximum values in summer, but they have different correlation coefﬁcients with the explanatory factors. The time series of the second mode (second col- umn in Table 2) shows a signiﬁcant but low correlation with multiple drivers, exceeding 0.4 only for surface chloro- The EOFs are interpreted considering the correlation of the EOF time series (Fig. 3a, c, e, g) with the time series of the forcing indicators shown in Fig. 2 (see Table 2), with heat ﬂuxes in the northern Adriatic Sea time-lagged by 2 months as the estimated time of entry of the NAdr dense water in the SAdr pit. https://doi.org/10.5194/sp-1-osr7-10-2023 State Planet, 1-osr7, 10, 2023 CHAPTER3.1 CHAPTER3.1 CHAPTER3.1 V. Di Biagio et al.: Dissolved oxygen in the southern Adriatic Sea 3.2 The 2021 anomaly The year 2021 shows an overall negative anomaly in the oxy- gen concentration proﬁle (Fig. 4b) compared to the 1999– 2020 climatological proﬁles (Fig. 4a). In particular, the anomaly affects a layer that thinned during the year, mov- ing from 0–600 m depth in winter–early spring to 30–400 m in late spring–summer and 0–80 m in autumn. The (absolute) maximum values correspond to 25–30 mmol m−3 at the sur- face in spring and at the SOM depth in summer. phyll (r = −0.41) and waters from the NAdr area (r = 0.48). The time series of the third mode (third column, same ta- ble) is moderately correlated with both surface chlorophyll (r = −0.61) and NAdr waters (r = 0.68 correlation), but also with heat ﬂuxes in the area (r = 0.51) and, to a lower extent, with mixed-layer depth (r = −0.41) and subsurface chloro- phyll (r = 0.48). We veriﬁed that, among the EOF modes, the negative anomaly of the ﬁrst mode is the main contributor to the 2021 negative oxygen anomaly (not shown). The time series of the ﬁrst mode (Fig. 3a) is actually negative from 2019 and cor- https://doi.org/10.5194/sp-1-osr7-10-2023 V. Di Biagio et al.: Dissolved oxygen in the southern Adriatic Sea Indeed, as our second result, the variability that is not ex- plained by the EOF decomposition appears to be connected with a possible regime shift, associated with the entrance of new water masses that are warmer, markedly saltier, and less oxygenated which were not previously observed in the anal- ysed time period. One of the causes of the decrease in total oxygen concen- tration in the SAdr could be due to the exceptional saliniza- tion observed in the SAdr since 2017 (Mihanovi´c et al., 2021; Menna et al., 2022b). This increase was related to the inﬂow of new, warmer, and noticeably saltier water masses from the north-eastern Ionian Sea (Mihanovi´c et al., 2021; Menna et al., 2022b). The inﬂow of saltier and warmer water masses is also evident by observing the temporal evolution of these parameters through the Strait of Otranto (Fig. B1). In par- ticular, in the upper layer (0–150 m) both temperature and salinity show an overall positive trend throughout the pe- riod 1999–2021, whereas the decrease observed in 2006– 2011 and 2017–2018 can be associated with the inﬂow of less saline AW, triggered by the anticyclonic circulation of the NIG (Fig. 2f). In the intermediate layer (150–600 m), salinity shows a positive trend in 1999–2021, while no clear trend is observed for temperature. Moreover, a sharp increase in salinity (∼0.1) is observed in 2019. This increase oc- curred after the NIG inversion from anticyclonic to cyclonic (Fig. 2f), resulting in a further increase in salinity due to both the decrease in AW advection and the increase in LIW inﬂow. The exceptional increase in salinity occurring after 2019 has already been documented (Mihanovi´c et al., 2021; Menna et al., 2022b) and also observed north of the SAdr pit. Further monitoring of such anomalously high salinity val- ues and assessment of their potential impact on the marine food web are of great importance, as picoplankton groups are sensitive to this environmental variable (Mella-Flores et al., 2011), and changes in biomass and production due to salinity have already been observed in previous studies in the Adriatic Sea (Beg Paklar et al., 2020; Mauri et al., 2021). V. Di Biagio et al.: Dissolved oxygen in the southern Adriatic Sea 7 Figure 4. Hovmöller diagrams of 1999–2020 mean of daily oxygen concentration computed from Copernicus biogeochemical reanaly- sis (prod. ref. nos. 1 and 2, Table 1) after the bias correction proce- dure based on in situ TAC data (prod. ref. no. 3) (a) and anomaly in 2021 with respect to the period of 1999–2020 (b). spect to the mean over 1999–2020. This study enriches our knowledge of the dissolved oxygen state and long-term dy- namics in the area by proposing a seamless time and space perspective that is complementary to previous climatologies and data aggregation information (e.g. Lipizer et al., 2014) and adding an explanatory framework for the driving mech- anisms in the marine environment. The EOF statistical analysis that we conducted on the ver- tical oxygen proﬁles yielded two key results. First, in contrast with a climatological view, the analysis was able to capture most of the interannual oxygen variability associated with variability in the main drivers (i.e. heat ﬂuxes affecting sol- ubility, biological productivity, vertical mixing). We do not detect a clear deoxygenation trend in the subsurface layer, while the multiannual variability is characterized by an al- ternation of enrichment and reduction phases, whose domi- nant correlations with the drivers for each EOF time series are in the (absolute) range of 0.40–0.70. The possibility of observing such cyclic signals is enhanced by the relatively small volume and short residence time of the SAdr pit wa- ters (Querin et al., 2016) with respect to other Mediterranean areas (Coppola et al., 2018). This feature makes the SAdr a potential efﬁcient probe to detect a rapid response to changes in its meteo-marine drivers, i.e. circulation and atmospheric patterns. Figure 4. Hovmöller diagrams of 1999–2020 mean of daily oxygen concentration computed from Copernicus biogeochemical reanaly- sis (prod. ref. nos. 1 and 2, Table 1) after the bias correction proce- dure based on in situ TAC data (prod. ref. no. 3) (a) and anomaly in 2021 with respect to the period of 1999–2020 (b). responds to the negative anomaly of only one of its drivers (Table 2), i.e. subsurface chlorophyll (Fig. 2d), and not heat ﬂuxes (Fig. 2a). In particular, we estimated a mean negative anomaly approximately equal to 6 % with respect to the cli- matological mean (1999–2020) for subsurface chlorophyll in 2021. V. Di Biagio et al.: Dissolved oxygen in the southern Adriatic Sea Moreover, if such a strong negative oxygen anomaly as that observed in 2021 were to persist, it could have direct im- pacts on local marine organisms, as well as on the cycling of dissolved chemical elements (Conley et al., 2009), poten- tially altering the energy ﬂux towards the higher trophic lev- els (Ekau et al., 2010). The importance of the relationship between dissolved oxygen and the catch distribution of some marine species has already been proved in the Adriatic Sea (Chiarini et al., 2022). CHAPTER3.1 CHAPTER3.1 V. Di Biagio et al.: Dissolved oxygen in the southern Adriatic Sea 4 Conclusions Merging the Copernicus biogeochemical reanalysis in the Mediterranean Sea with in situ TAC data of biogeochemical Argo ﬂoats allowed us to characterize the interannual vari- ability in dissolved oxygen in the southern Adriatic Sea in the time period of 1999–2021 and the 2021 anomaly with re- By integrating model and in situ data, our study demon- strates the importance of following up the oxygen content in a seamless spatial and temporal way, as it is a fundamental indicator of good environmental status (GES; Oesterwind et https://doi.org/10.5194/sp-1-osr7-10-2023 State Planet, 1-osr7, 10, 2023 CHAPTER3.1 CHAPTER3.1 V. Di Biagio et al.: Dissolved oxygen in the southern Adriatic Sea ctiv- V. Di Biagio et al.: Dissolved oxygen in the southern Adriatic Sea V. Di Biagio et al.: Dissolved oxygen in the southern Adriatic Sea 8 al., 2016) and a factor that signiﬁcantly affects ﬁshing activ- ities and the economy. Appendix A: Quantile mapping bias correction of DO concentration proﬁles Figures A1 and A2 show the modelled DO concentration pro- ﬁles and histogram distributions before and after the quantile mapping bias correction, respectively, conducted by using the BGC-Argo ﬂoat measurements available in 2014–2020 (Fig. 1c). The quantile mapping, better than other methods (i.e. additive delta change, multiplicative delta change, and variance scaling; results not shown), acted on the proﬁles by modifying the values of the concentrations (as indicated by the different colour bars in Fig. A1a and A1b) but, at the same time, maintaining the main dynamics observed before the correction: mixing and stratiﬁcation at the surface dur- ing the year, subsurface oxygen maximum onset in spring and development in summer, and interannual variability re- lated to the mixed-layer-depth dynamics in the intermediate layers. The distributions of the values of the model output before and after the quantile mapping bias correction and the values from BGC-Argo ﬂoats are displayed in Fig. A2. The correction actually changed the modelled values (Fig. A2a) to reproduce the shape of the distribution of the observations (Fig. A2c). In particular, after the correction (Fig. A2b) the modelled data show higher variability and a more skewed distribution toward the higher values, similarly to the obser- vations. Figure A1. Hovmöller diagram of the modelled oxygen concentrations spatially averaged within the area of autocorrelation equal to 0.9, Figure A1. Hovmöller diagram of the modelled oxygen concentrations spatially averaged within the area of autocorrelation equal to 0.9, indicated in Fig. 1b, before the bias correction by quantile mapping (a) and after the procedure (b). Figure A1. Hovmöller diagram of the modelled oxygen concentrations spatially averaged within the area of autocorrelation equal to 0.9, indicated in Fig. 1b, before the bias correction by quantile mapping (a) and after the procedure (b). CHAPTER3.1 State Planet, 1-osr7, 10, 2023 https://doi.org/10.5194/sp-1-osr7-10-2023 State Planet, 1-osr7, 10, 2023 https://doi.org/10.5194/sp-1-osr7-10-2023 CHAPTER3.1 V. Di Biagio et al.: Dissolved oxygen in the southern Adriatic Sea 9 Figure A2. Frequency histogram of modelled oxygen concentrations before the bias correction by quantile mapping (a) and after the procedure (b), compared with BGC-Argo observations (c). Figure A2. Frequency histogram of modelled oxygen concentrations before the bias correction by quantile mapping (a) and after the procedure (b), compared with BGC-Argo observations (c). Appendix B: Time series of surface and intermediate temperature and salinity at the Otranto Strait Figure B1. Time series of temperature (a) and salinity (b), averaged in the vertical layers 0–150 m (red lines) and 150–600 m (black lines) of the Otranto Strait (39.8◦N, 18.5–19.5◦E) in the time period of 1999–2021. In panel (a), light red and dark red indicate data before and after de-seasonalization, respectively. Data are provided by Copernicus physical reanalysis (prod. ref. nos. 4 and 5, Table 1). References Cushman-Roisin, B., Gacic, M., Poulain, P. M., and Arte- giani, A. (Eds.): Physical oceanography of the Adriatic Sea: past, present and future. Springer Science & Business Media, https://doi.org/10.1007/978-94-015-9819-4, 2013. Batisti´c, M., Gari´c, R., and Molinero, J. C.: Interannual vari- ations in Adriatic Sea zooplankton mirror shifts in circula- tion regimes in the Ionian Sea, Clim. Res., 61, 231–240, https://doi.org/10.3354/cr01248, 2014. 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In panel (a), light red and dark red indicate data before and after de-seasonalization, respectively. Data are provided by Copernicus physical reanalysis (prod. ref. nos. 4 and 5, Table 1). Disclaimer. Publisher’s note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Data availability. Publicly available datasets were analysed in this study. Modelling and in situ data can be found at the Copernicus Marine Service, with references and DOIs indicated in Table 1 of the paper. Acknowledgements. This study has been conducted using EU Copernicus Marine Service Information. Acknowledgements. This study has been conducted using EU Copernicus Marine Service Information. Author contributions. VDB and GC conceived the idea. VDB, RM, and MM conducted the analysis. VDB, RM, and GC wrote the ﬁrst draft, with contributions from the other co-authors. All the authors discussed and reviewed the submitted manuscript. Financial support. This study has been partly funded by the Copernicus Mediterranean – Monitoring Forecasting Centre (con- tract LOT reference: 21002L5-COP-MFC MED-5500, issued by Mercator Ocean) within the framework of the Marine Copernicus Service. Competing interests. The contact author has declared that none of the authors has any competing interests. State Planet, 1-osr7, 10, 2023 State Planet, 1-osr7, 10, 2023 https://doi.org/10.5194/sp-1-osr7-10-2023 https://doi.org/10.5194/sp-1-osr7-10-2023 V. Di Biagio et al.: Dissolved oxygen in the southern Adriatic Sea V. Di Biagio et al.: Dissolved oxygen in the southern Adriatic Sea V. Di Biagio et al.: Dissolved oxygen in the southern Adriatic Sea 10 Review statement. This paper was edited by Marilaure Grégoire and reviewed by three anonymous referees. Review statement. This paper was edited by Marilaure Grégoire and reviewed by three anonymous referees. 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Pujol, M.-I., Taburet, G., and SL-TAC team: EU Coper- nicus Marine Service Quality Information Docu- ment for the European Seas Gridded L 4 Sea Surface State Planet, 1-osr7, 10, 2023 https://doi.org/10.5194/sp-1-osr7-10-2023 CHAPTER3.1 Characterization of the organic vs. inorganic fraction of suspended particulate matter in coastal waters based on ocean color radiometry remote sensing n of the Copernicus Ocean State Report (OSR7) Hubert Loisel1, Lucile Duforêt-Gaurier1, Trung Kien Tran1, Daniel Schaffer Ferreira Jorge1, François Steinmetz2, Antoine Mangin3, Marine Bretagnon3, and Odile Hembise Fanton d’Andon3 1Université du Littoral Côte d’Opale, CNRS, Univ. Lille, IRD, UMR 8187 – LOG – Laboratoire d’Océanologie et de Géosciences, 62930 Wimereux, France 2HYGEOS, Euratechnologies, 165 avenue de Bretagne, 59000 Lille, France 3ACRI-ST, 260 Route du Pin Montard, 06904 Sophia-Antipolis, France Correspondence: Hubert Loisel (hubert.loisel@univ-litoral.fr) Received: 30 August 2022 – Discussion started: 30 September 2022 Revised: 14 February 2023 – Accepted: 27 February 2023 – Published: 27 September 2023 Hubert Loisel1, Lucile Duforêt-Gaurier1, Trung Kien Tran1, Daniel Schaffer Ferreira Jorge1, François Steinmetz2, Antoine Mangin3, Marine Bretagnon3, and Odile Hembise Fanton d’Andon3 1Université du Littoral Côte d’Opale, CNRS, Univ. Lille, IRD, UMR 8187 – LOG – Laboratoire d’Océanologie et de Géosciences, 62930 Wimereux, France 2HYGEOS, Euratechnologies, 165 avenue de Bretagne, 59000 Lille, France 3ACRI-ST, 260 Route du Pin Montard, 06904 Sophia-Antipolis, France Correspondence: Hubert Loisel (hubert.loisel@univ-litoral.fr) Received: 30 August 2022 – Discussion started: 30 September 2022 Revised: 14 February 2023 – Accepted: 27 February 2023 – Published: 27 September 2023 cus Ocean State Report (OSR7) Abstract. Knowledge of the organic and inorganic particulate fraction of suspended material in coastal waters is essential for the study of particle dynamics and biogeochemical cycles in these complex and highly variable environments. Thanks to the availability of appropriate spatial sensors and to the considerable improvements in algorithms dedicated to the satellite observation of coastal waters from ocean color radiometry (OCR) achieved in the last 2 decades, various optical and biogeochemical parameters can now be routinely monitored over coastal waters. Here we show that a proxy for particulate composition (PPC) can be estimated from OCR observations. The present algorithm, based on a neural network approach, has been validated using a broad range of biogeo- chemical data collected in various contrasted coastal waters and has been applied to MERIS observations over the global coastal ocean at a 1 km × 1 km spatial resolution from 2002 to 2012. The relevance of the temporal occurrence of PPC in a given water pixel has been illustrated over the global coastal ocean, and its pertinence has been discussed in depth for the English Channel and the southern North Sea, which are characterized by a well-documented variability in suspended particulate matter composition. V. Di Biagio et al.: Dissolved oxygen in the southern Adriatic Sea Teruzzi, A., Di Biagio, V., Feudale, L., Bolzon, G., Lazzari, P., Salon, S., Coidessa, G., and Cossarini, G.: EU Coper- nicus Marine Service Quality Information Document for the Mediterranean Sea Biogeochemistry Reanalysis, MED- SEA_MULTIYEAR_BGC_006_008, Issue 3.2, Mercator Ocean International,https://catalogue.marine.copernicus.eu/documents/ QUID/CMEMS-MED-QUID-006-008.pdf (last access: 6 March 2023), 2022. State Planet, 1-osr7, 10, 2023 State Planet, 1-osr7, 10, 2023 CHAPTER3.1 Chapter 3.2 – 7th edition of the Copernicus Ocean State Report (OSR7) Chapter 3.2 – 7th edition of the Copernicus Ocean State Planet, 1-osr7, 11, 2023 https://doi.org/10.5194/sp-1-osr7-11-2023 © Author(s) 2023. This work is distributed under the Creative Commons Attribution 4.0 License. ter 3.2 – 7th edition of the Copernicus Ocean State Report (O Characterization of the organic vs. inorganic fraction of suspended particulate matter in coastal waters based on ocean color radiometry remote sensing The present algorithm can directly be applied to all OCR sensors. Among the large set of water quality parameters sampled in coastal waters, the concentration of the suspended partic- ulate matter (SPM) or equivalent parameters (e.g., the tur- bidity) has been intensively studied by the scientiﬁc com- munity as a key parameter for understanding sediment trans- port, downstream sedimentation, and coastal geomorpholog- ical processes (Velegrakis et al., 1997; Lahet and Stramski, 2010; Vantrepotte et al., 2012; Loisel et al., 2014; Anthony et al., 2015; Marchesiello et al., 2019). For instance, combining SPM spatiotemporal products with wave data allows resus- pension areas to be identiﬁed (Loisel et al., 2014). While the SPM spatiotemporal patterns provide relevant information on the suspended particulate dynamics, the variation in the composition (i.e., chemical nature) of SPM may disclose rel- Published by Copernicus Publications. H. Loisel et al.: Organic vs. inorganic fraction of suspended particulate matter from remote sensing 2 Table 1. CMEMS and non-CMEMS products used in this study, including information on data documentation. Product Product ID and Data access Documentation ref. no. type 1 RRS (remote- sensing reﬂectance), satellite observations https://www.globcolour.info/ (last access: 28 April 2023) Product User Guide: https://www.globcolour.info/CDR_ Docs/GlobCOLOUR_PUG.pdf (last access: 28 April 2023) 2 PPC, satellite observations It will be introduced in GlobColour and in the Copernicus Marine Service (EU Coper- nicus Marine Service Product, 2022) Quality Information Document (QUID): Garnesson et al. (2022); Product User Manual (PUM): Colella et al. (2022) et al., 2016; Reynolds and Stramski, 2019) and satellite ocean color radiometry (OCR) observations (Vantrepotte et al., 2011). From in situ data collected in the near-shore ma- rine environment at Imperial Beach in California, Wo´zniak et al. (2010) set threshold values for the POC / SPM ratio to identify changes in the particulate assemblage from the dominance of mineral particles (POC / SPM < 0.06) to the dominance of organic particles (POC / SPM > 0.25) and for mixed particulate assemblages (0.25 < POC / SPM < 0.6). Neukermans et al. (2016) and Reynolds et al. (2016) fol- lowed the approach of Wo´zniak et al. (2010) to partition their Arctic seawater datasets into these three broad compositional classes. evant information on the complex chemical, physical, and bi- ological processes occurring in coastal waters. For instance, the portion of particulate organic matter (POM) in SPM is of particular interest when investigating the adsorption of trace elements on particles, abrupt changes in water quality due to pelagic or benthic blooms, or the fate of suspended matter between the water column and sediments. Modeling particulate transport requires speciﬁc modules coupled with hydrodynamical models. For example, in the SUBSTANCE module (Mengual et al., 2017), which can be coupled with CROCO, a French code built upon the well-known Regional Oceanic Modeling System (Shchepetkin and McWilliams, 2005), conservative and non-conservative (i.e., biological) particulate substances have to be deﬁned by their own den- sity and/or settling velocity, depending on the nature of the SPM (mineral vs. organic). The settling and ﬂocculation pro- cesses, closely linked to the organic fraction of SPM and to the transport of particulate variables in the water column, are also considered in these modules. H. Loisel et al.: Organic vs. inorganic fraction of suspended particulate matter from remote sensing Due to the high variability in the physical and biogeo- chemical processes occurring in coastal waters, traditional approaches such as oceanographic cruises and in situ time series, although essential, are very time-consuming and ex- pensive, and it is sometimes uncertain whether they will yield meaningful results for the studied phenomena. Satellite ob- servation of OCR is now well-recognized as a powerful tool to monitor the spatiotemporal distribution of biogeochemi- cal and optical parameters in coastal waters (IOCCG, 2000; Loisel et al., 2013; IOCCG, 2018; Groom et al., 2019). Over the past decades, various OCR bio-optical algorithms have shown that SPM (Ahn et al., 2006; Nechad et al., 2010; Feng et al., 2014; Han et al., 2016; Balasubramanian et al., 2020; Pahlevan et al., 2022) and POC concentrations (Liu et al., 2015; Hu et al., 2016; Wo´zniak et al., 2016; Le et al., 2017; Tran et al., 2019) can be estimated over coastal waters. Particulate organic carbon (POC) encompasses living (phytoplankton, heterotrophic bacteria, and viruses) and non- living (i.e., detritus) organic particles in suspension. Infor- mation on the SPM and POC variability represents crucial input for the initiation and validation of sediment transport models (Douillet et al., 2001; Ford and Fox, 2014; Wu et al., 2020) and biogeochemical models (Aumont et al., 2015). While the analysis of the variation in the absolute values of SPM and POC concentrations brings relevant information for our understanding of the carbon cycle and marine particulate dynamics in coastal waters, the dimensionless POC / SPM ratio is used in many studies to describe the temporal vari- ability in the particulate matter pool composition and ori- gin (Coynel et al., 2005; Emmerton et al., 2008; Doxaran et al., 2012, 2015; Ehn et al., 2019), which is often related to variation in the regional environmental forcing (e.g., wa- ter discharge, phytoplankton bloom dynamics). This ratio is also used to better interpret the in situ optical measurements (Babin et al., 2003; Loisel et al., 2007; Wo´zniak et al., 2011; Doxaran et al., 2012; Neukermans et al., 2016; Reynolds The objective of this study is to provide a proxy for partic- ulate composition (PPC) from remote sensing. For that pur- pose, the POC / SPM ratio value is used as an intermediate product which allows the PPC, composed of three different classes (i.e., organically dominated, mineral-dominated, and mixed), to be estimated. 1 Introduction Water quality parameters display large spatiotemporal vari- ability in coastal waters as these areas are the location of strong coupling between aquatic and terrestrial systems and are under pressure from great natural and anthropogenic forc- ing. The characterization of the spatiotemporal distribution of biological, biogeochemical, and physical parameters in coastal waters is of fundamental importance for a variety of applications dedicated to coastal management, which often involves economic interests, and for improving our under- standing of the dynamics of the coastal ecosystems and their associated biogeochemical cycles. CHAPTER3.2 CHAPTER3.2 2.1.1 In situ datasets In situ measurements were collected between 1997 and 2014 in the southeastern Beaufort Sea (Bélanger et al., 2008), French Guiana (Vantrepotte et al., 2012, 2015), European coastal waters (the English Channel, North Sea, Bay of Bis- cay) (Lubac and Loisel, 2007; Lubac et al., 2008; Neuker- mans et al., 2012; Bonato et al., 2016; Novoa et al., 2017), and the South China Sea/Vietnam East Sea (Loisel et al., 2014, 2017). The sampling strategies, the ﬁeld measurement protocols, and data processing are described in related pa- pers. Measurements include concentrations of POC and SPM (µgL−1), remote-sensing reﬂectances (Rrs, sr−1), the par- ticulate backscattering coefﬁcient (bbp), and the particulate attenuation coefﬁcient (cp) (m−1) at 650 nm. The samples cover a wide range of biogeochemical variability as POC and SPM concentrations span 4 and 3 orders of magnitude, re- spectively. The ﬁrst in situ database, named DS0, includes 300 coincident POC, SPM, bbp, and cp measurements (Ta- ble 2). In Sect. 2.3, DS0 will be used to examine the em- pirical relationship between bbp/cp and POC / SPM. The POC / SPM ratio ranges between 1.1×10−3 and 8.7×10−1. In comparison, Wo´zniak et al. (2010) collected 44 sam- ples in the near-shore area off Imperial Beach (California) with POC / SPM values between 2.3×10−2 and 4.2×10−1. The second in situ database, referred to as DS, is made of 325 coincident POC, SPM, and Rrs measurements, with 3.9 × 10−4 ≤POC/SPM ≤5.6 × 10−1 (Table 2). It will be used to test the performance of POC / SPM estimates from the neural network algorithm (Sect. 3.1). H. Loisel et al.: Organic vs. inorganic fraction of suspended particulate matter from remote sensing of data Years In situ POC SPM POC SPM derived from POC SPM derived from bbp(650) cp(550) satellite Rrs in situ Rrs DS0 European coastal 300 2010–2014 0.1136 – – 0.0162 waters [0.0001, 0.8700] – – [0.0013, 0.519] (0.1465) – – (0.0076) DS European coastal waters, 325 1997–2018 0.0895 – 0.0894 – French Guiana, Beaufort Sea, [0.0004, 0.5606] – [0.0026, 0.5382] – Vietnam East Sea (0.1046) – (0.1024) – DSM French 101 2002–2018 0.0801 0.1116 – – coastal waters, [< 0.0001, 0.5606] [0.0002, 0.5574] – – European coastal waters (0.1074) (0.1117) – – State Planet, 1-osr7, 1 H. Loisel et al.: Organic vs. inorganic fraction of suspended particulate matter from remote sensing We will re-examine the relevance of the POC / SPM threshold values of Wo´zniak et al. (2010), developed to assess the organic vs. mineral fraction of the bulk particulate matter. This will be done through the ex- amination of the relationship between the POC / SPM ratio State Planet, 1-osr7, 11, 2023 https://doi.org/10.5194/sp-1-osr7-11-2023 CHAPTER3.2 CHAPTER3.2 H. Loisel et al.: Organic vs. inorganic fraction of suspended particulate matter from remote sensing 3 H. Loisel et al.: Organic vs. inorganic fraction of suspended particulate matter from remote sensing and the bbp/cp optical ratio, which is an indicator of the bulk particulate assemblage chemical composition (Twardowski et al., 2001; Loisel et al., 2007). An extensive in situ dataset collected in a biogeochemically contrasted environment has been gathered for that purpose. A neural network (NN) al- gorithm will be developed and validated against a large in situ dataset collected in various coastal environments to as- sess PPC from OCR. The new algorithm will then be applied to MERIS (MEdium Resolution Imaging Spectrometer) ob- servations (2002–2012) over global coastal waters at a 1 km2 spatial resolution to discuss the signiﬁcance of this new prod- uct. A speciﬁc focus will be on the English Channel and the southern North Sea, for which the temporal occurrence of the organic vs. inorganic fraction for a given pixel, built from 10-year temporal series, will be discussed. CHAPT Table 2. Information on the in situ data used in this study: mean [minimum, maximum] and standard deviation (SD). Dataset Region No. of data Years In situ POC SPM POC SPM derived from POC SPM derived from bbp(650) cp(550) satellite Rrs in situ Rrs DS0 European coastal 300 2010–2014 0.1136 – – 0.0162 waters [0.0001, 0.8700] – – [0.0013, 0.519] (0.1465) – – (0.0076) DS European coastal waters, 325 1997–2018 0.0895 – 0.0894 – French Guiana, Beaufort Sea, [0.0004, 0.5606] – [0.0026, 0.5382] – Vietnam East Sea (0.1046) – (0.1024) – DSM French 101 2002–2018 0.0801 0.1116 – – coastal waters, [< 0.0001, 0.5606] [0.0002, 0.5574] – – European coastal waters (0.1074) (0.1117) – – State Planet, 1-osr7, 1 Table 2. Information on the in situ data used in this study: mean [minimum, maximum] and standard deviation (SD). Dataset Region No. 2.1.2 The global coastal MERIS Rrs and match-up datasets MERIS level 1 data were used to study the PPC spatial and temporal distribution (Sect. 3.2) and for the match-up ex- https://doi.org/10.5194/sp-1-osr7-11-2023 2.2 Algorithm development Two different approaches were initially tested to estimate PPC from Rrs. For the ﬁrst approach, SPM is estimated by the Han et al. (2016) algorithm (referred to as HA16) which consists of semi-analytical relationships between SPM and Rrs in the red or NIR bands, according to the level of turbid- ity. The typical band–ratio relationship using the red to blue– green bands is used to assess POC from the algorithm of Tran et al. (2019) (referred to as TR19). Once POC and SPM are derived, the POC / SPM ratio is calculated, and PPC is es- timated using the different threshold values (see Sect. 2.3). However, Tran et al. (2019) showed that POC concentration can be overestimated in the presence of mineral waters. The result is that the POC / SPM ratio is also overestimated when mineral-dominated waters dominate (not shown). Moreover, the errors in both POC and SPM estimations are additive when the POC / SPM ratio is ﬁnally calculated. To limit this error propagation, we focus on the development of a single algorithm to derive the POC / SPM ratio from Rrs in one step. The objective here is to re-examine the pertinence of the POC / SPM threshold values of Wo´zniak et al. (2010) on a larger in situ coastal dataset (DS0) (covering a wider range of optical and biogeochemical variability) through the examina- tion of the POC / SPM to bbp/cp relationship. As expected, bbp/cp decreases when POC / SPM increases, that is when we move from a mineral-dominated to an organically dom- inated environment, with bbp/cp values typically lower than 0.010–0.012 (Twardowski et al., 2001; Loisel et al., 2007) (Fig. 1). The regression line (bbp/cp vs. POC / SPM; black line) is plotted, and the estimated regression coefﬁcients are indicated with their standard error (Fig. 1). The threshold values are ﬁrst ﬁxed according to the bbp/cp values (as a given range of bbp/cp values corresponds to a given range of the refractive index of the bulk particulate matter) and then adjusted, with a careful examination of each data point for which ancillary data (i.e., chlorophyll a, counted cells, phy- toplankton to particulate absorption ratio, and Rrs spectra) are used to better characterize the bulk particulate matter. The ﬁrst threshold value has been shifted to 0.08 (correspond- ing to a bbp/cp value of 0.012) to encompass data points collected in mineral-dominated environments (close to river mouths). CHAPTER3.2 The cri- teria considered for the match-up selection are described in Bailey and Werdell (2006). Due to the absence of organically dominated match-up data points using the MERIS sensor, complementary match-up data points were added to DSM by looking at SeaWiFS (Sea-Viewing Wide Field-of-View Sensor) match-up with DS. We only kept the match-up data points with a good Rrs retrieval (only possible using DS). For that purpose, only data points with Rrs(in situ) /Rrs(satellite) values from 412 to 560 nm, below 0.5, or above 1.5 are se- lected. The DSM dataset is composed of 101 matched points after the application of these criteria. The POC / SPM mean value is 0.0801 for DSM instead of 0.1136 and 0.0895 for DS0 and DS, respectively. date this NN. Overall, 60 % of the observations were used to construct and train the NN, 20 % were used for its validation, and 20 % were used to test its performance independently. The training and validation phases are performed jointly, al- lowing us to stop the training procedure when the generaliza- tion of the NN stops improving. We tested several combina- tions of Rrs bands (412, 443, 490, 510, 560, and 665 nm) to best predict the POC / SPM ratio. The ﬁnal NN architecture was best trained using Rrs at 412, 490, 510, and 560 nm as the input layer, two hidden layers (8 and 10 neurons), and one output layer (POC / SPM ratio). The metrics used to evalu- ate the performance of the NN optimization are described in Portillo Juan and Negro Valdecantos (2022). 2.3 Determination of the organic, mineral, and mineral–organic mixed fraction As previously explained, the POC / SPM ratio is an indica- tor of the particle assemblage and can be used to partition in situ data into three water types as deﬁned by Wo´zniak et al. (2010). In addition, some theoretical and ﬁeld studies showed that the variability in the ratio bbp/bp can be related to the particle composition (Boss et al., 2004; Twardowski et al., 2001; Loisel et al., 2007; Duforêt-Gaurier et al., 2018). Low bbp/bp values are observed for a particle population domi- nated by low refractive-index material such as phytoplank- ton. In contrast, high bbp/bp values are generally observed in the presence of a relatively high concentration of inorganic particles. At 650 nm, cp is dominated by the scattering, and the optical ratio bbp/cp can be used instead of bbp/bp (Loisel et al., 2007). State Planet, 1-osr7, 11, 2023 CHAPTER3.2 H. Loisel et al.: Organic vs. inorganic fraction of suspended particulate matter from remote sensing 4 ercise (Sect. 3.1). MERIS level 1 data (∼1 km pixel res- olution) over the 2002–2012 period were processed using the polymer atmospheric correction algorithm (Steinmetz et al., 2011; Steinmetz and Ramon, 2018), which was adapted for coastal waters within the framework of the GlobCoast project. Following Mélin and Vantrepotte (2015), only pixels presenting a distance to the coast lower than 200 km and with a bottom depth not deeper than 4000 m are selected (Loisel et al., 2017). A third dataset, named DSM (for DataSet Match- up), was composed of collocated MERIS data with in situ data points of POC and SPM (Table 2), collected within the framework of the French Coastal Monitoring Network SOMLIT (Service d’Observation en Milieu Littoral, https: //www.somlit.fr/en/, last access: 22 March 2023). The cri- teria considered for the match-up selection are described in Bailey and Werdell (2006). Due to the absence of organically dominated match-up data points using the MERIS sensor, complementary match-up data points were added to DSM by looking at SeaWiFS (Sea-Viewing Wide Field-of-View Sensor) match-up with DS. We only kept the match-up data points with a good Rrs retrieval (only possible using DS). For that purpose, only data points with Rrs(in situ) /Rrs(satellite) values from 412 to 560 nm, below 0.5, or above 1.5 are se- lected. The DSM dataset is composed of 101 matched points after the application of these criteria. The POC / SPM mean value is 0.0801 for DSM instead of 0.1136 and 0.0895 for DS0 and DS, respectively. ercise (Sect. 3.1). MERIS level 1 data (∼1 km pixel res- olution) over the 2002–2012 period were processed using the polymer atmospheric correction algorithm (Steinmetz et al., 2011; Steinmetz and Ramon, 2018), which was adapted for coastal waters within the framework of the GlobCoast project. Following Mélin and Vantrepotte (2015), only pixels presenting a distance to the coast lower than 200 km and with a bottom depth not deeper than 4000 m are selected (Loisel et al., 2017). A third dataset, named DSM (for DataSet Match- up), was composed of collocated MERIS data with in situ data points of POC and SPM (Table 2), collected within the framework of the French Coastal Monitoring Network SOMLIT (Service d’Observation en Milieu Littoral, https: //www.somlit.fr/en/, last access: 22 March 2023). 2.2 Algorithm development An asymptote is reached at high POC / SPM values (above 0.2) and concerns data points with low bbp/cp (below For that purpose, a neural network approach has been se- lected as a second approach. We used a feed-forward network with log-sigmoid hidden neurons and linear output neurons coupled to a Levenberg–Marquardt algorithm allowing an ef- ﬁcient back propagation through the training procedure (Lv et al., 2018; Hagan et al., 1996). The DS dataset was ran- domly divided into three datasets to develop, train, and vali- https://doi.org/10.5194/sp-1-osr7-11-2023 CHAPTER3.2 CHAPTER3.2 H. Loisel et al.: Organic vs. inorganic fraction of suspended particulate matter from remote sensing 5 satellite Rrs (Fig. 2b). This strongly emphasizes the impact of atmospheric correction on PPC, which is a common fea- ture of OCR products. In Fig. 2b, all mineral-dominated data are from the SOMLIT dataset. As the in situ Rrs values are not available for SOMLIT data, we cannot apply the criteria described in Sect. 2.1.2 to remove inaccurate satellite Rrs re- trievals. Removing such Rrs values will certainly allow us to identify strongly misclassiﬁed patterns. In Fig. 2b, 68.4 % of mineral-dominated data, 44.4 % of mixed data, and 73.7 % of organically dominated data are well-classiﬁed. Due to at- mospheric correction uncertainties, a proper estimation of POC / SPM values from remote sensing is still very challeng- ing, while the estimation of PPC can still be performed with a reasonable accuracy. Extra match-up data points, including both in situ Rrs and POC / SPM measurements, are, however, needed to deﬁnitely support this conclusion. 0.075) values typical of phytoplankton-dominated environ- ments. The new threshold value of POC / SPM for organi- cally dominated waters is set to 0.2 (i.e., bbp/cp < 0.075) to encompass in situ data points collected during bloom events. This value is, however, very similar to the value previously obtained by Wo´zniak et al. (2010) (0.25). Data points located along the asymptote are therefore associated with organically dominated waters. The thresholds are applied to monthly POC / SPM values derived from MERIS data, and the frequency of dominance is computed for each pixel of the scene over the 10 years (2002–2012) as detailed below. A pixel geographically lo- cated at a given latitude and longitude is named the k pixel (k = 1, Stot). Stot is the total number of pixels over the se- lected geographical area. 3.2 Global and regional PPC patterns The PPC algorithm has been applied to MERIS monthly Rrs(λ) data over the 2002–2012 time period, and the max- imum occurrence of PPC has been calculated over this 10- year time period as described in Sect. 2.3 (Fig. 3a). As ex- pected, we observe a well-marked near-shore–off-shore gra- dient from mineral-dominated waters to organically domi- nated waters with a transition zone corresponding to mixed particulate assemblages. Large deltas and estuaries charac- terized by intense sediment discharge, such as the Ama- zon Estuary, Mekong Delta, Huanghe (Yellow River) Delta, Yangtze Estuary, or Ganges–Brahmaputra Delta, present a very high occurrence of mineral-dominated waters (70 %– 90 %). These areas can extend much further from the river outlet, depending on the bathymetry and the surface cur- rents, as for instance in the central part of the Yellow Sea at the Yangtze Estuary. Values of POC / SPM of 0.12–0.15, depicting mix-dominated particulate assemblages, have been measured in the East China Sea region (124–126◦E, 30– 32◦N) in great agreement with our present ﬁnding (Hung et al., 2007). The dominance of mineral-dominated waters is generally persistent throughout the considered time pe- riod as depicted by the relatively low (20 %–30 %) coefﬁ- cient of variation observed at the outlet of these different es- tuaries and deltas (Fig. 3b). In contrast, at the Amazon and Yangtze estuaries and the Huanghe Delta, some off-shore areas with a high (70 %) coefﬁcient of variation depict the impact of seasonal and interannual interactions between the continental (river and sediment discharges) and oceanic forc- ing (tide, wind, surface current, sub-mesoscale structures) and the bathymetry. For example, close interaction between the Amazon plume dominated by mineral particles and the retroﬂection of the North Brazil Current carrying oceanic particulate organic matter occurs in the region at the Amazon Estuary where the slope in the continental shelf drastically changes (Gensac et al., 2016; Varona et al., 2019), causing a signiﬁcant temporal variation in the PPC. In contrast to the Dk = maximum nk 1,nk 2,nk 3 Nk tot × 100(%). (1) (1) Eq. (1) is repeated Stot times to compute the frequency of dominance over the whole scene and provide maps in Sect. 3.2. 2.2 Algorithm development For each k pixel, we computed the number of valid pixels (Nk tot) over the period that corresponds to 120 months (Nk tot ≤120). The term “valid pixel” means a non-ﬂag pixel for which the Rrs value is provided in order to derive POC / SPM with the neural network. Then, for the k pixel, we calculate how many times the class i is identiﬁed over Nk tot: nk i is the class occurrence, and i = 1,2, or 3 with 1 for mineral-dominated, 2 for organically dominated, and 3 for mixed water classes. The frequency of dominance is de- ﬁned as the ratio of the occurrence to the number of valid pixels: H. Loisel et al.: Organic vs. inorganic fraction of suspended particulate matter from remote sensing Comparison of in situ and derived POC / SPM from neural network algorithm (NN POC / SPM) (a) for DS (i.e., in situ Rrs values are used by NN POC / SPM) and (b) for DSM (i.e., satellite Rrs values are used by NN POC / SPM). The dashed line is the 1 : 1 line. Red, blue, and green colors are for mineral-dominated, organically dominated, and mixed waters, respectively. For both panels, the vertical and horizontal solid lines indicate the thresholds (0.08 and 0.2) used to partition the water types. Dots are color-coded according to the values of the in situ POC / SPM, whereas the circles are color-coded according to the values of POC / SPM derived from NN. When circles and dots are of the same color, it means that the retrieved values belong to the same class as the in situ ones. pertinence of the PPC product is conducted over the eastern English Channel (EEC) and the southern North Sea (SNS), for which the biogeochemical and physical processes driving the suspended particulate matter composition are relatively well-documented. The eastern English Channel and southern North Sea coastal region is known for its strong hydrody- namics marked by intense tidal currents. This shallow-water region, therefore largely impacted by resuspension effects, is also largely inﬂuenced by freshwater discharges from differ- ent rivers, the most important ones being the Seine and the Somme along the French coast of the EEC and the Thames and the Scheldt along the SNS. Strong spring and early sum- mer phytoplankton blooms of diatoms and Phaeocystis glo- bosa occurred in this region at different times of the year, depending on the nutrient and light (i.e., season and turbidity previously mentioned rivers and despite the Congo River be- ing the world’s second-largest river in terms of both drainage area and water discharge, only a small PPC signature (mainly organically dominated) can be seen at the outlet of its estuary (not visible in Fig. 3a), except in the coefﬁcient of variation (Fig. 3b). This is coherent with the fact that the sediment dis- charge of the Congo River is very low, in contrast to the dis- solved part of the discharge, and that most of the particulate assemble is organic (Coynel et al., 2005). H. Loisel et al.: Organic vs. inorganic fraction of suspended particulate matter from remote sensing 6 Figure 1. Relationships between bbp/cp (650 nm) and POC / SPM from DS0. The black curve is the power regression. The vertical dashed lines represent the thresholds delimiting the water types according to Wo´zniak et al. (2010). The vertical solid lines show the new thresholds deﬁned in the present study. The linear regression (in log space) between derived (named y) and in situ bbp/cp (named x) is given, as are the regression coefﬁcient (r), the median absolute percentage difference (MAPD), the median ratio (MR), and the mean bias (MB). These statistical indicators (MAPD, MR, and MB) are calculated in normal space as described in Jorge et al. (2021). Figure 1. Relationships between bbp/cp (650 nm) and POC / SPM from DS0. The black curve is the power regression. The vertical dashed lines represent the thresholds delimiting the water types according to Wo´zniak et al. (2010). The vertical solid lines show the new thresholds deﬁned in the present study. The linear regression (in log space) between derived (named y) and in situ bbp/cp (named x) is given, as are the regression coefﬁcient (r), the median absolute percentage difference (MAPD), the median ratio (MR), and the mean bias (MB). These statistical indicators (MAPD, MR, and MB) are calculated in normal space as described in Jorge et al. (2021). Figure 2. Comparison of in situ and derived POC / SPM from neural network algorithm (NN POC / SPM) (a) for DS (i.e., in situ Rrs values are used by NN POC / SPM) and (b) for DSM (i.e., satellite Rrs values are used by NN POC / SPM). The dashed line is the 1 : 1 line. Red, blue, and green colors are for mineral-dominated, organically dominated, and mixed waters, respectively. For both panels, the vertical and horizontal solid lines indicate the thresholds (0.08 and 0.2) used to partition the water types. Dots are color-coded according to the values of the in situ POC / SPM, whereas the circles are color-coded according to the values of POC / SPM derived from NN. When circles and dots are of the same color, it means that the retrieved values belong to the same class as the in situ ones. Figure 2. State Planet, 1-osr7, 11, 2023 3.1 Validation of the classiﬁcation In situ measurements of Rrs from DS are used as input for the neural network algorithm to test the performance of the esti- mation of algorithm-derived values, named NN POC / SPM (Fig. 2a). The NN algorithm achieves a good performance over the whole range of POC / SPM (Fig. 2a). The median absolute percentage difference (MAPD) is 24 %, the median ratio (MR) is 1.06, and the bias is 0.004. The slope of the type II (log-transformed) regression is 0.90. The same clas- siﬁcation is obtained for 88.5 % of data points between the in situ and model-derived values. About 10.9 % of data points are misclassiﬁed in the adjacent group and only 0.62 % are misclassiﬁed in a non-adjacent group. SeaWiFS and MERIS match-ups on both the independent SOMLIT in situ dataset (for which no in situ Rrs values are available) and DS in situ data (for which in situ Rrs values are available) are shown in Fig. 2b. Compared to the results obtained using in situ measurements (Fig. 2a), the data points are much more scat- tered around the 1 : 1 line when POC / SPM is derived from https://doi.org/10.5194/sp-1-osr7-11-2023 State Planet, 1-osr7, 11, 2023 CHAPTER3.2 CHAPTER3.2 H. Loisel et al.: Organic vs. inorganic fraction of suspended particulate matter from remote sensing H. Loisel et al.: Organic vs. inorganic fraction of suspended particulate matter from remote sensing A speciﬁc zoom on this area reveals the dominance of organic matter even at the outlet of the estuary, in good agreement with in situ data reported in Coynel et al. (2005). While an extensive investigation of the different spa- tiotemporal patterns presented in Fig. 3 are is beyond the scope of the present paper, a speciﬁc focus illustrating the State Planet, 1-osr7, 11, 2023 https://doi.org/10.5194/sp-1-osr7-11-2023 CHAPTER3.2 H. Loisel et al.: Organic vs. inorganic fraction of suspended particulate matter from remote sensing 7 Figure 3. (a) Frequency (%) of dominant water type (red for mineral-dominated, green for mixed, blue for organically dominated) over the MERIS time period of 2002–2012. The yellow color corresponds to unclassiﬁed pixels, which means pixels for which the retrieved value of POC / SPM is non-valid (i.e., negative). (b) Coefﬁcient of variation in POC / SPM over the MERIS period of 2002–2012. level) availability (Breton et al., 2000; Antajan et al., 2004; Sazhin et al 2007) For these different reasons the com- monthly occurrence of each PPC class is in excellent agree- ment with our present knowledge of this area (Fig 4) Be- Organic vs. inorganic fraction of suspended particulate matter from remote sensing H. Loisel et al.: Organic vs. inorganic fraction of suspended particulate matter from remote sensing 7 Figure 3. (a) Frequency (%) of dominant water type (red for mineral-dominated, green for mixed, blue for organically dominated) over th MERIS time period of 2002–2012. The yellow color corresponds to unclassiﬁed pixels, which means pixels for which the retrieved value o POC / SPM is non-valid (i.e., negative). (b) Coefﬁcient of variation in POC / SPM over the MERIS period of 2002–2012. Figure 3. (a) Frequency (%) of dominant water type (red for mineral-dominated, green for mixed, blue for organically dominated) over the MERIS time period of 2002–2012. The yellow color corresponds to unclassiﬁed pixels, which means pixels for which the retrieved value of POC / SPM is non-valid (i.e., negative). (b) Coefﬁcient of variation in POC / SPM over the MERIS period of 2002–2012. level) availability (Breton et al., 2000; Antajan et al., 2004; Sazhin et al., 2007). For these different reasons, the com- position of the suspended particulate matter in this area is strongly variable, as already stressed by different ﬁeld studies based on optical and biogeochemical measurements (Loisel et al., 2007; Vantrepotte et al., 2007; Lubac et al., 2008). H. Loisel et al.: Organic vs. inorganic fraction of suspended particulate matter from remote sensing H. Loisel et al.: Organic vs. inorganic fraction of suspended particulate matter from remote sensing 8 Figure 4. Frequency of dominance (%) for mineral-dominated (red), mixed (green), organically dominated (blue), or unclassiﬁed (yellow) waters over the MERIS period of 2002–2012 in the eastern English Channel and the southern North Sea. Figure 4. Frequency of dominance (%) for mineral-dominated (red), mixed (green), organically dominated (blue), or unclassiﬁed (yellow) waters over the MERIS period of 2002–2012 in the eastern English Channel and the southern North Sea. 4 Concluding remarks physical (i.e., waves) forcing (Lesourd et al., 2003; Lancelot et al., 2007; Gohin, 2011; Renosh et al., 2014). This is con- sistent with the results of Vantrepotte et al. (2012), who ob- served that the relative contribution of the particulate matter assemblage with a strong proportion of mineral particles in- creases signiﬁcantly during the winter time. In March, organ- ically dominated pixels appear in the SNS coastal region and at the Somme River area in the EEC. Then, while the organ- ically dominated particle assemblage areas are developing in the SNS from March to July, similar particulate assemblages start developing from May to July in off-shore waters of the EEC and along the EEC coast from June. The timing of the development of organic matter areas in the EEC and the SNS is in perfect agreement with phytoplankton bloom dynamics in these regions, which ﬁrst develop in the Bay of the Somme area and the SNS, followed by another bloom which develops south in the area of the Bay of the Seine (Brunet et al., 1996; Vantrepotte et al., 2007; Joint and Pomroy, 1993; Van der Zande et al., 2012; Gohin et al., 2019). Finally, areas covered by organically dominated pixels are at a maximum in June and July, and then mixed assemblages start developing in the northern part of the domain to fully cover the whole region in October. Mixed assemblages are more likely dominated by aggregates of mineral particles and detrital matter (non-living organic particles) which developed during the senescence of the bloom (Vantrepotte et al., 2007; Loisel et al., 2007; Go- hin, 2011; Grattepanche et al., 2011). physical (i.e., waves) forcing (Lesourd et al., 2003; Lancelot et al., 2007; Gohin, 2011; Renosh et al., 2014). This is con- sistent with the results of Vantrepotte et al. (2012), who ob- served that the relative contribution of the particulate matter assemblage with a strong proportion of mineral particles in- creases signiﬁcantly during the winter time. In March, organ- ically dominated pixels appear in the SNS coastal region and at the Somme River area in the EEC. Then, while the organ- ically dominated particle assemblage areas are developing in the SNS from March to July, similar particulate assemblages start developing from May to July in off-shore waters of the EEC and along the EEC coast from June. H. Loisel et al.: Organic vs. inorganic fraction of suspended particulate matter from remote sensing The monthly occurrence of each PPC class is in excellent agree- ment with our present knowledge of this area (Fig. 4). Be- tween mid-fall and winter (from October to February), the particulate matter pool of the EEC and the SNS is character- ized by mineral and mix-dominated assemblages, mainly due to sediment river inputs and resuspension effects from strong State Planet, 1-osr7, 11, 2023 https://doi.org/10.5194/sp-1-osr7-11-2023 CHAPTER3.2 CHAPTER3.2 4 Concluding remarks inorganic fraction of suspended particulate matter from remote sensing H. Loisel et al.: Organic vs. inorganic fraction of suspended particulate matter from remote sensing 9 FS provided the Polymer algorithm to perform atmospheric correc- tions to process satellite images in coastal waters. AM, MB, and OHFd’A gave suggestions and comments. 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V., Pahlevan, N., Smith, B., Binding, C., Schalles, J., Loisel, H., Gurlin, D., Greb, S., Alikas, K., Randla, M., Bunkei, M., Moses, W., Nguy\^{e}˜n, H., Lehmann, M. K., O’Donnell, D., Ondrusek, M., Han, T.-H., Fichot, C. G., Moore, T., and Boss, E.: Robust algorithm for estimating total suspended solids (TSS) in inland and nearshore coastal waters, Remote Sens. Environ., 246, 111768, https://doi.org/10.1016/j.rse.2020.111768, 2020. Acknowledgements. This study was realized with support from the French Space Agency (CNES) through the COYOTE project (CNES/TOSCA program). MERIS data were processed within the framework of the GlobCoast project funded by the French Agence Nationale de la Recherche (grant no. ANR-11-BS56-0018). Some of the in situ measurements used in this study were collected from different cruises performed in Vietnam coastal waters, French Guiana, and the eastern English Channel through various projects funded by CNES. The authors are grateful to all the people that contributed to data provided by the Service d’Observation en Mi- lieu Littoral (SOMLIT), INSU-CNRS. We also thank our many col- leagues who participated in the collection of the various datasets cited in this study. We thank Arnaud Cauvin and Xavier Mériaux, who conducted ﬁeld missions at LOG, processed ﬁeld biogeophys- ical data, and were in charge of LOG database management. 4 Concluding remarks We express our gratitude to Roy Elhourany for his advice concerning the neural network algorithm. The authors thank the reviewers, who helped to improve the paper with their comments and suggestions. Bonato, S., Breton, E., Didry, M., Lizon, F., Cornille, V., Lécuyer, E., Christaki, U., and Artigas, L. F.: Spatio- temporal patterns in phytoplankton assemblages in inshore– offshore gradients using ﬂow cytometry: A case study in the eastern English Channel, J. Marine Syst., 156, 76–85, https://doi.org/10.1016/j.jmarsys.2015.11.009, 2016. Boss, E., Pegau, W. S., Lee, M., Twardowski, M., Shybanov, E., Korotaev, G., and Baratange, F.: Particulate backscatter- ing ratio at LEO 15 and its use to study particle com- position and distribution, J. Geophys. Res., 109, C01014, https://doi.org/10.1029/2002JC001514, 2004. Breton, E., Brunet, C., Sautour, B., and Brylinski, J.-M.: Annual variations of phytoplankton biomass in the East- ern English Channel: comparison by pigment signatures and microscopic counts, J. Plankton Res., 22, 1423–1440, https://doi.org/10.1093/plankt/22.8.1423, 2000. Financial support. This research has been supported by the Cen- tre National d’Etudes Spatiales (TOSCA/COYOTE project) and the Agence Nationale de la Recherche (grant no. ANR-11-BS56-0018). Brunet, C., Brylinski, J., Bodineau, L., Thoumelin, G., Bentley, D., and Hilde, D.: Phytoplankton Dynamics During the Spring Bloom in the South-eastern English Channel, Estuar. Coast. Shelf S., 43, 469–483, https://doi.org/10.1006/ecss.1996.0082, 1996. Review statement. This paper was edited by Griet Neukermans and reviewed by two anonymous referees. Bélanger, S., Babin, M., and Larouche, P.: An empirical ocean color algorithm for estimating the contribution of chro- mophoric dissolved organic matter to total light absorption in optically complex waters, J. Geophys. Res., 113, C04027, https://doi.org/10.1029/2007JC004436, 2008. 4 Concluding remarks The timing of the development of organic matter areas in the EEC and the SNS is in perfect agreement with phytoplankton bloom dynamics in these regions, which ﬁrst develop in the Bay of the Somme area and the SNS, followed by another bloom which develops south in the area of the Bay of the Seine (Brunet et al., 1996; Vantrepotte et al., 2007; Joint and Pomroy, 1993; Van der Zande et al., 2012; Gohin et al., 2019). Finally, areas covered by organically dominated pixels are at a maximum in June and July, and then mixed assemblages start developing in the northern part of the domain to fully cover the whole region in October. Mixed assemblages are more likely dominated by aggregates of mineral particles and detrital matter (non-living organic particles) which developed during the senescence of the bloom (Vantrepotte et al., 2007; Loisel et al., 2007; Go- hin, 2011; Grattepanche et al., 2011). The PPC can be used as an appropriate indicator for charac- terizing the nature of bulk suspended particulate matter from satellite OCR remote sensing. The MERIS spatiotemporal patterns of this new indicator are consistent with general fea- tures documented in the literature. Due to the visible bands used, common to all OCR sensors, the PPC algorithm can be applied to all OCR satellite sensors, allowing us to as- sess the long-term trend of suspended particulate matter over the global coastal ocean. The application of this algorithm to the satellite Copernicus Marine Environment Monitoring Service (CMEMS) data is underway to assess the different temporal patterns (seasonal, trend, and irregular) using the Census X-11 approach (Vantrepotte et al., 2011). Data availability. DS0, DS, and DSM can be obtained on request. Author contributions. HL, as the recipient of most of the fund- ing, proposed the idea of the work, gave permission to use data in LOG, was involved in the algorithm development and data anal- ysis, and co-wrote the paper. LDG was involved in the algorithm development, supervised the analysis and visualization of the data, and was involved in paper organization and co-writing. TKT ana- lyzed and visualized the in situ and satellite data, developed and tested the algorithms, and developed the codes to process images. DSFJ supported the development of the MATLAB-processed im- ages, provided the ﬁnal plots and statistics, and revised the paper. 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Shelf Sci., 72, 201– 212, https://doi.org/10.1016/j.ecss.2006.10.016, 2007. https://doi.org/10.5194/sp-1-osr7-11-2023 State Planet, 1-osr7, 11, 2023 CHAPTER3.2 CHAPTER3.2 Chapter 3.3 – 7th edition of the Copernicus Ocean State Report (OSR7) Chapter 3.3 – 7th edition of the Copernicus Ocean State Planet, 1-osr7, 12, 2023 https://doi.org/10.5194/sp-1-osr7-12-2023 © Author(s) 2023. This work is distributed under the Creative Commons Attribution 4.0 License. ter 3.3 – 7th edition of the Copernicus Ocean State Report (O Recent changes in extreme wave events in the south-western South Atlantic n of the Copernicus Ocean State Report (OSR7) rnicus Ocean State Report (OSR7) Institute of Environmental Research, Secretariat of Environment, Infrastructure and Logistics of São Paulo State (SEMIL/SP), Rua Joaquim Távora 822, 04015-011, São Paulo – SP, Brazil 3Department of Physical Geography, Faculty of Philosophy, Literature and Human Sciences, University of São Paulo (FFLCH/USP), Av. Prof. Lineu Prestes, 338, 05508-000, São Paulo – SP, Brazil 4Department of Atmospheric Sciences, Institute of Astronomy, Geophysics and Atmospheric Science, University of São Paulo, Rua do Matão, 1226, 05508-090, São Paulo – SP, Brazil an State Report (OSR7) Correspondence: Carolina B. Gramcianinov (carolina.gramcianinov@hereon.de) 1 Introduction In recent years, several extreme events have been reported in the South Atlantic Ocean (e.g. Marcello et al., 2018; Dalagnol et al., 2022), thus directly reﬂecting hazards along the coast. One of the regions that are facing relevant changes is the south-western South Atlantic (SWSA), with an in- crease in extreme wave and storm surge occurrences (Souza et al., 2019; Gramcianinov et al., 2023). With high economic relevance, the SWSA region hosts strategic harbours in South America, where 7.55 × 108 t of goods were transported in 2021 (ANTAQ, 2022), and promising oil and gas exploration ﬁelds. In addition, the region also holds rich biodiversity, in- cluding coral reefs and 856 km2 of mangroves that are cru- cial for coastal hazard protection, economic activities (e.g. ﬁshery), and the cultural identity of the coastal communi- ties (ICMBio, 2018; Pereira-Filho et al., 2021). The SWSA coastal cities have a dense population, with approximately 20 million people who are extremely vulnerable to coastal erosion and infrastructure damage (Zamboni and Nicolodi, 2008). Assessing the extreme waves and wave trends in the SWSA with traditional approaches has proven to be chal- lenging for several reasons. The difﬁculties remain mostly in the still-limited understanding of the local physical pro- cesses (e.g. wave–current interaction) and climate variabili- ties (e.g. the overlapping effect teleconnections). The limited accuracy of long-term integrations and the scarce data avail- ability can make these analyses even more difﬁcult. Some recent studies have revealed changes in the wave pattern in the South Atlantic, usually ascribed to the increase in the ex- treme waves in the Southern Ocean (SO). In general, pre- vious global or hemisphere-based studies have reported in- creases in wave height extremes in the Southern Hemisphere (SH) over the past 41 years, and this tendency is expected to continue in the future (Caires and Sterl, 2005; Dobrynin et al., 2012; Lemos et al., 2019). However, when focusing on the SWSA, the mean and extreme wave climate trends present larger uncertainties. In addition to understanding the signiﬁcant wave height (Hs) trends, assessing changes in wave event characteristics, such as the mean wave direction and peak period, is of ut- most importance. Silva et al. (2020) showed how the oscil- lation between the southern and eastern dominant wave en- ergy ﬂux directions has led to changes in the coastal mor- phodynamics at both regional and local scales. Received: 31 July 2022 – Discussion started: 23 September 2022 Revised: 8 March 2023 – Accepted: 31 March 2023 – Published: 27 September 2023 (2021b); numerical models PUM: Law-Chune (2022) 2 GLOBAL_ANALYSIS_FORECAST_WAV_001_027 (GLO-NRT), EU Copernicus Marine Service Product (2022) QUID: Aouf (2022); numerical models PUM: Dalphinet et al. (2022) 3 Baixada Santista Coastal Hazards database (BDe-BS), Personal contact Souza et al. (2019, 2022) dataset Received: 31 July 2022 – Discussion started: 23 September 2022 Revised: 8 March 2023 – Accepted: 31 March 2023 – Published: 27 September 2023 Received: 31 July 2022 – Discussion started: 23 September 2022 Revised: 8 March 2023 – Accepted: 31 March 2023 – Published: 27 September 2023 Abstract. Over the past decades, the South Atlantic Ocean has experienced several changes, including a re- ported increase in coastal erosion and ﬂoods. This study aims to investigate the recent changes in the extreme wave events over the south-western South Atlantic (SWSA) – which hosts the most economically important har- bours in South America, high oil and gas production demands, and rich biodiversity. This investigation considers not only the occurrence of extreme wave events but also extreme wave indicators that characterise the potential wave impact on offshore and coastal areas. Extreme wave events are obtained using the averaged monthly 95th percentile of signiﬁcant wave height (Hs) from 1993 to 2021, combining the CMEMS global wave reanalysis and near-real-time products. Annual and seasonal statistics are derived to analyse mean and extreme wave climate and trends in the study region, focusing on Hs, peak period, and wave power. The analysis gives an overview of the wave climate in the study domain, including a discussion about seasonal differences. For a more direct application to future risk assessment and management, we perform an analysis considering the regional moni- toring and warning system division established by the Brazilian Navy. We used a coastal hazard database that covers a portion of the coast to investigate how the trends given by the CMEMS wave products may impact the coastal zone. Our ﬁndings showed signiﬁcant changes in the SWSA mainly associated with an increase in mean values of Hs, wave period, and consequently the wave power. Narrowing down to the coastal impact, we found an increase in the number of coastal hazards in the State of São Paulo associated with waves, which agrees with the increase in the number of extreme wave events in the adjacent ocean sector. However, the increased number of coastal events is also driven by local factors. Published by Copernicus Publications. CHAPTER3.3 CHAPTER3.3 C. B. Gramcianinov et al.: Extreme waves in the south-western South Atlantic 2 Table 1. Copernicus Marine Service (CMEMS) and non-CMEMS products used in this study, including the Quality Information Document (QUID) and Product User Manual (PU Product Product ID and type Data access Documentation ref. no. 1 GLOBAL_MULTIYEAR_WAV_001_032 (WAVERYS), EU Copernicus Marine Service Product (2021) QUID: Law-Chune et al. State Planet, 1-osr7, 12, 2023 1 Introduction Some previ- ous works reported wave power changes under the present climate (Odériz et al., 2021; 1979–2018) and mean wave di- rection and period changes in both present (Hemer et al., 2010; 1989–2005) and future climate (Lobeto et al., 2021; 2081–2100). These changes directly affect naval and coastal risk assessments, requiring special efforts to properly link the global-scale ﬁndings to regional and local wave extremes. Against such a background, this section aims to report and investigate the recent extreme wave climate trends (1993– https://doi.org/10.5194/sp-1-osr7-12-2023 State Planet, 1-osr7, 12, 2023 CHAPTER3.3 C. B. Gramcianinov et al.: Extreme waves in the south-western South Atlantic 3 .2 Percentile computation 2.2 2021) in the SWSA while focusing on wave event char- acteristics such as event frequency, intensity, duration, and peak period. We examined the seasonal statistics and cli- matic trends using both traditional (i.e. percentile-based) and storm-based approaches to provide new insights into the re- gional wave climate changes. To obtain results with more di- rect application to future risk assessment and management, we performed an analysis considering the regional monitor- ing and warning system, as well as the impact of the recent wave climate changes on the coast. In this work, the percentiles were computed using the em- pirical distribution of the Hs peaks (Hspeaks) within a given period, thus allowing us to obtain a more detailed view of in- dividual wave events’ occurrence. The selected Hspeaks must be separated by a minimum of 48 h to guarantee the inde- pendence of the peaks. This time window has been widely applied in past studies to ensure the collection of one peak per storm (e.g. Caires and Sterl, 2005; Meucci et al., 2020). Besides that, 48 h is a suitable but not so restrictive time threshold for extreme-wave analysis in the region, partic- ularly considering the differences among the seasons. The 95th percentile was computed based on the monthly Hspeaks distribution at each grid point. Using these monthly 95th percentiles, we calculated the annual and seasonal means to analyse trends and proceed with the wave event analy- sis (Sect. 2.4). The seasonal mean of the monthly 95th per- centiles was computed for the summer and winter, using the average December–January–February (DJF) and June–July– August (JJA), respectively, thus having one value per year. The annual percentiles were computed by the average of all monthly percentiles within the year. 2.3 Trend estimation and testing Trends were estimated based on Sen’s slope estimator (Sen, 1968), which evaluates the magnitude of a time series trend. The signiﬁcance of Sen’s slope was calculated by the Mann– Kendall test (Mann, 1945; Kendall, 1975), considering a p value lower than 0.05. Both methods are non-parametric (distribution-free) procedures and consider the monotonic increase upward or downward of the time series, thus be- ing more robust to climate-based analysis (e.g. Wang et al., 2020). 2.1 Datasets The main dataset used in this work was the Copernicus Marine Service (CMEMS) global reanalysis, named WAV- ERYS (product ref. no. 1; Table 1; Law-Chune et al., 2021a), available from 1993 to 2020. To include 2021 in the analy- sis, the WAVERYS was complemented with data from the CMEMS Global Ocean Waves Analysis Near Real Time product (GLO-NRT; product ref. no. 2; Table 1). The combi- nation (in time) of these two products is hereafter referred to as CMEMS wave products. WAVERYS is available at a 1/5◦ horizontal grid as 3-hourly outputs from 1993 to 2020, while the wave analysis has a 1/12◦horizontal grid as 3-hourly instantaneous output ﬁelds. The GLO-NRT product was in- terpolated to the 0.20◦horizontal grid, so a more consis- tent analysis can be achieved despite using different sources. Both products are produced using the Météo-France Wave Model (MFWAM) with the dissipation terms developed by Ardhuin et al. (2010). WAVERYS is forced by hourly surface winds and daily sea-ice fraction ﬁelds derived from the ﬁfth- generation reanalysis from the European Centre for Medium- Range Weather Forecasts (ECMWF) (ERA5; Hersbach et al., 2020) and ocean currents obtained from the ocean reanalysis Global Ocean Reanalysis and Simulation (GLORYS). The GLO-NRT is forced only by a 6-hourly wind analysis from the IFS-ECMWF atmospheric system. 1 Introduction As a ﬁnal result, we have a mean annual and seasonal percentile time series at each grid point. 2.5 Wave power calculation Following Staneva et al. (2022), we also calculated the wave power in the study domain. Wave power or wave energy ﬂux was obtained following Eq. (1): et al., 2021; Souza et al., 2022). The deﬁnition of coastal hazards is mainly based on processes such as coastal ero- sion and/or coastal inundation, the latter also forced by conti- nental ﬂooding (heavy rainfall) in estuarine areas. Therefore, the coastal hazards registered in the BDe-BS represent events with high intensity since they were brought to the attention of the public due to their signiﬁcant impact on the beaches, destruction of urban structures, and public and private prop- erties, as well as disruption of the city’s day-to-day and port activities. More details regarding the database can be found in Souza et al. (2019). P = ρ g2 64 π Hs2 Te, (1) (1) where P is the wave energy ﬂux per unit of wave-crest length (kW m−1), ρ is water density, g is the acceleration due to gravity, Hs is the signiﬁcant wave height, and Te is the wave energy period. Te is directly available in the WAV- ERYS products (named VTM10) and is deﬁned as the mean wave period obtained by the Te ≡Tm−1,0 = m−1/m0, based on the −1th and 0th moment of the wave spectrum. Currently, the BDe-BS initiative is maintained by the São Paulo state government through the “Preventive Plan for Coastal Erosion, Coastal Inundation, and Flooding” (adapted from the Portuguese: Plano Preventivo de Defesa Civil para Erosão Costeira, Inundações Costeiras e Enchen- tes/Alagamentos causadas por Eventos Meteorológicos- Oceanográﬁcos Extremos como Ressacas do Mar e Marés Altas). Despite representing a small portion of the coastal area of the SWSA, the number of intense/extreme events reaching the São Paulo state coast can be considered repre- sentative of most of the coastal extension of the domain in this study, except for subarea D (see Fig. 1). The reason for this extrapolation is mainly the lack of long-term records in other locations. In addition, the database covers the central portion of the study region in a region with high economic importance. C. B. Gramcianinov et al.: Extreme waves in the south-western South Atlantic 4 Figure 1. The south-western South Atlantic Ocean with subareas A to D deﬁned by the Brazilian Navy for warning and monitoring op- erations within MetArea V. Shaded values are the bathymetry from ETOPO1. Following Weisse and Günther (2007), the intensity is equal to the difference between the maximum Hs of the event and the SET at that point. The wave event statistics, such as the number of events, intensity, mean wave direc- tion, and peak period, are presented herein as annual and seasonal means to build the spatial distribution and trends and obtain the spatially averaged time series. The intensity and wave parameters were calculated by averaging all indi- vidual events (above SET) within the year or season. The same analysis was applied successfully in the Black Sea by Staneva et al. (2022), allowing a better understanding of the extreme wave events’ spatial distribution and trends. More details about these methods can be found in Weisse and Gün- ther (2007). Figure 1. The south-western South Atlantic Ocean with subareas A to D deﬁned by the Brazilian Navy for warning and monitoring op- erations within MetArea V. Shaded values are the bathymetry from ETOPO1. 2.6 Coastal risk assessment Warnings and risk assessment in this region are supervised by the Centre of Hydrography of the Brazilian Navy (CHM, from Centro de Hidrograﬁa da Marinha), which is recog- nised by the World Meteorological Organization (WMO) as the issue service for the MetArea V (Atlantic waters west of 7◦N–35.8◦S, 20◦W). According to the CHM monitoring system, the coastal region of SWSA can be divided into four subareas (Fig. 1). These subareas were used to analyse the wave climate trends in the domain, considering the regional speciﬁcity and facilitating future discussions about risk man- agement. This analysis may facilitate the applicability of the results found here to improve future monitoring and warning system development. We used a historical database of coastal hazards in the State of São Paulo, within subarea C, to further investi- gate coastal impacts. The Baixada Santista Coastal Hazards database (BDe-BS; product ref. no. 3; Table 1) covers the period from 1928 to 2021 and is obtained using the hemero- graphic method (mostly newspapers) and material from so- cial media (mostly videos), showing coastal impacts caused by strong waves and anomalously high tides (either meteo- rological or astronomical tides) (Souza et al., 2019; Linhares 2.4 Extreme-wave-event analysis An evaluation of WAVERYS for the western South At- lantic wave climate was made by Crespo et al. (2022). The authors compared Hs from the WAVERYS, ERA5, and the National Centers for Environmental Prediction (NCEP) wave reanalysis (Chawla et al., 2013) against wave buoy mea- surements at three locations along the Brazilian coast and found that WAVERYS presented the highest correlation and the lowest root mean square deviation (RMSD). The ERA5 performance in representing the winds is also relevant as the quality of the forcing ﬁeld is crucial in a wave simula- tion. Previous works have shown that ERA5 can represent the wind climate, extreme percentiles, and storm variability (e.g. Belmonte Rivas and Stoffelen, 2019; Gramcianinov et al., 2020a; Crespo et al., 2022). The wave event statistics were derived following the methods developed by Weisse and Günther (2007), in which consecu- tive points over a speciﬁc threshold within a given time series are considered to deﬁne extreme wave events. This event- counting process was performed for each grid cell consider- ing its unique severe event threshold (SET), deﬁned herein as the average of the monthly 95th percentile of Hspeaks con- sidering the whole period (1993–2021). Notably, there is no widely accepted method for selecting threshold values, and values between the 90th and 99th percentile Hs are of- ten used (De Leo et al., 2020; Gramcianinov et al., 2020b). Moreover, the use of the averaged monthly percentile results in a smoothed ﬁeld, especially due to the Hspeaks variabil- ity among the year. In this way, for some locations, the ex- ceedance of events above SET was larger than 5 %. State Planet, 1-osr7, 12, 2023 State Planet, 1-osr7, 12, 2023 https://doi.org/10.5194/sp-1-osr7-12-2023 CHAPTER3.3 CHAPTER3.3 State Planet, 1-osr7, 12, 2023 3.2 Extreme wave event trends The SASH mostly inﬂuences the wave climate by generating easterly waves towards central Brazil, northward from 23◦S. How- ever, the wave events in this location are associated with rela- tively small Hs, as is possible to see by the local Hsp95 values (Fig. 2a, i) and wave event intensities (Fig. 2c, k). For in- stance, the Hsp95 values in the northern portion of the domain do not reach 3.5 m in the winter (Fig. 2i). p95 Figure 3 also shows the spatial trends of the mean dis- tribution of the wave peak period and wave power during the events (Fig. 3d, e, i, j). There is a general increase in the peak period in the study domain, conﬁned to the cen- tral portion of the coast in the winter (Fig. 3i). The increase in wave period and Hs can lead to important changes in the wave power (Eq. 1). Note that for the wave power calcula- tion, Te was used, and the trends presented in Fig. 3d and i are based on the Tp. However, considering a JONSWAP wave spectrum, Te is directly related to Tp (Te = 0.9×Tp; Guillou, 2020). The wave power presents a small but signiﬁcant in- crease along the coast in the whole period and wintertime (Fig. 3e, j), reaching maximum values (> 0.2 kW m−1 yr−1) offshore of the southern Brazilian and Uruguayan coast (30– 40◦S) in the winter. Following the mean Hs behaviour, the increase in the wave power is larger in the winter than in the whole period – as expected, as wave power is proportional to Hs2 (Eq. 1). Other extreme event indicators, such as in- tensity, mean wave direction, and lifetime, did not present a robust trend signal and, therefore, are not shown. The overall pattern and values presented in Fig. 2 agree with previous studies, even though methodological differ- ences exist, thus making a straightforward comparison dif- ﬁcult. For instance, Gramcianinov et al. (2020b), using the 90th percentile computed through a spatially varying time window, found a mean of 1.3 and 5.5 extratropical cyclones per year associated with extreme wave events in the region in the summer and winter, respectively. These values are com- parable with the number of events presented in the maps of Fig. 2b, g, and l. 3.2 Extreme wave event trends The monthly Hsp95 trends present a sparse and weak signal in the study domain, except for the winter (Fig. 3f). The southern coast presents a signiﬁcant increase in the Hsp95 values, which are greater than 2 cm yr−1 in some locations during the wintertime. When looking at the mean Hs trend, it is possible to see a general increase in this wave parame- ter along the Brazilian continental shelf, covering the coastal and offshore regions. The magnitude of the mean Hs increase is small (< 0.2 cm yr−1) but signiﬁcant in the whole period (Fig. 3b). The mean Hs increase in winter is relatively greater (between 0.4 and 0.8 cm yr−1; Fig. 3g). The differences be- tween the mean and Hsp95 trend signal are in agreement with the ﬁndings of Young and Ribal (2019), who showed that the Hs distribution changes in the last years were skewed to the left, with an increase in small waves – which can change the mean without changing the extreme percentiles. The trends in the number of extreme wave events also present sparse behaviour, but with signiﬁcant increases along the Brazilian coast (Fig. 3c, h). The event increase occurs on most of the coast in the whole-period analysis, while it is conﬁned to some portions of the southern and south-eastern coast dur- ing the winter (Fig. 3h). It is important to note that the rise in the number of events does not follow the Hsp95 trend pattern. During the winter, the main storm track is in its north- ernmost position (Hoskins and Hodges, 2005), resulting in more wave events than in other seasons (Fig. 2j). Typically, in winter, the region presents relatively long fetches along the coast (south-west-/north-east-oriented) associated with cy- clones generated at approximately 35◦S (Gramcianinov et al., 2021). These fetches can be widely intensiﬁed by rear anticyclones on the western side of the cyclone, thus caus- ing this conﬁguration to be widely related to the most severe cases observed in the domain (e.g. da Rocha et al., 2004; Machado et al., 2010; Dragani et al., 2013). The high number of events in the northern boundary of the domain (Fig. 2b, j) can be associated with the South At- lantic Subtropical High (SASH), which is also a generating system in the study region (Pianca et al., 2010). 3.1 Extreme-wave-climate characterisation The 95th percentile distribution, with its gradient towards the south (Fig. 2a, e, i) followed by the concentration of more intense extreme events to the southern portion of the domain (Fig. 2c, g, k), reﬂects the inﬂuence of the South Atlantic https://doi.org/10.5194/sp-1-osr7-12-2023 CHAPTER3.3 C. B. Gramcianinov et al.: Extreme waves in the south-western South Atlantic 5 extreme wave events in the coastal region between 30◦S and 32◦S and found a mean of 1.33 events per year above the 90th percentile between 1979 and 2008. We also reported this rel- atively small value at this exact location in Fig. 2b, g, and l. In this way, the method applied herein presents robust results according to what is reported in the region. storm track in the region. The storm track controls the ex- treme wave climate in the SWSA due to the strongest winds associated with the cyclones (e.g. Campos et al., 2018), and it is located between 55 and 30◦S (Hoskins and Hodges, 2005; Gramcianinov et al., 2019). Despite sharing a common extreme-wave-generation source, as is possible to see by the similar mean-wave- direction distribution (Fig. 2d, h, l), summer (DJF) and win- ter (JJA) present distinct wave patterns due to the south- ward shift in the storm track in the summertime (Hoskins and Hodges, 2005). Thus, the summer presents smaller val- ues of Hs – and consequently 95th percentile Hspeaks (Hsp95 hereafter) values (Fig. 2e) – in the study domain, which is re- ﬂected a lower number of events (Fig. 2f) and weaker events (Fig. 2g) than winter and the whole period. Climatologically, the austral autumn (MAM) presents behaviour closer to the summer pattern, while spring (SON) and winter patterns are similar. In this study, we further analyse the whole period and winter since summer does not present many cases. C. B. Gramcianinov et al.: Extreme waves in the south-western South Atlantic C. B. Gramcianinov et al.: Extreme waves in the south-western South Atlantic 6 Figure 2. (a, f, k) Averaged monthly 95th percentile Hspeaks value (m), (b, g, l) extreme-wave-event number, (c, h, m) intensity (Hs–95th Hspeaks) (m), (d, i, n) mean direction (degrees), and (e, j, o) mean wave power (10−1 kW m−1) in (a–e) the whole period (1993–2021), (f–j) summer (DJF), and (k–o) winter (JJA) based on the CMEMS wave products (product ref. nos. 1 and 2; Table 1). Figure 2. (a, f, k) Averaged monthly 95th percentile Hspeaks value (m), (b, g, l) extreme-wave-event number, (c, h, m) intensity (Hs–95th Hspeaks) (m), (d, i, n) mean direction (degrees), and (e, j, o) mean wave power (10−1 kW m−1) in (a–e) the whole period (1993–2021), (f–j) summer (DJF), and (k–o) winter (JJA) based on the CMEMS wave products (product ref. nos. 1 and 2; Table 1). 2021), which make it difﬁcult to correlate climate indexes with wave parameters directly. However, even considering these variabilities, most parameters present a positive trend, although not always signiﬁcant. As explained in Sect. 2.3, we consider the Mann–Kendall test to assess the signiﬁcance of the trends. The sensitivity of the Mann–Kendall test may be related to the large variance of the time series, which di- rectly inﬂuences the trend detected by this method (Wang et al., 2020). analysis can produce sparse results that may not be easily applied to more practical and operational tasks. Therefore, the trends in some event parameters were analysed for each Brazilian Navy monitoring and warning subarea (Fig. 4). For this analysis, we focused on the parameters with signiﬁcant trends in at least one region and season, although both whole- period and winter trends are presented in Fig. 4 for consis- tency. Both subareas C and D present a signiﬁcant increase in the number of events in the whole period. The trends of 0.2 and 0.28 events per year represent an increase of ∼20 % in subareas C and D in 29 years (based on the increase in the annual mean of their series). Together with subarea B, these regions also present an increase in the mean wave power – despite no signiﬁcant change in the peak period. In the win- ter, subareas A and C present signiﬁcant trends in the number of events per year, representing a 27.2 % and 20 % increase, respectively. C. B. Gramcianinov et al.: Extreme waves in the south-western South Atlantic Subarea C presents a small but signiﬁcant in- crease in peak period in the winter, as well as in the wave power. The wave power also increases in subarea D in the winter. 3.2 Extreme wave event trends Regarding the intensity, the same au- thors found a mean Hs of 6.5 m associated with these cy- clone events, which is also comparable to the intensity values (above the percentile) in some locations of the study domain (Fig. 2c, h, m). Moreover, Machado et al. (2010) accessed The extreme-event analysis based on each grid point in a high-resolution hindcast provides a more detailed view of pattern changes along the coast. On the other hand, such an https://doi.org/10.5194/sp-1-osr7-12-2023 State Planet, 1-osr7, 12, 2023 CHAPTER3.3 CHAPTER3.3 CHAPTER3.3 State Planet, 1-osr7, 12, 2023 C. B. Gramcianinov et al.: Extreme waves in the south-western South Atlantic 7 Figure 3. Trends in the (a, f) 95th percentile Hspeaks (cm per year), (b, g) mean Hs (cm per year), (c, h) number of extreme wave events (number per year), (d, i) peak period of the events (10−1 s yr−1), and (e, j) mean wave power (10−1 kW m−1 yr−1) in the (a–e) whole period and (f–j) winter (JJA) based on the CMEMS wave products (product ref. nos. 1 and 2; Table 1). Grey crosses represent points where the trend is signiﬁcant (Mann–Kendall test, p value ≤0.05). Figure 3. Trends in the (a, f) 95th percentile Hspeaks (cm per year), (b, g) mean Hs (cm per year), (c, h) number of extreme wave events (number per year), (d, i) peak period of the events (10−1 s yr−1), and (e, j) mean wave power (10−1 kW m−1 yr−1) in the (a–e) whole period and (f–j) winter (JJA) based on the CMEMS wave products (product ref. nos. 1 and 2; Table 1). Grey crosses represent points where the trend is signiﬁcant (Mann–Kendall test, p value ≤0.05). Most of the coastal hazards reported by the BDe-BS are as- sociated with waves, combined or not with a tidal rise. This is revealed by the numbers not only in Table 2 but also in the time series (Fig. 5a). The trends of all events and events associated with waves are similar, especially considering the trend error. It is possible to compare the trends in the coastal hazards forced by waves (0.22 events per year; Table 2) with the trends of the number of extreme wave events in subarea C (Fig. 5b; 0.20 events per year in Fig. 4a). However, con- sidering the mean number of coastal events forced by waves (4.4 events per year), the increase on the coast corresponds to 145 % in 29 years. pared to the whole period (30 %). Thus, the hazards forced exclusively by anomalous tides are rare in the winter, which may be related to the high wave event frequency in this sea- son. The number of events on the coast increased both for the whole period and in winter (JJA). The increase in the number of coastal hazards was mainly led by wave events since events caused only by tidal inﬂuence did not present any signiﬁcant trend. 3.3 Coastal risk analysis The Charlies subarea (C) is one of the most affected loca- tions, experiencing an increased number of extreme wave events, peak periods, and wave power in the last years. How- ever, linking the changes in the regional wave climate with coastal hazards is not a straightforward task as the wave sys- tems are modiﬁed by bathymetry, and their impact depends on the coastal morphodynamics. Table 2 presents the number of events recorded by the BDe-BS and the computed trends for each type of hazard. The São Paulo coast was affected by 163 hazards between 1993 and 2021, of which 48 % (78) were caused exclusively by storm waves and 30 % (49) by the combination of waves and tides (as a result of either as- tronomical or meteorological tides). In the winter, 93 % (51) of events are associated with waves, with combined events showing a similar proportion (35 %) of total events com- By the time series, it is possible to note a high interannual variability due to large-scale climate modes that affect the re- gional wave climate through storm track shifts (e.g. Sasaki et al., 2021). The SWSA is affected by many large-scale vari- ability modes that interact, which are widely studied in the at- mosphere but not well understood in the wave ﬁelds (Godoi et al., 2020; Godoi and Torres Júnior, 2020; Sasaki et al., State Planet, 1-osr7, 12, 2023 https://doi.org/10.5194/sp-1-osr7-12-2023 CHAPTER3.3 https://doi.org/10.5194/sp-1-osr7-12-2023 C. B. Gramcianinov et al.: Extreme waves in the south-western South Atlantic 8 Figure 4. Annual time series and trends for the (a, b) number of extreme wave events (number per month), (c, d) mean peak period of the events (s), and (e, f) mean wave power (10−1 kW m−1) in the (a, c, e) whole period and (b, d, f) winter (JJA) computed for each Brazilian Navy warning subarea along the study domain’s coast (Fig. 1) based on the CMEMS wave products (product ref. nos. 1 and 2; Table 1). The trends are presented in the legend, and the units are number per year, 10−1 s yr−1, and 10−1 kW m−1 yr−1, respectively. The abbreviations “n.s.” and “sig.” in the legend mean “not signiﬁcant” and “signiﬁcant”, respectively, according to the Mann–Kendall test (p value ≤0.05). Figure 4. Annual time series and trends for the (a, b) number of extreme wave events (number per month), (c, d) mean peak period of the events (s), and (e, f) mean wave power (10−1 kW m−1) in the (a, c, e) whole period and (b, d, f) winter (JJA) computed for each Brazilian Navy warning subarea along the study domain’s coast (Fig. 1) based on the CMEMS wave products (product ref. nos. 1 and 2; Table 1). The trends are presented in the legend, and the units are number per year, 10−1 s yr−1, and 10−1 kW m−1 yr−1, respectively. The abbreviations “n.s.” and “sig.” in the legend mean “not signiﬁcant” and “signiﬁcant”, respectively, according to the Mann–Kendall test (p value ≤0.05). Moreover, coastal hazards can also occur after a sequence of events that result in a more vulnerable coast due to the lack of recovery time (Souza et al., 2019). For instance, these additional elements to coastal erosion can explain why the total increase in wave-forced events recorded on the coast in 29 years (145 %) is much larger than the increase in subarea C (∼20 %). In this case, the human-made modiﬁcation of the shoreline may intensify the damaging effects of the increase in extreme wave events (Muehe, 2018). et al., 2018; Vettor and Guedes Soares, 2020). Our ﬁnd- ings show important changes in the SWSA mainly associ- ated with an increase in the mean Hs values and wave period. These changes directly impact the offshore and coastal zone, increasing the wave power reaching the region and, conse- quently, aggravating the coastal hazards along the coast. C. B. Gramcianinov et al.: Extreme waves in the south-western South Atlantic Even though extreme waves have a major role in coastal ﬂooding and coastal erosion (e.g. Parise et al., 2009; Machado and Calliari, 2016), there still exists a lack of knowledge about how observed large- to regional-scale trends will affect the coast. Most of the Brazilian Navy’s monitoring and warning subareas within MetArea V (WMO) require attention regarding wave climate changes. According to the WAVERYS hindcast analysis, the number of extreme wave events (above the 95th percentile Hspeaks) increased in subareas A, C, and D, and the mean wave power increased in subareas B, C, and D. The trends vary depending on whether the whole period or only wintertime is considered. In this work, we analyse the winter (JJA) since it shows the most extreme wave patterns, but an extension of the analysis to other seasons is recommended for the future as the autumn C. B. Gramcianinov et al.: Extreme waves in the south-western South Atlantic The results show an increase of 120 % and 145 % of total events and wave-forced events on the coast in 29 years, considering the whole-period mean. This high increase is in agreement with Souza et al. (2019), who found a pronounced increase in wave-forced hazards after the decades 2000–2009 and 2010–2019 (226 % compared with the 1928–1999 period) when analysing a longer period of the same database (1928–2016). Although there is no total agreement between the extreme events detected in subarea C and hazards reported on the coast in the year-by-year analysis, the trend behaviour is sim- ilar. The number of combined events (wave + tides) and the total event trend are superposed in Fig. 5b. These overlaid pictures show that differences in the wave-forced coastal haz- ards and extreme wave events in subarea C may result from the inﬂuence of sea-level-elevation events forced by storms or astronomical tides. For instance, 2002 and 2009 do not present a peak in the extreme wave events within subarea C, but they were marked by a high number of wave-forced coastal hazards related to a higher percentage of combined events. These wave events would not become a hazard if the local sea level elevation did not allow waves to reach fur- ther into the continental area. In addition, the disagreement between the coastal and offshore event time series can be ad- dressed for bathymetry and morphology reasons. Figure 5 shows the time series of the yearly events of coastal hazards from the BDe-BS against the spatial sum of the number of events in subarea C obtained by the wave event analysis (described in Sect. 2.4). Due to the small number of coastal hazards per year, we show the time series and trends for the whole period to obtain more robust statistics. Never- theless, the winter trends presented the same signal and re- lation among the event types as the whole period trends (see Table 2). Note that the absolute numbers of events recorded on the coast and obtained in subarea C are not comparable. This analysis aims to evaluate the similarity in the variability and trends of the time series. The number of events presented for subarea C in Fig. 5 is the sum of events in all grid points within deﬁned boundaries. https://doi.org/10.5194/sp-1-osr7-12-2023 State Planet, 1-osr7, 12, 2023 State Planet, 1-osr7, 12, 2023 CHAPTER3.3 CHAPTER3.3 CHAPTER3.3 C. B. State Planet, 1-osr7, 12, 2023 C. B. Gramcianinov et al.: Extreme waves in the south-western South Atlantic Gramcianinov et al.: Extreme waves in the south-western South Atlantic C. B. Gramcianinov et al.: Extreme waves in the south-western South Atlantic Sea level rise, both in climatic and synoptic scales, and astronomical tides play a large role, potentially turning moderate waves into damaging ones as a high sea level allows waves to propagate and break further into the continent. Souza et al. (2019) highlighted that the most se- vere coastal hazards reported in the region do not present the highest values of Hs or sea level elevation but a combination of factors. Many other elements, such as coastal vulnerabil- ity, precipitation, morphology, and coastline orientation, af- fect the establishment of a coastal hazard (e.g. Muehe, 2018; Souza et al., 2019), mainly when the hazard is deﬁned by its impact on the coast and not by some pure meteorological and/or oceanographic parameter. Figure 5. (a) Number of coastal hazards per year caused by any forcing (blue) and waves (red) based on BDe-BS (product ref. no. 3; Table 1). (b) Numbers and trends of coastal hazards per year asso- ciated with waves (grey bars and black line) and combined events (waves + tides; yellow bars and lines) based on BDe-BS and total number and trend of wave events in subarea C (red lines) based on the CMEMS wave products (product ref. nos. 1 and 2; Table 1). The trends presented in these panels are statistically signiﬁcant (Mann– Kendall test, p value ≤0.05), and the values are shown in Table 2. Figure 5. (a) Number of coastal hazards per year caused by any forcing (blue) and waves (red) based on BDe-BS (product ref. no. 3; Table 1). (b) Numbers and trends of coastal hazards per year asso- ciated with waves (grey bars and black line) and combined events (waves + tides; yellow bars and lines) based on BDe-BS and total number and trend of wave events in subarea C (red lines) based on the CMEMS wave products (product ref. nos. 1 and 2; Table 1). The trends presented in these panels are statistically signiﬁcant (Mann– Kendall test, p value ≤0.05), and the values are shown in Table 2. Therefore, a complete assessment of coastal impacts needs more speciﬁc analysis considering local information and data, which is impracticable in this work, considering the study domain size. However, the trends derived herein are a valuable factor in identifying areas potentially vulnerable to climate change hazards and are also useful for engineers and stakeholders working towards the sustainable development of maritime activities. C. B. Gramcianinov et al.: Extreme waves in the south-western South Atlantic 9 Table 2. The number of coastal hazards reported by the BDe-BS (product ref. no. 3; Table 1) in the whole period (1993–2021) and in the winter (JJA). The percentage calculation is based on the total number of events, which represents the sum of events forced only by waves, only by anomalous tides (by either astronomical or meteorological components), or by a combination of both (“waves + tides”). The last row highlights all events associated with waves, with or without tidal inﬂuence (“waves”). The trend unit is events per year or season, and bold values denote signiﬁcance (Mann–Kendall test, p value ≤0.05). Table 2. The number of coastal hazards reported by the BDe-BS (product ref. no. 3; Table 1) in the whole period (1993–2021) and in the winter (JJA). The percentage calculation is based on the total number of events, which represents the sum of events forced only by waves, only by anomalous tides (by either astronomical or meteorological components), or by a combination of both (“waves + tides”). The last row highlights all events associated with waves, with or without tidal inﬂuence (“waves”). The trend unit is events per year or season, and bold values denote signiﬁcance (Mann–Kendall test, p value ≤0.05). Whole period Winter (JJA) Number of events Trend (number per year) Number of events Trend (number per season) Total 163 (100 %) 0.23 55 (100 %) 0.14 Wave + tides 49 (30 %) 0.10 19 (35 %) 0.06 Only waves 78 (48 %) 0.12 32 (58 %) 0.06 Only tides 36 (22 %) 0.01 4 (7 %) 0.01 Waves 127 (78 %) 0.22 51 (93 %) 0.12 this location is mainly associated with wave forcing and can be related to the increase in the number of extreme wave events in subarea C. Despite the well-known limitation in wave modelling, particularly to extreme waves (e.g. Cam- pos et al., 2018), this ﬁnding provides evidence that the WA- VERYS hindcast may be useful to assess not only extreme wave climate in the study domain (as shown by Crespo et al., 2022) but also the events reaching the coast in the long to medium term. However, more care is needed for interannual and interseasonal analyses that require year-by-year assess- ment, especially because a coastal hazard depends not only on the waves. 4 Conclusion The present work aims to assess changes in the extreme wave climate in the SWSA, giving new insights into offshore coastal risk assessment and management in this domain. Un- derstanding the extreme wave changes is crucial for support- ing future projections, which are indispensable for the de- sign and safety control of ship vessels, offshore and coastal structures and maintenance (e.g. oil/gas platforms, aquacul- ture, wind and wave farms), and coastal infrastructure (e.g. ports, roads, and touristic facilities) (e.g. Bitner-Gregersen https://doi.org/10.5194/sp-1-osr7-12-2023 CHAPTER3.3 State Planet, 1-osr7, 12, 2023 References ANTAQ: Anuário da Agência Nacional de Transporttes Aqua- viários (ANTAQ), Annual Report of the National Water Trans- portation Agency, Brazil, https://anuario.antaq.gov.br, last ac- cess: 12 May 2022 (in Portuguese). y g Aouf, L.: EU Copernicus Marine Service Quality Information Document for Global Ocean Waves Analysis and Forecasting Product, GLOBAL_ANALYSISFORECAST_WAV_001_027, Issue 3, Mercator Ocean International, https: //catalogue.marine.copernicus.eu/documents/QUID/ CMEMS-GLO-QUID-001-027.pdf (last access: 5 April 2023), 2022. Author contributions. CBG: conceptualisation, formal analysis, methodology, visualisation, writing – original draft. JS: conceptu- alisation, methodology, writing – review and editing, supervision. CRGS and PLS: methodology, writing – review. RC and PLdSD: writing – editing, supervision. Ardhuin, F., Rogers, E., Babanin, A. V., Filipot, J.-F., Magne, R., Roland, A., van der Westhuysen, A., Queffeulou, P., Lefevre, J.-M., Aouf, L., and Collard, F.: Semiempirical Dissipation Source Functions for Ocean Waves. Part I: Deﬁnition, Cal- ibration, and Validation, J. Phys. Oceanogr., 40, 1917–1941, https://doi.org/10.1175/2010jpo4324.1, 2010. Competing interests. The contact author has declared that none of the authors has any competing interests. Disclaimer. Publisher’s note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Belmonte Rivas, M. and Stoffelen, A.: Characterizing ERA-Interim and ERA5 surface wind biases using ASCAT, Ocean Sci., 15, 831–852, https://doi.org/10.5194/os-15-831-2019, 2019. Bitner-Gregersen, E. M., Vanem, E., Gramstad, O., Hørte, T., Aarnes, O. J., Reistad, M., Breivik, Ø., Magnus- son, A. K., and Natvig, B.: Climate change and safe design of ship structures, Ocean Eng., 149, 226–237, https://doi.org/10.1016/j.oceaneng.2017.12.023, 2018. Acknowledgements. The authors would like to acknowledge Marcel Ricker for his support with the wave event method im- plementation. The study was supported by the European Green Deal project “Large scale RESToration of COASTal ecosystems through rivers to sea connectivity” (REST-COAST) (grant no. 101037097). We gratefully acknowledge the project DOORS (grant no. 101000518) and DAM Mission project CostalFuture. Carolina B. Gramcianinov is funded by the Helmholtz European Partner- ing “Research Capacity Building for healthy, productive and re- silient Seas” (SEAReCap) (grant no. PIE-0025). This study also used data and resources from the projects “Resposta Morfodinâmica de Praias do Sudeste Brasileiro aos Efeitos da Elevação do Nível do Mar e Eventos Meteorológico-Oceanográﬁcos Extremos até 2100” (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brazil (CAPES) – ﬁnance code 88887.139056/2017-00), “Sistema de Aviso de Ressacas e Inundações Costeiras para o Litoral de São Paulo, com foco em Impactos das Mudanças Climáticas” (São Paulo Research Foundation, FAPESP; grant no. C. B. Gramcianinov et al.: Extreme waves in the south-western South Atlantic These changes may require adaptation measures, such as enhancing coastal protection (e.g. build- ing dikes and establishing harbour protection measures). The ﬁndings reported in this work may also support the design of new projects and future assessments that will allow the advancement of the association of large-scale wave climate with coastal impacts. (MAM) and spring (SON) weather patterns are also able to produce severe waves (e.g. Crespo et al., 2022). By our ﬁnd- ings, we recommend special attention to subareas C and D as they present changes in both the number of events and wave power. Regarding the coastal assessment, we found an increase in the number of coastal hazards in the State of São Paulo. According to our analysis, the increase in coastal hazards in State Planet, 1-osr7, 12, 2023 https://doi.org/10.5194/sp-1-osr7-12-2023 CHAPTER3.3 CHAPTER3.3 CHAPTER3.3 C. B. Gramcianinov et al.: Extreme waves in the south-western South Atlantic 10 Review statement. This paper was edited by Marta Marcos and reviewed by Alejandro Orﬁla and one anonymous referee. Code and data availability. The data products used in this arti- cle, as well as their names and documentation, are summarised in Table 1. The wave products are available through the Copernicus Marine Service (https://doi.org/10.48670/moi-00022, EU Coperni- cus Marine Service Product, 2021; https://doi.org/10.48670/moi- 00017, EU Copernicus Marine Service Product, 2022). The Baix- ada Santista Coastal Hazards database (BDe-BS) is available upon request by email to Celia R. G. Souza (celia@sp.gov.br). Codes are available upon request by email to the corresponding author. Code and data availability. The data products used in this arti- cle, as well as their names and documentation, are summarised in Table 1. The wave products are available through the Copernicus Marine Service (https://doi.org/10.48670/moi-00022, EU Coperni- cus Marine Service Product, 2021; https://doi.org/10.48670/moi- 00017, EU Copernicus Marine Service Product, 2022). 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B., Castro, B., and Dottori, M.: Intraseasonal variability of ocean surface wind waves in the western South Atlantic: the role of cyclones and the Paciﬁc South American pattern, Weather Clim. Dynam., 2, 1149–1166, https://doi.org/10.5194/wcd-2-1149-2021, 2021. https://doi.org/10.5194/sp-1-osr7-12-2023 State Planet, 1-osr7, 12, 2023 CHAPTER3.3 Unusual coccolithophore blooms in Scottish waters n of the Copernicus Ocean State Report (OSR7) Richard Renshaw1, Eileen Bresnan2, Susan Kay1,3, Robert McEwan4, Peter I. Miller3, and Paul Tett5 1Hadley Centre, Met Ofﬁce, FitzRoy Road, Exeter EX1 3PB, UK 2Marine Scotland Marine Laboratory, 375 Victoria Rd, Aberdeen AB11 9DB, UK 3Plymouth Marine Laboratory, Prospect Place, Plymouth PL1 3DH, UK 4Cefas, Barrack Rd, Weymouth DT4 8UB, UK 5Scottish Association for Marine Science, Oban, Argyll PA37 1QA, UK Richard Renshaw1, Eileen Bresnan2, Susan Kay1,3, Robert McEwan4, Peter I. Miller3, and Paul Tett5 1Hadley Centre, Met Ofﬁce, FitzRoy Road, Exeter EX1 3PB, UK 2Marine Scotland Marine Laboratory, 375 Victoria Rd, Aberdeen AB11 9DB, UK 3Plymouth Marine Laboratory, Prospect Place, Plymouth PL1 3DH, UK 4Cefas, Barrack Rd, Weymouth DT4 8UB, UK 5Scottish Association for Marine Science, Oban, Argyll PA37 1QA, UK Correspondence: Richard Renshaw (richard.renshaw@metofﬁce.gov.uk) us Ocean State Report (OSR7) Received: 22 August 2022 – Discussion started: 30 September 2022 Revised: 19 April 2023 – Accepted: 2 May 2023 – Published: 27 September 2023 Abstract. Two unusual blooms were observed in Scottish waters during summer 2021: one in the Clyde Sea and the other by the east coast of the Shetland Islands. Both had the appearance of coccolithophore blooms. Transmission electron microscopy of a sample from the Clyde Sea conﬁrmed the presence there of the coccol- ithophore Emiliania huxleyi. We examine the conditions that led to these unusual blooms. In situ data are scarce, and so we draw inference from satellite data and reanalysis. For Shetland, the bloom can be seen to originate further north on the edge of the continental shelf. It is advected south and then west towards the Shetland coast by surface currents. For the Clyde Sea region, April 2021 was the coldest April of the last 30 years (National Climate Information Centre). We hypothesise that this cold weather restricted the usual spring bloom of diatoms. A restricted spring bloom would mean higher-than-usual concentrations of nutrients in the summer. It might also mean reduced numbers of grazers. These factors would provide ideal conditions for coccolithophores to ﬂourish as temperatures and sunlight increase. CHAPTER3.3 Chapter 4.1 – 7th edition of the Copernicus Ocean State Report (OSR7) Chapter 4.1 – 7th edition of the Copernicus Ocean State Planet, 1-osr7, 13, 2023 https://doi.org/10.5194/sp-1-osr7-13-2023 © Author(s) 2023. This work is distributed under the Creative Commons Attribution 4.0 License. ter 4.1 – 7th edition of the Copernicus Ocean State Report (O Published by Copernicus Publications. 1 Introduction turn the sea a milky turquoise colour, visible to the human eye and in satellite imagery. The function of the coccoliths is unclear. They are believed to be protective, either against grazing, against viral or bacte- rial attack, or as a refractor of light that acts as a sunshade in excessively bright conditions (Monteiro et al., 2016). Johns et al. (2023) show that coccoliths initially provide some pro- tection from viral attack but once shed can mediate such at- tacks. Müller (2019) suggests that coccolith production may have evolved originally as an efﬁcient mechanism for intra- cellular Ca2+ detoxiﬁcation at a time of elevated seawater Ca2+ concentrations (e.g. during the Cretaceous and Jurassic periods). Coccolithophores belong to a diverse group of phytoplank- ters (class Prymnesiophyceae) that is widespread in the oceans. The ecological group that includes Emiliana is par- ticularly abundant in upwelling and temperate sub-polar re- gions (Balch et al., 2019). Most coccolithophores are not themselves harmful or toxic, but they are of ecological importance, particularly for carbon cycling and sequestra- tion (Rost and Riebesell, 2004). They typically produce an exoskeleton consisting of several calcium carbonate plates called coccoliths (Young et al., 1999). These coccoliths are not opaque (phytoplankton require light for photosynthesis), but they scatter and polarise light. Coccolith shedding oc- curs during the later stages of the bloom life cycle, when the cells are threatened, for example by pathogen pressure (Frada et al., 2008). During this stage, the coccoliths are shed and accumulate in the surrounding water. The visual effect is to Summer 2021 saw milky, turquoise-coloured waters caused by algal blooms in two locations off Scotland, in the Clyde Sea on the west coast and also to the east of the Shet- land Islands (Fig. 1). These blooms were visually striking and so unusual that they were reported in the news (e.g. Brad- shaw, 2021). Transmission-electron-microscope analysis of Published by Copernicus Publications. CHAPTER4.1 CHAPTER4.1 R. Renshaw et al.: Coccolithophore blooms R. Renshaw et al.: Coccolithophore blooms 2 Table 1. CMEMS and non-CMEMS products used in this study. Table 1. CMEMS and non-CMEMS products used in this study. Product Data ref. no. Product ID and type access Documentation 1 NWSHELF_MULTIYEAR_PHY _004_009 (1993–2022), numerical models EU Copernicus Service Product (2021a) Quality Information Document (QUID): Renshaw et al. (2021); Product User Manual (PUM): Tonani et al. 2 Data The North West Shelf (NWS) reanalysis (product ref. no. 1, Table 1) is based on the physical ocean model NEMO (Madec and Team, 2008) at 7 km horizontal resolution and 51 vertical levels, with tides represented, over a domain that encompasses the North West Shelf (Renshaw et al., 2021). Atmospheric forcing is from the ERA5 atmospheric reanal- ysis (Hersbach et al., 2023). River discharge volumes for the year 2018 onwards come from a daily climatology. The re- analysis uses NEMOVAR (Mogensen et al., 2012) to assim- ilate observations of physical variables (satellite sea surface temperature, SST, as well as in situ temperature and salinity proﬁles; Waters et al., 2015). 1 Introduction (2022) 2 OCEANCOLOUR_ATL_BGC_L3 _MY_OBSERVATIONS_009_013 (1998–2021), satellite observations EU Copernicus Service Product (2022) QUID: Garnesson et al. (2022); PUM: Collela et al. (2022) 3 SST_ATL_SST_L4_REP _OBSERVATIONS_010_026 (1998–2021), satellite observations EU Copernicus Service Product (2021b) QUID: Autret et al. (2021a); PUM: Autret et al. (2021b) 4 ERA5 atmospheric reanalysis (1979–2022) Copernicus Climate Change Ser- vice, Climate Data Store (2023) (2023) Hersbach et al. (2023) a water sample from the Clyde Sea (Fig. 1d) conﬁrmed the algae to be a morphotype of coccolithophore, Emiliania hux- leyi (morphotype B). Blooms of this organism are common in spring and early summer in the North Atlantic and occur in some years in the northern North Sea and the western En- glish Channel. However, such striking occurrences have not been reported from the Clyde Sea for many years. Colleagues of one of the authors (PT) recall no such event since 1983, when they remember sampling turquoise waters and coccol- ithophores in sea lochs of the Firth of Clyde. In this paper we use observations and reanalysis data to look for unusual envi- ronmental conditions in 2021 that might have allowed these blooms to thrive. optically active constituent in the Clyde Sea. Ocean colour algorithms are designed to minimise errors due to suspended sediment and DOM. The presence of large numbers of coc- coliths would also have a strong impact on backscattered ra- diation (Voss et al., 1998). The estimation process does mask for cloud, sun glint, and coccoliths (OC-CCI, 2020). This masking means there are few OC chlorophyll estimates avail- able for the Clyde Sea during the period of the Clyde bloom (12 June to 7 July 2021). We use the OC product in Fig. 5 not as a measure of absolute value but to show the timing of growth and also how values in 2021 compare to other years. State Planet, 1-osr7, 13, 2023 2.1 Ocean colour Ocean colour (OC) instruments measure water-leaving radi- ation at various wavelengths in the visible and near-infrared spectrum. Satellite measurements of ocean colour are used here in two forms. One is imagery. The enhanced colour maps from Sentinel-3 OLCI (Ocean and Land Colour Im- ager) and Sentinel-2 MSI (Multi-Spectral Imager) in Fig. 2 provide visual indications of algal blooms. The other is ob- servation products, point estimates of near-surface chloro- phyll concentration derived from multiple sensors. The CMEMS (Copernicus Marine Environment Monitoring Ser- vice) product used here (product ref. no. 2, Table 1) estimates chlorophyll concentration for several distinct phytoplankton functional types, including diatoms and nanophytoplankton. The nanophytoplankton category includes E. huxleyi. Here we use the reanalysis for sea surface temperature and also for near-surface currents in the region of the Euro- pean Slope Current. Reanalysis SST is strongly constrained by high-quality satellite observations. Variations in surface currents are driven predominantly by surface winds (Röhrs et al., 2023), provided here by ERA5. The slope current it- self is forced also by meridional density gradient and steep bathymetry (Marsh et al., 2017). Validation of the reanalysis Several factors make chlorophyll estimation difﬁcult for coastal waters. Bowers et al. (2000) ﬁnd that dissolved or- ganic matter (DOM) from fresh water is usually the largest State Planet, 1-osr7, 13, 2023 https://doi.org/10.5194/sp-1-osr7-13-2023 CHAPTER4.1 3 R. Renshaw et al.: Coccolithophore blooms Figure 1. (a) Sentinel-2 MSI image of Clyde Sea on 21 June 2021 at 11:35 UTC, true colour with enhanced contrast. Processed by the Natural Environment Research Council Earth Observation Data Acquisition and Analysis Service (NEODAAS) using ACOLITE atmospheric correction. (b) Sentinel-2 MSI image of Shetland Islands on 1 July 2021, processed by ESA (https://www.esa.int/ESA_Multimedia/Images, last access: 30 May 2023). (c) Bathymetry map with locations of (a) and (b) marked in red. Shelf edge is visible as transition from light blue (less than 200 m depth) to dark blue (deep water). (d) Scanning electron micrograph of sample from the Clyde Sea, June 2021, identiﬁed as E. huxleyi, morphotype B. Credit: Eileen Bresnan (Marine Scotland Science). Figure 1. (a) Sentinel-2 MSI image of Clyde Sea on 21 June 2021 at 11:35 UTC, true colour with enhanced contrast. Processed by the Natural Environment Research Council Earth Observation Data Acquisition and Analysis Service (NEODAAS) using ACOLITE atmospheric correction. (b) Sentinel-2 MSI image of Shetland Islands on 1 July 2021, processed by ESA (https://www.esa.int/ESA_Multimedia/Images, last access: 30 May 2023). 2.1 Ocean colour (c) Bathymetry map with locations of (a) and (b) marked in red. Shelf edge is visible as transition from light blue (less than 200 m depth) to dark blue (deep water). (d) Scanning electron micrograph of sample from the Clyde Sea, June 2021, identiﬁed as E. huxleyi, morphotype B. Credit: Eileen Bresnan (Marine Scotland Science). shows that it does produce a realistic slope current (Renshaw et al., 2021). Kondrik et al. (2019) show that often blooms develop fur- ther south and east in the North Sea in spring or early summer and are advected by an anti-clockwise circulation sometimes reaching as far north as Shetland. Sometimes a bloom orig- inates in the north, along the northern edge of the continen- tal shelf, and is advected southwards. Bathymetry in Fig. 1c shows the location of this shelf edge. The European Slope Current ﬂows eastward along this edge, bringing North At- lantic water into the Norwegian Sea. Some of this water ﬂows south past Shetland into the North Sea. Imagery for July 2021 (Fig. 2c, d) seems to show the bloom is of the latter kind, originating along the northern edge of the shelf. State Planet, 1-osr7, 13, 2023 3 Shetland bloom Figure 1 shows a bloom on the eastern side of Shetland. There is no information on the species present, but the brightness and turquoise colour of the bloom suggest coc- colithophores. Coccolithophore blooms in the North Sea are not unusual. An unusual feature of the 2021 bloom was that it came so close inshore. Examination of imagery for the years 2017 to 2020 from Plymouth Marine Laboratory (PML) (de- scribed in Highﬁeld et al., 2014) and of a dataset of coccol- ithophore blooms for 1998 to 2016 in Kondrik et al. (2019) ﬁnds no other examples where blooms intrude among the is- lands and bays on the eastern side of Shetland. To conﬁrm the bloom’s origins and to understand why 2021 was unusual, we used the OceanParcels software pack- age (Delandmeter and van Sebille, 2019) to simulate the tra- jectory of virtual particles in the ocean. Particles were ini- tially positioned at 1 m depth along the slope current (Fig. 3). https://doi.org/10.5194/sp-1-osr7-13-2023 State Planet, 1-osr7, 13, 2023 CHAPTER4.1 CHAPTER4.1 CHAPTER4.1 R. Renshaw et al.: Coccolithophore blooms Figure 2. Enhanced ocean colour satellite imagery (provided by PML) from the Sentinel-3 OCLI and Sentinel-2 MSI instruments for 12 and 19 June 2021 and 2 and 7 July 2021. The brightest pixels are indicative of high numbers of coccoliths. Land and cloud are coloured black. Figure 2. Enhanced ocean colour satellite imagery (provided by PML) from the Sentinel-3 OCLI and Sentinel-2 MSI instruments for 12 and 19 June 2021 and 2 and 7 July 2021. The brightest pixels are indicative of high numbers of coccoliths. Land and cloud are coloured black. These locations were chosen based on where the speed of the current (from a reanalysis mean climatology for 1993 to 2021) exceeded 0.2 ms−1. OceanParcels modelled 3D move- ment of the particles, advected by daily mean currents from the NWS reanalysis (product ref. no. 1, Table 1) and using a 3 h time step. This was done separately for each year from 1998–2022, starting with particles in initial positions on 3 April and running forward 3 months to predict positions on 3 July (Fig. 3). The years 2012, 2016, and 2019 also experienced easterly winds in spring or early summer (based on ERA5 reanaly- sis) and similarly show large numbers of red particles still within the plot region on 3 July. https://doi.org/10.5194/sp-1-osr7-13-2023 State Planet, 1-osr7, 13, 2023 3 Shetland bloom Top plot shows 3 April starting positions for particles positioned along the European Slope Current. The area around Sh ith a grey line. Other plots show particle positions in this area advected forward 3 months, using reanalysis surface cu from 1998–2022. Particles are coloured according to longitude of starting position. Figure 3. Top plot shows 3 April starting positions for particles positioned along the European Slope Current. The area around Shetland is marked with a grey line. Other plots show particle positions in this area advected forward 3 months, using reanalysis surface currents for each year from 1998–2022. Particles are coloured according to longitude of starting position. ter near the coast. We suggest that coincidences of timing and weather in 2021 created the unusual phenomenon of a visible coccolithophore bloom on the eastern Shetland coast. on the atmospheric conditions. Satellite instruments provide estimates of SST and of chlorophyll, with caveats discussed below. We have found no in situ measurements of conditions within the Clyde Sea itself. Biogeochemical reanalysis data are available (Kay et al., 2021), but their ability to accurately simulate the Clyde Sea is hampered by a lack of data on river discharge. Freshwater input and nutrient input from rivers are important variables for the biogeochemistry here. In this pa- per we analyse the data that are available and build from them 3 Shetland bloom Other years see large num- bers of other-coloured particles come close inshore, in par- ticular 2007, 2017, and 2020. In the satellite imagery (2017– 2020) and Kondrik catalogue (1998–2016), none of these years show coccolithophore blooms that reach into the bays and inlets of eastern Shetland. Imagery for 2020 (not shown) comes the closest, with a bloom 10–20 km away from the coast. The 2020 particle tracking has orange particles in this region, originating from further west on the shelf edge. In some years nearly all the particles move off beyond the edge of the plots (1999, 2002, 2003, 2009, 2022). In most years all the red particles (those initially at the eastern end of the slope current) disappear in this way. In 2021 it is these red particles that end up close to the eastern side of Shetland on 3 July. The year 2021 was unusual, although not unique, in that easterly winds during spring drove surface currents that pushed particles westward for part of that time. Figure 4 shows 10 m winds (from ERA5) and surface currents (from NWS reanalysis) for May 2021 and for a May climatology. To the east and north of Shetland, climatological winds are westerly, and the surface current is westerly or northerly. For May 2021, winds are north-easterly. These winds induce easterly surface currents (Ekman transport effect). We conclude that in some years blooms around Shetland form in water coming from the shelf edge. We cannot con- clude that bloom development is always linked to speciﬁc locations and timings of source water along the shelf edge. To understand how the bloom appeared so close inshore, we examined the daily particle trajectories for 2021 (not shown). Particles move south down the eastern side of Shet- land during the second half of June. In late June and early July there is a brief period of easterly winds, and the parti- cles are driven in towards the coast. Brief easterlies are not unusual, but these coincided with coccolithophore-laden wa- https://doi.org/10.5194/sp-1-osr7-13-2023 CHAPTER4.1 5 R. Renshaw et al.: Coccolithophore blooms Figure 3. Top plot shows 3 April starting positions for particles positioned along the European Slope Current. The area around Shetland is marked with a grey line. Other plots show particle positions in this area advected forward 3 months, using reanalysis surface currents for each year from 1998–2022. Particles are coloured according to longitude of starting position. 4.1 The Clyde Sea and the annual cycle of diatoms The Clyde Sea comprises a large tidal estuary with several islands and fjord-like sea lochs. It is the outlet of the River Clyde and other rivers into the Irish Sea. It has a maximum depth of 164 m, with a sill (the “Great Plateau”) of approxi- mately 40 m depth where it meets the Irish Sea. Freshwater outﬂow from rivers and from land drainage tends to main- tain stable stratiﬁcation in the basin (Simpson and Rippeth, 1993). This together with the sill restricts tidal mixing to mostly near-surface waters. Edwards et al. (1986) estimate a residence time of 2 months for surface water in the main body of the Clyde Sea. Nutrient content in the Clyde Sea tends to be higher than adjacent coastal waters (based on measure- ments of nitrate in Slesser and Turrell, 2013). Tidal currents within the Clyde Sea tend to be weak. Water in the deeper waters below the sill can stagnate, leading to nutrient build- up near the sea bed. Simpson and Rippeth (1993) show that strong winds can sometimes overcome the vertical stability and mix the water column. This would act to replenish nutri- ents in the surface layers in the event of an algal bloom. Hannah and Boney (1983) assessed extensive and more recent surveys (1976–1978) of the Inner Firth. They found rapid growth in diatoms from late March or early April in each year of the study, dominated by Skeletonema spp. and Thalassiosira nordenskioldii. They also found Nitzschia seriata (now called Pseudo-nitzschia “seriata type”) and Chaetoceros spp. at those times present in considerable numbers. During these spring blooms total chlorophyll was dominated by diatoms. For 1977 they found evidence that the Skeletonema were being grazed by microzooplankton (Ebria). Bresnan et al. (2016) also report an intense spring bloom dominated by Skeletonema during the monitoring pe- riod 2005–2013. a plausible storyline. That storyline starts with diatom growth in early spring. a plausible storyline. That storyline starts with diatom growth in early spring. A diatom bloom will consume nutrients (Stief et al., 2022), which will tend to inhibit further phytoplankton growth (Elser et al., 2007). Marshall and Orr (1927) also observed a second, smaller summer maximum but noted that diatoms near the surface were less healthy than those several metres deeper. Tests with samples left in direct sunlight and in shade showed that summer light levels were injurious for these di- atoms. 4 Clyde Sea bloom Analysis of the 2021 bloom in the Clyde Sea is hampered by a paucity of observations. Weather stations provide data State Planet, 1-osr7, 13, 2023 https://doi.org/10.5194/sp-1-osr7-13-2023 CHAPTER4.1 CHAPTER4.1 R. Renshaw et al.: Coccolithophore blooms 6 Figure 4. Top: ERA5 monthly mean 10 m winds (product ref. no. 4, Table 1) for (a) May 2021 and (b) May 1979–2020 climatology. Bottom: surface current for May from reanalysis (product ref. no. 1, Table 1) for (c) May 2021 and (d) May 1983–2020 climatology. Figure 4. Top: ERA5 monthly mean 10 m winds (product ref. no. 4, Table 1) for (a) May 2021 and (b) May 1979–2020 climatology. Bottom: surface current for May from reanalysis (product ref. no. 1, Table 1) for (c) May 2021 and (d) May 1983–2020 climatology. 4.2 Timing and source of 2021 Clyde coccolithophore bloom 4.2 Timing and source of 2021 Clyde coccolithophore bloom Marshall and Orr (1927) sampled the Clyde Sea and its lochs extensively, ﬁnding the following: Figure 1 shows the Clyde bloom on 21 June 2021. There are earlier visual reports of bright patches in the sea around the Isle of Arran in the centre of the Clyde Sea on 12 June (Evers- King et al., 2021). Satellite imagery (Fig. 2a) has no bright patch in the Clyde Sea that day, but the sea around Arran is There is a well-marked spring diatom maximum which starts at the end of March or the beginning of April. State Planet, 1-osr7, 13, 2023 https://doi.org/10.5194/sp-1-osr7-13-2023 CHAPTER4.1 R. Renshaw et al.: Coccolithophore blooms 7 Figure 5. Daily mean values averaged over the Clyde Sea basin of (a) reanalysis sea surface temperature for individual years. The year 2021 is in black. Chlorophyll concentrations (product ref. no. 2, Ta- ble 1) for the Clyde Sea from ocean colour products for (b) diatoms and (c) nanophytoplankton are also given. Black dots and lines are ocean colour estimates for 2021. Blue shows mean estimates for 1998 to 2020. Smaller purple dots are values for individual years, showing the year-to-year spread. partly obscured by cloud. Imagery for 13–16 June is almost wholly obscured by cloud, and the ﬁrst clear satellite image of a bright patch across the whole of the Clyde Sea is from 18 June (Fig. 2b). This patch persists until 5 July and then fades. Figure 2c shows that by this time the bloom is apparent even in the northernmost reaches of Loch Fyne. This sea loch ﬂows into the Clyde Sea but is tidal along its 65 km length. y g g Transmission electron microscopy of a sample from Mill- port collected at the end of June revealed the bloom to be comprised of liths and cells of E. huxleyi – morphotype B (Fig. 1d), providing the ﬁrst conﬁrmation of this species in high abundance at this site. E. huxleyi morphotypes A and B have been recorded in the waters around Scotland (van Bleijswijk et al., 1991; León et al., 2018). Little is known about the seasonality of E. huxleyi morphotypes on the west coast of Scotland. A study at the Marine Scotland Scottish Coastal Observatory (SCObs) monitoring site at Stonehaven on the east coast from 2010–2013 showed a distinct repeated seasonality in the occurrence of different E. 4.2 Timing and source of 2021 Clyde coccolithophore bloom huxleyi mor- photypes (León et al., 2018). Morphotype B was commonly recorded in spring, with morphotype A occurring from June to August followed by an overcalciﬁed form of morphotype A (type AO) in autumn and winter months. The dominance of E. huxleyi morphotype B in the 2021 Clyde bloom differs in timing from the seasonality recorded on the east coast. Coccolithophores have a haplodiplontic life cycle (Keuter et al., 2021). New cells are haploid (one set of chromosomes in the nucleus). These haploid cells develop into diploid cells (two sets of chromosomes in the nucleus). For the genus Emiliania, it is only the diploid form that produces coccol- iths. Frada et al. (2008) explain its “Cheshire Cat” strategy for resisting viral attack. Giant phycodnaviruses (Emiliania huxleyi viruses, EhVs) infect and lyse diploid-phase cells and are heavily implicated in the termination of blooms. The diploid cells transition to haploid cells that are resistant to EhVs, shedding coccoliths as they do. Thus the bloom in the Clyde Sea may have started some time before sufﬁcient coc- coliths had accumulated to make it visible. Figure 5. Daily mean values averaged over the Clyde Sea basin of (a) reanalysis sea surface temperature for individual years. The year 2021 is in black. Chlorophyll concentrations (product ref. no. 2, Ta- ble 1) for the Clyde Sea from ocean colour products for (b) diatoms and (c) nanophytoplankton are also given. Black dots and lines are ocean colour estimates for 2021. Blue shows mean estimates for 1998 to 2020. Smaller purple dots are values for individual years, showing the year-to-year spread. end of the range. Statistics from a high-resolution satellite SST product in Table 2 (product ref. no. 3, Table 1) conﬁrm this. Monthly means for April, May, and June 2021 are, re- spectively, at the 10th, 5th, and 52nd percentiles for those months over the period 1982–2021. It is possible that E. huxleyi was introduced by tidal mix- ing into the Clyde Sea from the Irish Sea or that it was already resident. Reverse particle tracking (not shown) ex- cludes immediate seeding from blooms at the Malin shelf break as a likely cause. The satellite imagery shows the bloom mostly conﬁned to the Clyde Sea. We conclude that conditions within the Clyde basin in late May or early June were particularly favourable for E. huxleyi to thrive. 4.2 Timing and source of 2021 Clyde coccolithophore bloom We aim here to understand exactly which aspects were favourable and what brought them about. Table 2a has monthly statistics of SST from a satellite SST product (product ref. no. 3, Table 1) for a point within the Clyde Sea (55.27◦N, 5.11◦W), close to the lower right intersection of grid lines in Fig. 1. April and May 2021 SSTs were unusually cold compared to climatology. June was close to the median. The cold SST can be linked to the weather (Table 2b, c, d). April saw anticyclonic weather that was cold, dry, and exceptionally sunny (Weather Maga- zine, 2021a). May 2021 had anomalously low atmospheric pressure over the UK, bringing storms, high rainfall, and high winds (Weather Magazine, 2021b). June was drier and warmer than average. https://doi.org/10.5194/sp-1-osr7-13-2023 4.3 Physical environment in 2021 Figure 5a shows daily values of sea surface temperature (SST) from the NWS reanalysis averaged over the Clyde Sea for the years 1998–2021. June 2021 values are in the middle of the range. Values for April and May are towards the cold State Planet, 1-osr7, 13, 2023 State Planet, 1-osr7, 13, 2023 https://doi.org/10.5194/sp-1-osr7-13-2023 CHAPTER4.1 CHAPTER4.1 CHAPTER4.1 R. Renshaw et al.: Coccolithophore blooms 8 Table 2. Statistics of monthly means from (a) CMEMS European area level 4 SST analysis at 55.27◦N, 5.11◦W, and (b, c, d) from weather stations in the Clyde catchment area (National Climate In- formation Centre, NCIC). ues drop rapidly during April, rising again towards the end of that month. Both types also show a fall immediately fol- lowing the two May storms, around 10 and 20 May. g y y As discussed in Sect. 2.1, chlorophyll estimation can be difﬁcult in coastal waters. Vertical mixing and river discharge due to the May storms might increase levels of dissolved organic matter and sediment in the water. Vertical mixing might dilute plankton in the surface layers that are sensed by the ocean colour instruments. The presence of coccoliths and cloud mean that much of the data for June have been masked in the estimation process. The ocean colour prod- uct includes an estimate of root mean square error (RMSE) following Brewin et al. (2017). For the Clyde Sea, values of RMSE for diatoms and nanophytoplankton in both April and May 2021 are given as approximately 0.5 mgm−3. This is similar in size to the estimates themselves for nanophyto- plankton, and so we avoid drawing conclusions from Fig. 5c. Estimated concentrations for diatoms are somewhat larger, and so we have more conﬁdence in drawing conclusions from the diatom time series (Fig. 5b). 4.5 Possible causes of Clyde bloom Mayers et al. (2019) assessed coccolithophore growth and mortality rates based on samples from the Celtic Sea in April 2015. They identiﬁed several conditions that favour coccolithophore blooms. These are considered individually below. (a) Warm, stratiﬁed waters. SST in June 2021 was close to average for that month (Table 2a). Vertical proﬁles of temperature from the reanalysis for 2021 (not shown) are stably stratiﬁed, but this is typical for June. The fol- lowing conclusion was reached: June 2021 temperatures were not unusual. (a) Warm, stratiﬁed waters. SST in June 2021 was close to average for that month (Table 2a). Vertical proﬁles of temperature from the reanalysis for 2021 (not shown) are stably stratiﬁed, but this is typical for June. The fol- lowing conclusion was reached: June 2021 temperatures were not unusual. Anomalies and percentiles are relative to yearly climatology: (a) SST is CMEMS reprocessed level 4 satellite product 1982–2020 (product ref. no. 3, Table 1). (b, c, d) Weather station data are for the Clyde catchment area from NCIC for 1980–2021. (b) Sunlight. June 2021 was sunnier than average (76th percentile, Table 2c). The following conclusion was reached: sunshine might have been a contributory fac- tor in 2021. There were two severe storms in May 2021, one on 9, 10, and 11 May and a stronger one on 20 and 21 May. These appear to coincide with periods when there is a pause in the rate of increase in SST (Fig. 5a). This suggests the strong winds are mixing the water column. (c) Availability of nutrients. We have no direct measure- ments of nutrients in the Clyde Sea. April 2021 was a dry month (5th percentile, Table 2d), and so river discharge in April would have been low. Rainfall in May was above average. May was also a stormy month, which may have mixed the water column, bringing nutrients from deep water into the photic zone (Pin- gree et al., 1977). Each of these factors could lead to higher-than-usual nutrient availability by the end of May. Ocean colour estimates of diatoms suggest lower- than-usual diatom growth in April and increased growth during May (Fig. 5b). The following conclusion was reached: observations show low chlorophyll mass in spring 2021. This could be due to the cold water tem- peratures and limited nutrient input in April 2021. The 4.3 Physical environment in 2021 (a) Sea surface temperature Monthly mean (◦C) Anomaly (◦C) Percentile April 7.53 −0.65 10 % May 8.81 −1.20 5 % June 12.24 0.05 52 % (b) Air temperature Monthly mean (◦C) Anomaly (◦C) Percentile April 5.5 −1.4 10 % May 8.4 −1.4 10 % June 13.4 1.0 83 % (c) Sunshine hours Percentage Total of climatology Percentile April 236 159 % 100 % May 151 80 % 19 % June 174 111 % 76 % (d) Rainfall Percentage Total (mm) of climatology Percentile April 16 20 % 5 % May 96 115 % 64 % June 42 47 % 12 % Anomalies and percentiles are relative to yearly climatology: (a) SST is CMEMS reprocessed level 4 satellite product 1982–2020 (product ref. no. 3, Table 1). (b, c, d) Weather station data are for the Clyde catchment area from NCIC for 1980–2021. State Planet, 1-osr7, 13, 2023 4.4 Ocean colour estimates of chlorophyll The following conclusion was reached: this could be a contributory factor, though we have no evidence for this. The ability of microzooplankton to multiply rapidly suggests at least that other factors were also involved. Our explanations are based on limited evidence (SST and chlorophyll estimates from satellites, modelling by the re- analysis). We do not have in situ measurements from within the blooms to conﬁrm our hypotheses. For both blooms, we propose the weather as a key fac- tor. Other studies also identify the importance of the weather for algal blooms. Whyte et al. (2014) looked at unusually strong blooms of the biotoxin-producing dinoﬂagellate Dino- physis on the western side of the Shetland Islands in the sum- mers of 2006 and 2013. They found these blooms coincided with periods where the winds, usually more southerly, be- came westerly. They suggested the westerly winds advected Dinophysis populations onshore, resulting in an increase in diarrhetic shellﬁsh toxin levels in farmed mussels (Mytilus edulis). Advection of a bloom into the region is another possible cause. We consider this unlikely for two reasons. The Clyde Sea is semi-enclosed, with an estimated residence time of 2 months for surface water in the main body of the Clyde Sea (Edwards et al., 1986). This was consistent with reverse tracking (not shown) of a set of virtual particles placed in the Clyde Sea in June and tracked backwards for 60 d to ﬁnd their source. The majority remained within the Clyde Sea. Also, satellite imagery shows a bloom in the Clyde Sea but not in the adjoining Irish Sea. There is evidence that the distribution of coccolithophores has expanded polewards in recent decades (Beaugrand et al., 2013; Winter et al., 2013; Rivero-Calle et al., 2015), due ei- ther to changes in ocean temperature or dissolved inorganic carbon. Growth of E. huxleyi is also known to be impacted by major changes in ocean pH (Riebesell et al., 2017). Changes in pH observed in seas around the UK (Findlay et al., 2022) are not large enough to explain recent variability in coccol- ithophore abundance in Scottish waters. Changing weather patterns have the potential to inﬂuence the occurrence of unusual phytoplankton blooms in coastal waters. These changes have the potential to impact higher trophic levels in the marine ecosystem. 4.4 Ocean colour estimates of chlorophyll Figure 5 shows a time series of estimates of chlorophyll a from the CMEMS ocean colour product (product ref. no. 2, Table 1) for two plankton functional types, diatoms and nanophytoplankton. Mean values for 1998–2020 (blue line) show similar patterns for diatoms and nanophytoplankton: concentrations rising to a peak in late March and a smaller second peak in early May, although with considerable year- to-year variation (purple dots). Estimates for 2021 are again similar between both functional types. Both have strong peaks in early April, well above the 1998–2020 mean. Val- https://doi.org/10.5194/sp-1-osr7-13-2023 CHAPTER4.1 9 R. Renshaw et al.: Coccolithophore blooms wet and stormy conditions in May of both years likely increased nutrient levels in near-surface layers in the Clyde Sea. Both these factors would help produce suit- able conditions for a bloom. pears to have developed in place. We hypothesise that envi- ronmental factors may have combined to create suitable con- ditions in the Clyde Sea. A cold and dry April could have re- stricted spring growth of diatoms, leaving nutrients available for a summer bloom of coccolithophores. A wet and stormy May might also have added to the nutrients. (d) Scarcity of predatory microzooplankton. Mayers et al. (2019) found that microzooplankton exert strong top-down control on coccolithophore populations, grazing up to 80 % of daily production in a bloom of E. huxleyi. We might hypothesise that fewer diatoms in April and May led to low numbers of microzooplankton during that time, reducing the grazing pressure on E. huxleyi in late May and early June. However, growth rates for microzooplankton can be rapid, sometimes more than three doublings per day for tintinnids (Verity, 1986). The following conclusion was reached: this could be a contributory factor, though we have no evidence for this. The ability of microzooplankton to multiply rapidly suggests at least that other factors were also involved. (d) Scarcity of predatory microzooplankton. Mayers et al. (2019) found that microzooplankton exert strong top-down control on coccolithophore populations, grazing up to 80 % of daily production in a bloom of E. huxleyi. We might hypothesise that fewer diatoms in April and May led to low numbers of microzooplankton during that time, reducing the grazing pressure on E. huxleyi in late May and early June. However, growth rates for microzooplankton can be rapid, sometimes more than three doublings per day for tintinnids (Verity, 1986). 4.4 Ocean colour estimates of chlorophyll A better understand- ing of the processes and dynamics involved will help in fore- warning, preparation, and development of adaptation mea- sures for these changes. https://doi.org/10.5194/sp-1-osr7-13-2023 5 Discussion plankton to climate change, Nature Climate Change, 3, 263–267, https://doi.org/10.1038/nclimate1753, 2013. Bo Bowers, D., Harker, G., Smith, P., and Tett, P.: Optical Prop- erties of a Region of Freshwater Inﬂuence (The Clyde Sea), Estuarine, Coastal and Shelf Science, 50, 717–726, https://doi.org/10.1006/ecss.1999.0600, 2000. p g Bradshaw, H.: Marine algae turned Scotland’s water bright blue, BBC News Online, https://www.bbc.co.uk/news/ uk-scotland-57720790 (last access: 30 May 2023), 2021. Competing interests. The contact author has declared that none of the authors has any competing interests. Bresnan, E., Cook, K., Hindson, J., Hughes, S., Lacaze, J. P., Walsham, P., Webster, L., and Turrell, W. R.: The Scottish coastal observatory 1997-2013 part 2: description of Scotland’s coastal waters, Scottish Marine and Freshwater Science, 7, 278, https://doi.org/10.7489/1881-1, 2016. Disclaimer. Publisher’s note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Brewin, R. J. W., Ciavatta, S., Sathyendranath, S., Jack- son, T., Tilstone, G., Curran, K., Airs, R. L., Cummings, D., Brotas, V., Organelli, E., Dall’Olmo, G., and Raitsos, D. E.: Uncertainty in Ocean-Color Estimates of Chloro- phyll for Phytoplankton Groups, Front. Mar. Sci., 4, 104, https://doi.org/10.3389/fmars.2017.00104, 2017. Acknowledgements. We gratefully acknowledge use of the OceanParcels code (Delandmeter and van Sebille, 2019) in cal- culating backward trajectories. We also acknowledge use of col- lated station statistics from the UK National Climate Informa- tion Centre and ERA5 reanalysis data (Hersbach et al., 2023) downloaded from the Copernicus Climate Change Service (C3S) Climate Data Store. We thank ESA for the satellite image in Fig. 1, made available under Creative Commons License BY-SA 3.0 IGO (https://creativecommons.org/licenses/by-sa/3.0/igo/, last access: 30 May 2023). Transmission electron analysis of the Mill- port water sample was performed at the Microscopy Unit, Institute of Medical Sciences, University of Aberdeen. Collela, S., Böhm, E., Cesarini, C., Garnesson, P., Netting, J., and Calton, B.: EU Copernicus Marine Service Product User Manual for North Atlantic Ocean Colour Plankton, Reﬂectance, Trans- parency and Optics MY L3 daily observations, Issue 3.0, Merca- tor Ocean International, https://catalogue.marine.copernicus.eu/ documents/PUM/CMEMS-OC-PUM.pdf (last access: 12 April 2023), 2022. Copernicus Climate Change Service, Climate Data Store: ERA5 hourly data on single levels from 1940 to present, Copernicus Climate Change Service (C3S) Climate Data Store (CDS) [data set], https://doi.org/10.24381/cds.adbb2d47, 2023. Review statement. This paper was edited by Griet Neukermans and reviewed by David Bowers and two anonymous referees. 5 Discussion Davidson, K., Tett, P., and Gowen, R.: Chapter 4 Harm- ful Algal Blooms, in: Marine Pollution and Human Health, pp. 95–127, The Royal Society of Chemistry, https://doi.org/10.1039/9781849732871-00095, 2011. 5 Discussion Phytoplankton are of special interest in the waters around Scotland, where aquaculture and ﬁshing are major industries. Through primary production, phytoplankton form the base of a food chain that sustains marine fauna (Frederiksen et al., 2006). Blooms of phytoplankton can be harmful to other ma- rine life and can produce toxins dangerous for human con- sumers of seafood (Davidson et al., 2011). There is thus in- creasing interest from policy-makers to understand the diver- sity dynamics of phytoplankton communities in Scotland and other parts of the North West Shelf (NWS) and to understand its inﬂuence on industries and diversity status assessments (Siemering et al., 2016; McQuatters-Gollop et al., 2019). This paper shows how use of satellite data and model re- analysis can help to meet the challenge of assessing major events in UK waters, despite a sparsity of in situ observa- tions. However we could be more conﬁdent in our ﬁndings if we had more information about environmental conditions. More complete data on river discharge would help in simulat- ing biogeochemical and ecosystem variables in the Clyde Sea and other inshore water bodies. Widespread routine monitor- ing of nutrient levels and phytoplankton components could help greatly in understanding future blooms. This paper presents hypotheses to explain two unusual blooms. We suggest that the bloom on the eastern side of Shetland originated in Atlantic water brought north of Shet- land by the European Slope Current. The water’s passage eastward was retarded by a period of anomalous easterly winds in May, and it was later steered towards the Shetland coast by a shorter period of easterly winds. The timing was such that there were abundant coccoliths present when this water was close inshore. The bloom within the Clyde Sea ap- Data availability. The data products used in this article, as well as their names, availability, and documentation, are summarised in Table 1. State Planet, 1-osr7, 13, 2023 State Planet, 1-osr7, 13, 2023 https://doi.org/10.5194/sp-1-osr7-13-2023 CHAPTER4.1 CHAPTER4.1 CHAPTER4.1 10 R. Renshaw et al.: Coccolithophore blooms plankton to climate change, Nature Climate Change, 3, 263–267, https://doi.org/10.1038/nclimate1753, 2013. Author contributions. RR processed the reanalysis data and wrote much of the text. 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S., Franco-Cisterna, B., García-Robledo, E., Holtap- pels, M., John, U., Maciute, A., Magee, M. J., Pors, R., Santl- Temkiv, T., Scherwass, A., Sevilgen, D. S., de Beer, D., Glud, R. N., Schramm, A., and Kamp, A.: Intracellular nitrate stor- age by diatoms can be an important nitrogen pool in freshwater and marine ecosystems, Commun. Earth Environ., 3, 154–164, https://doi.org/10.1038/s43247-022-00485-8, 2022. https://doi.org/10.5194/sp-1-osr7-13-2023 State Planet, 1-osr7, 13, 2023 Recent variations in oceanic transports across the Greenland–Scotland Ridge n of the Copernicus Ocean State Report (OSR7) Michael Mayer1,2,3, Takamasa Tsubouchi4, Susanna Winkelbauer1, Karin Margretha H. Larsen5, Barbara Berx6, Andreas Macrander7, Doroteaciro Iovino8, Steingrímur Jónsson7,9, and Richard Renshaw10 1Department of Meteorology and Geophysics, University of Vienna, 1090 Vienna, Austria 2Research Department, European Centre for Medium-Range Weather Forecasts (ECMWF), 53175 Bonn, Germany 3b.geos, 2100 Korneuburg, Austria 4Atmosphere and Ocean Department, Japan Meteorological Agency (JMA), Tokyo, 105-8431, Japan 5Faroe Marine Research Institute, Tórshavn, 100, Faroe Islands 6Marine Scotland, Aberdeen, AB11 9DB, United Kingdom 7Environmental division, Marine and Freshwater Research Institute, Hafnarfjörður, 220, Iceland 8Ocean Modeling and Data Assimilation Division, Centro Euro-Mediterraneo sui Cambiamenti Climatici (CMCC), Bologna, 40127, Italy 9Faculty of Natural Resource Sciences, University of Akureyri, Akureyri, 600, Iceland 10Met Ofﬁce, Exeter, EX1 3PB, United Kingdom Correspondence: Michael Mayer (michael.mayer@univie.ac.at) Received: 25 July 2022 – Discussion started: 7 October 2022 Revised: 20 January 2023 – Accepted: 20 February 2023 – Published: 27 September 2023 rnicus Ocean State Report (OSR7) ean State Report (OSR7) 9Faculty of Natural Resource Sciences, University of Akureyri, Akureyri, 600, Iceland 10 10Met Ofﬁce, Exeter, EX1 3PB, United Kingdom 10Met Ofﬁce, Exeter, EX1 3PB, United Kingdom Correspondence: Michael Mayer (michael.mayer@univie.ac.at) Received: 25 July 2022 – Discussion started: 7 October 2022 Revised: 20 January 2023 – Accepted: 20 February 2023 – Published: 27 September 2023 Abstract. Oceanic exchanges across the Greenland–Scotland Ridge (GSR) play a crucial role in shaping the Arctic climate and linking with the Atlantic meridional overturning circulation. Most considered ocean reanal- yses underestimate the observed 1993–2020 mean net inﬂow of warm and saline Atlantic Water of 8.0 ± 0.5 Sv by up to 15 %, with reanalyses at 0.25◦resolution additionally exhibiting larger biases in the single inﬂow branches compared to higher-resolution products. The underestimation of Atlantic Water inﬂow translates into a low bias in mean oceanic heat ﬂux at the GSR of 5 %–22 % in reanalyses compared to the observed value of 280 ± 18 TW. Interannual variations in reanalysis transports correlate reasonably well with observed trans- ports in most branches crossing the GSR. Observations and reanalyses with data assimilation show a marked reduction in oceanic heat ﬂux across the GSR of 4 %–9 % (compared to 1993–2020 means) during a biennial (2-year-long) period centered on 2018, a record low for several products. CHAPTER4.1 CHAPTER4.1 CHAPTER4.1 Chapter 4.2 – 7th edition of the Copernicus Ocean State Report (OSR7) Chapter 4.2 – 7th edition of the Co Chapter 4.2 – 7th edition of the Copernicus Oce State Planet, 1-osr7, 14, 2023 https://doi.org/10.5194/sp-1-osr7-14-2023 © Author(s) 2023. This work is distributed under the Creative Commons Attribution 4.0 License. ter 4.2 – 7th edition of the Copernicus Ocean State Report (O M. Mayer et al.: Recent variations in oceanic transports across the Greenland–Scotland Ridge M. Mayer et al.: Recent variations in oceanic transports across the Greenland–Scotland Ridge 2 Table 1. CMEMS and non-CMEMS products used in this study, including information on data documentation. Product Product ID and type Data access Documentation ref. no. 1 GLOBAL_REANALYSIS_PHY_001_031 (GREPv2), numerical models EU Copernicus Marine Service Product (2022a) Quality Information Document (QUID): Desportes et al. (2022) Product User Manual (PUM): Gounou et al. (2022) 2 GLOBAL_MULTIYEAR_PHY_001_030 (GLORYS12V1), numerical models EU Copernicus Marine Service Product (2022b) Quality Information Document (QUID): Drévillon et al. (2022a) Product User Manual (PUM): Drévillon et al. (2022b) 3 Mooring-derived ocean heat transport into Arctic Mediter- ranean from 1993 updated to July 2021 Data available at http://metadata.nmdc.no/metadata-api/ landingpage/0a2ae0e42ef7af767a920811e83784b1 (last access: 21 March 2023); updated time series are available from the authors upon reasonable request. Tsubouchi et al. (2020, 2021) Observational input data for product no. 3 Iceland–Faroe branch: January 1993 to December 2020 Data for I-F branch, F-S branch, NIIC, and Faroe Bank Channel are available at OceanSITES (http://www.oceansites.org/tma/gsr.html, last access: 22 March 2023). Data updates are available from the authors upon reasonable request. Hansen et al. (2015) Faroe–Shetland branch: January 1993 to June 2021 Berx et al. (2013) North Icelandic Irminger Current: October 1994 to July 2021 Jónsson and Valdimarsson (2012) Faroe Bank Channel: December 1995 to April 2021 Hansen et al. (2016) Denmark Strait: May 1996 to 2021 Jochumsen et al. (2017) Bering Strait: August 1997 to August 2019 Woodgate (2018) 4 SEALEVEL_GLO_PHY_L4_MY_008_047 (DUACS), satellite observations EU Copernicus Marine Service Product (2023) Quality Information Document (QUID): Pujol et al. (2023) Product User Manual (PUM): Pujol (2022) 5 Hindcast ocean simulations (no data assimilation) at 0.25◦ horizontal resolution and 50 vertical levels (GLOB4) and 0.0625◦horizontal resolution and 98 vertical levels (GLOB16) provided by CMCC Data available from the authors upon reasonable request. Iovino et al. (2023) 6 Ocean reanalysis at 0.25◦horizontal resolution and 75 verti- cal levels provided by UKMO (GloRanV14) Data available from the authors upon reasonable request. MacLachlan et al. (2015) PTER Table 1. CMEMS and non-CMEMS products used in this study, including information on data documentation. Product Product ID and type Data access Documentation ref. no. 1 GLOBAL_REANALYSIS_PHY_001_031 (GREPv2), numerical models EU Copernicus Marine Service Product (2022a) Quality Information Document (QUID): Desportes et al. (2022) Product User Manual (PUM): Gounou et al. (2022) 2 GLOBAL_MULTIYEAR_PHY_001_030 (GLORYS12V1), numerical models EU Copernicus Marine Service Product (2022b) Quality Information Document (QUID): Drévillon et al. Recent variations in oceanic transports across the Greenland–Scotland Ridge The anomaly was associated with a temporary reduction in geostrophic Atlantic Water inﬂow through the Faroe–Shetland branch and was aug- mented by anomalously cool temperatures of Atlantic Water arriving at the GSR. The latter is linked to a recent strengthening of the North Atlantic subpolar gyre and illustrates the interplay of interannual and decadal changes in modulating transports at the GSR. Published by Copernicus Publications. CHAPTER4.2 CHAPTER4.2 1 Introduction The Greenland–Scotland Ridge (GSR), encompassing the Denmark Strait, Iceland–Faroe (I-F) branch, Faroe–Shetland (F-S) branch, and European shelf, represents the main oceanic gateway to the so-called Arctic Mediterranean (the ocean bounded by the GSR, Davis Strait, and Bering Strait). Oceanic transports across the GSR play an important role in the Arctic and global climate systems. In the surface layer, the warm and saline Atlantic Water moves northward across the GSR and the light Polar Water ﬂows southward mainly through the Denmark Strait. In the lower layer, the cold and dense water is transported southward at depth into the North Atlantic, contributing to the lower limb of the Atlantic merid- ional overturning circulation (Hansen and Østerhus, 2000; Buckley and Marshall, 2016). y ) Transports across the GSR exhibit pronounced interan- nual variability and thereby play an important role in mod- ulating the heat budget of the Arctic Mediterranean (e.g., Muilwijk et al., 2018; Mayer et al., 2016; Asbjørnsen et al., 2019). Speciﬁcally, the inﬂow of warm and saline Atlantic Water (AW) exhibits a strong co-variability with ocean heat content, especially in the AW layer of the Arctic Mediter- ranean (M. Mayer et al., 2022). Tsubouchi et al. (2021), us- ing observation-based oceanic transport data (1993–2016), revealed a step change towards stronger oceanic heat trans- ports (OHTs) across the GSR around 2002, suggesting an enhanced contribution of OHT to the observed warming of the Arctic Ocean. M. Mayer et al. (2022) temporally ex- tended the monitoring of OHT at the GSR using ocean re- analyses and found a pronounced reduction in OHT around 2018, which could not be veriﬁed with observational data at that time and the causes of which were not explored in detail. Observational mass-consistent estimates of oceanic trans- ports (product ref 3) are updated to July 2021 following Tsubouchi et al. (2021; i.e., using the same strategy to in- ﬁll data gaps, estimate uncertainty, and create a box inverse model to close the volume budget). Temporal coverage and references for the single observational estimates used as in- put are provided in the data table (Table 1). Surface freshwa- ter inputs by river discharge and precipitation minus evapo- ration for 1993–2021 used as input to the box inverse model are based on Winkelbauer et al. (2022). 1 Introduction The used reanalyses assimilate temperature and salinity proﬁles available from databases such as Hadley EN4 (Good et al., 2013), which according to our investigations include only a small subset of the mooring data used for our observational transport es- timates. Currents are generally not assimilated in ocean re- analyses. Hence, the observation-based volume ﬂuxes repre- sent fully independent data, while temperature ﬂuxes repre- sent largely independent validation data. Here, we use observational oceanic transport data at the boundaries of the Arctic Mediterranean updated to 2021 and an extended set of ocean reanalyses to explore the pro- nounced reduction in OHT in more detail, track it to the main contributing oceanic branch, and relate these changes to larger-scale climate variations on interannual and decadal timescales. An additional aspect of this study is a more de- tailed validation of reanalysis-based oceanic transports at the GSR at the scale of single branches to further build trust in the usefulness of these products for monitoring Arctic cli- mate and its oceanic drivers. We note that quantiﬁcation methods of oceanic trans- ports in reanalyses and observations are fundamentally dif- ferent, which needs to be kept in mind when intercomparing. The reanalysis-based estimate is based on surface-to-bottom, coast-to-coast temperature and velocity sections across the Arctic Mediterranean. This ensures conservation of volume and avoids projection of potentially biased positioning of cur- rents in the reanalyses onto the transport estimates. The ob- servational estimate is based on the sum of 11 major ocean current transport estimates categorized into three major wa- ter masses – Atlantic Water (AW), Polar Water (PW), and Overﬂow Water (OW) (Tsubouchi et al., 2021). An assump- tion is that the 11 major ocean currents represent the ma- jor water mass exchanges well across the Arctic Mediter- ranean. This means it is important that no recirculation, e.g., of AW waters, remains unobserved, as this would introduce M. Mayer et al.: Recent variations in oceanic transports across the Greenland–Scotland Ridge M. Mayer et al.: Recent variations in oceanic transports across the Greenland–Scotland Ridge M. Mayer et al.: Recent variations in oceanic transports across the Greenland–Scotland Ridge 3 Copernicus Marine Environment Monitoring Service Global Reanalysis Ensemble Product (CMEMS GREPv2, prod- uct ref 1), consisting of ORAS5, CGLORS, GLORYS2V4, and GloRanV14 (an improvement of GloSea5, also known as the FOAM product, product ref 6; MacLachlan et al., 2015). These are all run at 0.25◦horizontal resolution with 75 vertical levels and use atmospheric forcing from ERA- Interim (Dee et al., 2011). The ensemble is complemented with GLORYS12 version 1 (product ref 2), a reanalysis at 0.083◦horizontal resolution with 50 vertical levels driven by ERA-Interim atmospheric forcing. Furthermore, two hind- cast ocean simulations (i.e., with no data assimilation) at 0.25◦horizonal resolution with 50 vertical levels (GLOB4) and 0.0625◦horizontal resolution and 98 vertical levels (GLOB16; Iovino et al., 2023) driven by JRA55-do (Tsujino et al., 2018) are employed to investigate the impact of resolu- tion on oceanic volume ﬂuxes. Heat transports from GLOB4 and GLOB16 are not assessed as these are biased due to their setup as forced runs without data assimilation. Transports are computed on the native grid through line integrals similar to Pietschnig et al. (2018). State Planet, 1-osr7, 14, 2023 M. Mayer et al.: Recent variations in oceanic transports across the Greenland–Scotland Ridge (2022a) Product User Manual (PUM): Drévillon et al. (2022b) 3 Mooring-derived ocean heat transport into Arctic Mediter- ranean from 1993 updated to July 2021 Data available at http://metadata.nmdc.no/metadata-api/ landingpage/0a2ae0e42ef7af767a920811e83784b1 (last access: 21 March 2023); updated time series are available from the authors upon reasonable request. Tsubouchi et al. (2020, 2021) Observational input data for product no. 3 Iceland–Faroe branch: January 1993 to December 2020 Data for I-F branch, F-S branch, NIIC, and Faroe Bank Channel are available at OceanSITES (http://www.oceansites.org/tma/gsr.html, last access: 22 March 2023). Data updates are available from the authors upon reasonable request. Hansen et al. (2015) Faroe–Shetland branch: January 1993 to June 2021 Berx et al. (2013) North Icelandic Irminger Current: October 1994 to July 2021 Jónsson and Valdimarsson (2012) Faroe Bank Channel: December 1995 to April 2021 Hansen et al. (2016) Denmark Strait: May 1996 to 2021 Jochumsen et al. (2017) Bering Strait: August 1997 to August 2019 Woodgate (2018) 4 SEALEVEL_GLO_PHY_L4_MY_008_047 (DUACS), satellite observations EU Copernicus Marine Service Product (2023) Quality Information Document (QUID): Pujol et al. (2023) Product User Manual (PUM): Pujol (2022) 5 Hindcast ocean simulations (no data assimilation) at 0.25◦ horizontal resolution and 50 vertical levels (GLOB4) and 0.0625◦horizontal resolution and 98 vertical levels (GLOB16) provided by CMCC Data available from the authors upon reasonable request. Iovino et al. (2023) 6 Ocean reanalysis at 0.25◦horizontal resolution and 75 verti- cal levels provided by UKMO (GloRanV14) Data available from the authors upon reasonable request. MacLachlan et al. (2015) lanet, 1-osr7, 14, 2023 https://doi.org/10.5194/sp-1-osr7-14-2 CHAPTER4.2 CHAPTER4.2 M. Mayer et al.: Recent variations in oceanic transports across the Greenland–Scotland Ridge 2 Data and methods We use monthly data from a comprehensive set of ocean reanalyses to compute oceanic transports across the GSR, Davis Strait, and Bering Strait. The latter two straits are cal- culated to close the volume budget and obtain unambiguous net heat transport into the Arctic Mediterranean (Schauer and Beszczynska-Möller, 2009). Inﬂow (positive) has been deﬁned as positive towards the Arctic Mediterranean. The employed products are an updated ensemble based on the https://doi.org/10.5194/sp-1-osr7-14-2023 State Planet, 1-osr7, 14, 2023 CHAPTER4.2 CHAPTER4.2 M. Mayer et al.: Recent variations in oceanic transports across the Greenland–Scotland Ridge M. Mayer et al.: Recent variations in oceanic transports across the Greenland–Scotland Ridge Figure 1. Map of the Greenland–Scotland Ridge region, schematically depicting shallow shelf water and deep water, major AW inﬂow (red arrows) and OW outﬂow (blue arrows) branches (DS stands for Denmark Strait, and FBC stands for Faroe Bank Channel), the location of oceanic moorings used for deriving the observation-based transport estimates (yellow bars), and the section used for the computation of transports from ocean reanalyses (dashed green line). Adapted from OceanSITES (http://www.oceansites.org/tma/gsr.html, last access: 20 March 2023, Fig. 1). Figure 1. Map of the Greenland–Scotland Ridge region, schematically depicting shallow shelf water and deep water, major AW inﬂow (red arrows) and OW outﬂow (blue arrows) branches (DS stands for Denmark Strait, and FBC stands for Faroe Bank Channel), the location of oceanic moorings used for deriving the observation-based transport estimates (yellow bars), and the section used for the computation of transports from ocean reanalyses (dashed green line). Adapted from OceanSITES (http://www.oceansites.org/tma/gsr.html, last access: 20 March 2023, Fig. 1). biases into the observational estimate. This assumption has been assessed and conﬁrmed many times over the last 2 decades from establishment of sustained hydrographic sec- tions in the GSR in the 1990s (e.g., Dickson et al., 2008) to recent oceanographic surveys to capture ocean circulation in the GSR for AW (e.g., Berx et al., 2013; Hansen et al., 2017; Rossby et al., 2018; Jónsson and Valdimarsson, 2012) and OW (Hansen et al., 2018). We also note that remaining uncertainties arising from potential undersampling are taken into account in the observational estimate obtained through the inverse model. For reference, Fig. 1 shows pathways of major ﬂows across the GSR and the locations of considered oceanic moorings and the sections used for evaluation of re- analyses. biases into the observational estimate. 2 Data and methods This assumption has been assessed and conﬁrmed many times over the last 2 decades from establishment of sustained hydrographic sec- tions in the GSR in the 1990s (e.g., Dickson et al., 2008) to recent oceanographic surveys to capture ocean circulation in the GSR for AW (e.g., Berx et al., 2013; Hansen et al., 2017; Rossby et al., 2018; Jónsson and Valdimarsson, 2012) and OW (Hansen et al., 2018). We also note that remaining uncertainties arising from potential undersampling are taken into account in the observational estimate obtained through the inverse model. For reference, Fig. 1 shows pathways of major ﬂows across the GSR and the locations of considered oceanic moorings and the sections used for evaluation of re- analyses. for observational products (see references for more details, e.g., Eldevik et al 2009). We additionally use sea level anomaly (SLA) data pro- vided through CMEMS (product ref 4) for investigating drivers of observed OHT anomalies. The global mean SLA trend is removed before computation of the presented diag- nostics. Deseasonalized anomalies are based on the 1993–2019 cli- matologies, i.e., the period for which all data are available. Anomaly time series have a 12-monthly temporal smoother applied to emphasize interannual variations. Conﬁdence lev- els (95 % is set as threshold for signiﬁcance testing) for tem- poral correlations take auto-correlation of the involved time series into account (see Oort and Yienger, 1996). As in M. Mayer et al. (2022), we assume total uncertainties in monthly mean observations (provided in Tsubouchi et al., 2021) to consist of roughly half systematic and half random errors; i.e., the two contributions are the total uncertainty re- duced by a factor of 1/ √ 2. Consequently, the contribution of random errors to uncertainties in long-term mean obser- vational estimates is further reduced by a factor of 1/ √ N, where N is the number of years, and deseasonalized anoma- lies only include the random errors. State Planet, 1-osr7, 14, 2023 3.1 General evaluation of transports of water masses across the GSR The main discrepancy is the under- estimation of I-F inﬂow and overestimation of F-S inﬂow by all GREP reanalyses, while the high-resolution products GLORYS12 and GLOB16 are in much better agreement with observations. Direct comparison of GLOB16 to GLOB4 con- ﬁrms that overestimation of F-S volume ﬂux is reduced when going from 0.25 to 0.0625◦resolution. This suggests that increased resolution, along with more realistic bathymetry, improves representation of inﬂow pathways in the reanaly- ses. We also note that temporal anomaly correlations with observed I-F volume ﬂuxes are low (Pearson correlation co- efﬁcients r range from −0.03 to 0.26 and are statistically in- signiﬁcant) for all reanalyses but are substantially higher for F-S volume ﬂuxes (r ranges from 0.30 to 0.71 and is statisti- cally signiﬁcant for all products with data assimilation). Figure 2e shows deseasonalized anomalies of AW vol- ume ﬂux, with a 12-monthly smoother applied to emphasize interannual variability. Typical variability is similar across observations (temporal standard deviation σ = 0.35 Sv) and reanalyses (σ ranges between 0.22 and 0.47 Sv). Temporal correlations between reanalyzed and observed AW inﬂow anomalies are moderately high (r ranges between 0.39 and 0.60 and is statistically signiﬁcant for all products; see the legend of the plot for values). Figure 2f shows anomalies of total oceanic heat ﬂux across the GSR, which show similar variability to AW volume ﬂux; i.e., the strength of AW inﬂow modulates not only the sea- sonal cycle of the total GSR heat ﬂux but also its interannual variations (r ranges from 0.86 to 0.91). GSR total heat ﬂux from reanalyses is in slightly better agreement with observa- tions than AW volume ﬂuxes (r ranges between 0.51 and 0.63 and is statistically signiﬁcant for all products; see the legend of the plot for values). Figure 2f also shows a prominent neg- ative heat ﬂux anomaly centered around the year 2018, which has already been noted by M. Mayer et al. (2022) for net heat transport into the Arctic Mediterranean. Figure 2d shows the mean annual cycle of heat ﬂux across the GSR, i.e., the sum of sensible heat transported by all wa- ters crossing the GSR. The mean annual cycle of GSR heat ﬂuxes generally follows that of AW volume ﬂuxes, with a minimum between boreal spring and early summer and a maximum in fall and early winter. 3.1 General evaluation of transports of water masses across the GSR The AW net ﬂow from high- resolution products does not stand out compared to the 0.25◦ products and is close to the GREP mean. Agreement is also good for the PW outﬂow, where all products show the ob- served seasonal maximum ﬂow in boreal winter. The range of reanalysis-based estimates is large in a relative sense, with the observations lying in the middle of the range. There is less coherence across products concerning the OW trans- ports. GLORYS12, GLORYS2V4, and CGLORS are close to observations, with rather persistent overﬂow on the order of −5.4 Sv and seasonal variations that agree with the obser- vations. Other products tend to have overﬂows that are too weak (most notably ORAS5), and some also exhibit biases in the representation of the annual cycle (e.g., GloRanV14). The net heat transport is clearly related to the strength of AW inﬂow, but even CGLORS, which has a higher AW mean volume ﬂux (8.3 Sv) than observations, has a negative net heat ﬂux bias. The reason is that all reanalyses exhibit a warm bias in outﬂowing OW (not shown) and a cold bias in Davis Strait inﬂow (see Pietschnig et al., 2018). Based on M. Mayer et al. (2022) and taking oceanic storage into account, the energy-budget-based estimate of the net heat boundary transport suggests even higher values (∼348 TW) than observations. This inferred value appears high, but we note that this indirect approach has been applied successfully to infer observation-based oceanic transports in the North At- lantic (Trenberth and Fasullo, 2017; Liu et al., 2020; J. Mayer et al., 2022) and the central Arctic (Mayer et al., 2019), and hence it is deemed credible. This estimate at least adds con- ﬁdence to the conclusion that the observational estimate of oceanic heat transport is not biased high. Table 3 also con- ﬁrms that long-term averages for heat ﬂux across the GSR are qualitatively very similar to the net heat transport; i.e., heat ﬂuxes across the GSR are the dominant contributor to oceanic heat transport into the Arctic Mediterranean. Table 2 additionally includes long-term average volume ﬂuxes in the main AW inﬂow branches (North Icelandic Irminger Current (NIIC), I-F branch, F-S branch, and Euro- pean shelf). The reanalysis-based estimates generally agree well with observations. 3.1 General evaluation of transports of water masses across the GSR Figure 2 presents mean annual cycles and anomaly time se- ries of relevant oceanic transport quantities at the GSR. It is complemented with long-term averages shown in Table 2 (volume ﬂuxes) and Table 3 (heat ﬂuxes). Observations show seasonally varying AW inﬂow across the GSR (8.0 ± 0.5 Sv; mean ± standard deviation of the mean reported through- out, unless explicitly stated) with a maximum in Decem- ber and minimum in June–July (Fig. 2a). The AW inﬂow is largely balanced by PW (Fig. 2c; −1.8± 0.7 Sv) and OW (Fig. 2b; −5.6 ± 0.3 Sv) outﬂow, yielding a relatively small net volume ﬂux across the GSR of 0.7 Sv (balanced by ﬂows through Bering and Davis straits). The PW outﬂow exhibits Transported water masses at the GSR are decomposed into AW, PW, and OW, largely following Eldevik et al. (2009). PW is deﬁned as T < 4 ◦C and σθ < 27.7 kg m−3. OW is deﬁned as σθ > 27.8 kg m−3. The rest (i.e., waters with σθ < 27.8 kg m−3 with PW taken out) is considered AW. Note that these deﬁnitions have been revised from M. Mayer et al. (2022). Water mass decomposition is performed each month based on the monthly temperature and salinity ﬁelds in the reanalyses. These deﬁnitions are similar to those used State Planet, 1-osr7, 14, 2023 https://doi.org/10.5194/sp-1-osr7-14-2023 CHAPTER4.2 M. Mayer et al.: Recent variations in oceanic transports across the Greenland–Scotland Ridge 5 an annual cycle balancing the AW inﬂow (i.e., maximum out- ﬂow in boreal winter), while the OW exchange is more stable throughout the year (i.e., small annual cycle). all reanalyses exhibit lower net heat transport (by ∼16 % for the GREP mean of 256 ± 19 TW) than that observed (306 ± 19 TW; 311 ± 20 TW when including sea ice). GLO- RYS12 exhibits a mean net heat ﬂux similar to the GREP mean, but we note that all GREP reanalyses have AW in- ﬂux that is too strong in the F-S branch (Table 2), where the climatologically warmest waters cross the GSR, and thus en- hance the heat ﬂux in those products for the wrong reason. All reanalyses but one (CGLORS) underestimate AW in- ﬂow across the GSR when compared to observations, but the shape of the annual cycle of all estimates is in good agreement with observations. https://doi.org/10.5194/sp-1-osr7-14-2023 M. Mayer et al.: Recent variations in oceanic transports across the Greenland–Scotland Ridge 6 n annual cycle of (a) AW volume ﬂux, (b) OW volume ﬂux, (c) PW volume ﬂux, and (d) GSR total (AW + PW + OW) heat anomalies of (e) AW volume ﬂux and (f) GSR total heat ﬂux. The red shading indicates ±1 standard error in the observational correlations of reanalyses with observations are provided in the legends. re 2 Mean annual cycle of (a) AW volume ﬂux (b) OW volume ﬂux (c) PW volume ﬂux and (d) GSR total (AW + PW + OW) heat Figure 2. Mean annual cycle of (a) AW volume ﬂux, (b) OW volume ﬂux, (c) PW volume ﬂux, and (d) GSR total (AW + PW + OW) heat ﬂux. Temporal anomalies of (e) AW volume ﬂux and (f) GSR total heat ﬂux. The red shading indicates ±1 standard error in the observational data. Temporal correlations of reanalyses with observations are provided in the legends. State Planet, 1-osr7, 14, 2023 3.1 General evaluation of transports of water masses across the GSR Seasonal minima and max- ima in GSR heat ﬂux co-occur with those of AW volume ﬂux; i.e., seasonal variations in heat ﬂux are largely volume ﬂux driven, and the seasonal cycle in volume-weighted tempera- tures is in phase. Since net volume ﬂux across the GSR is small, the ambi- guity arising from the choice of reference temperature can be considered small as well. However, for the long-term av- erages we focus on net heat transport into the Arctic Mediter- ranean, i.e., the sum of heat ﬂuxes across the GSR, plus those through Bering and Davis straits. Values in Table 3 show that State Planet, 1-osr7, 14, 2023 https://doi.org/10.5194/sp-1-osr7-14-2023 CHAPTER4.2 CHAPTER4.2 CHAPTER4.2 M. Mayer et al.: Recent variations in oceanic transports across the Greenland–Scotland Ridg 3.2 Spatial structure of the July 2017–June 2019 transport anomaly in the biennial (2-year-long) signal of average anomalies in July 2017–June 2019. Figure 3c shows that during this period strong warm anomalies were present over 0–400 m depth in the eastern Denmark Strait, and warm anomalies are also seen in the F-S branch. The latter suggests a temporary deep- ening of the AW layer. Velocity anomalies for the July 2017– June 2019 period (Fig. 3d) suggest that the positive temper- ature anomalies in the eastern Denmark Strait are driven by enhanced NIIC transports. The strongest and deepest veloc- ity anomaly in July 2017–June 2019 is located in the eastern part of the F-S branch, where reduced inﬂow is present from the surface down to the interface at ∼600 m, and hence we focus on this branch next. Negative anomalies are also seen in To set the scene for further investigation, we present climato- logical temperatures and currents at the GSR based on GLO- RYS12 in Fig. 3a and b, respectively. Comparison with anal- ogous ﬁgures based on the GREP (shown in M. Mayer et al., 2022) allows us to appreciate the beneﬁts of increased reso- lution (0.08 vs. 0.25◦resolution), including a more distinct representation of inﬂow and outﬂow branches and a more spatially variable bathymetry, especially in the I-F branch. Next, we investigate the recent reduction in AW volume and GSR heat ﬂuxes in more detail. This is most prominent State Planet, 1-osr7, 14, 2023 https://doi.org/10.5194/sp-1-osr7-14-2023 CHAPTER4.2 M. Mayer et al.: Recent variations in oceanic transports across the Greenland–Scotland Ridge M. Mayer et al.: Recent variations in oceanic transports across the Greenland–Scotland Ridge .: Recent variations in oceanic transports across the Greenland–Scotland Ridge tude–depth sections of (a) mean temperature (with water mass boundaries indicated), (b) mean velocity, (c) temperature, and (d) July 2017–June 2019 anomalous velocity across the GSR based on GLORYS12 (stippl malies are > 2σ of biennial anomalies). Note that the section does not go along the shallowest part of the Den where, leading to deeper trenches in some places (see also Fig. 1). Time series of (e) anomalous volume an through the Faroe–Shetland branch, where the red shading indicates ±1 standard error in the observational eanalyses with observations are provided in the legends. /10.5194/sp-1-osr7-14-2023 State Planet, 1-o 7 Figure 3. 3.2 Spatial structure of the July 2017–June 2019 transport anomaly GSR total PW OW AW AW NIIC AW I-F AW F-S Shelf Observations 0.7 ± 0.9 −1.8 ± 0.7 −5.6 ± 0.3 8.0 ± 0.5 0.9 ± 0.1 3.8 ± 0.3 2.7 ± 0.3 0.6 ± 0.3 GREP mean 1.2 ± 0.6 −1.2 ± 0.5 −5.0 ± 0.5 7.4 ± 0.6 0.9 ± 0.2 2.5 ± 0.4 3.5 ± 0.6 0.5 ± 0.2 ORAS5 1.8 −0.6 −4.4 6.8 1.2 2.6 2.8 0.2 CGLORS 1.4 −1.5 −5.4 8.3 0.8 2.8 3.9 0.8 GLORYS2V4 1.0 −0.9 −5.4 7.3 0.8 2.6 3.3 0.6 GloRanV14 0.4 −1.8 −5.0 7.2 0.9 1.9 4.1 0.3 GLORYS12 1.0 −1.0 −5.3 7.3 1.0 3.3 2.4 0.6 GLOB16 −0.2 −2.6 −4.9 7.4 0.6 3.2 3.0 0.6 GLOB4 1.2 −2.7 −4.4 8.3 0.8 3.2 3.7 0.6 Table 3. Long-term mean of total heat ﬂux across the GSR and into the Arctic Mediterranean (total GSR transports plus Bering Strait and Davis Strait transports). The energy-budget-based transport es- timate is taken from M. Mayer et al. (2022) and by deﬁnition can only be provided for a closed area like the Arctic Mediterranean. All values are based on 1993–2020 data. Values are given in TW. to be related to a shift towards generally higher AW volume ﬂux around 2016 (see Fig. 2e). This is not seen in any of the other products. Temperature transport anomalies in the F-S branch are strongly correlated with volume ﬂux anomalies (compare Fig. 3e and f), and there is a clear reduction in F-S branch temperature ﬂux in July 2017–June 2019 of −21.9, −24.1, and −16.1 TW in the observations, GREP_mean, and GLORYS12, respectively. The contribution of temperature anomalies in the F-S branch during that time was small, with biennial anomalies of observed volume-weighted tem- peratures between −0.2 and −0.1 K (similar for all prod- ucts). The total AW heat ﬂux anomaly for July 2017–June 2019 was −11.0, −22.5, and −19.4 TW in observations, GREP_mean, and GLORYS12, respectively. Thus, the re- duction in GSR heat transports during that period was mainly driven by a reduction in volume inﬂow through the F-S branch. It was only partly offset by compensating trans- port anomalies in other branches. Very similar biennial heat transport anomalies for the total GSR (−11.1, −20.9, and −19.3 TW for observations, GREP_mean, and GLORYS12, respectively) conﬁrm AW as the main driver of heat ﬂux vari- ability across the GSR. 3.2 Spatial structure of the July 2017–June 2019 transport anomaly Longitude–depth sections of (a) mean temperature (with water mass boundaries indicated), (b) mean velocity, (c) July 2017–June 2019 anomalous temperature, and (d) July 2017–June 2019 anomalous velocity across the GSR based on GLORYS12 (stippling denotes grid cells where anomalies are > 2σ of biennial anomalies). Note that the section does not go along the shallowest part of the Denmark Strait and I-F Ridge everywhere, leading to deeper trenches in some places (see also Fig. 1). Time series of (e) anomalous volume and (f) anomalous temperature ﬂux through the Faroe–Shetland branch, where the red shading indicates ±1 standard error in the observational data. Temporal correlations of reanalyses with observations are provided in the legends. Figure 3. Longitude–depth sections of (a) mean temperature (with water mass boundaries indicated), (b) mean velocity, (c) July 2017–June 2019 anomalous temperature, and (d) July 2017–June 2019 anomalous velocity across the GSR based on GLORYS12 (stippling denotes grid cells where anomalies are > 2σ of biennial anomalies). Note that the section does not go along the shallowest part of the Denmark Strait and I-F Ridge everywhere, leading to deeper trenches in some places (see also Fig. 1). Time series of (e) anomalous volume and (f) anomalous temperature ﬂux through the Faroe–Shetland branch, where the red shading indicates ±1 standard error in the observational data. Temporal correlations of reanalyses with observations are provided in the legends. CHAPTER4.2 https://doi.org/10.5194/sp-1-osr7-14-2023 State Planet, 1-osr7, 14, 2023 State Planet, 1-osr7, 14, 2023 https://doi.org/10.5194/sp-1-osr7-14-2023 CHAPTER4.2 M. Mayer et al.: Recent variations in oceanic transports across the Greenland–Scotland Ridg 8 Table 2. Long-term mean of volume ﬂux in different water masses and branches across the GSR. Observational European shelf volume ﬂuxes are based on Østerhus et al. (2019). All values are based on 1993–2020 (since observational data do not completely cover 2021) data, except for GLOB16 and GLOB4 (based on 1993–2019 data). Values are given in Sv. Table 2. Long-term mean of volume ﬂux in different water masses and branches across the GSR. Observational European shelf volume ﬂuxes are based on Østerhus et al. (2019). All values are based on 1993–2020 (since observational data do not completely cover 2021) data, except for GLOB16 and GLOB4 (based on 1993–2019 data). Values are given in Sv. 3.2 Spatial structure of the July 2017–June 2019 transport anomaly GSR total Arctic Mediterranean Observations 280 ± 18 306 ± 19 GREP mean 243 ± 21 256 ± 19 ORAS5 219 239 CGLORS 265 276 GLORYS2V4 234 242 GloRanV14 255 269 GLORYS12 243 252 Based on the energy budget – 348 the overﬂow from 600 to 1000 m depth. There is some com- pensation by positive velocity anomalies in the western F-S (meaning reduced southward ﬂow there), but the effect of the eastern F-S anomaly dominates, and the net F-S volume ﬂux anomaly was clearly reduced during this period (see below). We note that these main features are also similar in anomaly sections based on the GREP ensemble mean (not shown). State Planet, 1-osr7, 14, 2023 4 Conclusions with higher SLA in the SPG (i.e., a weaker gyre) associated with higher volume-weighted temperature and vice versa. Although correlations are high (r up to 0.69) in the SPG re- gion, they are not statistically signiﬁcant. The cause may be the low number of degrees of freedom, as the SLA in the SPG exhibits high temporal auto-correlation (see Fig. 4d dis- cussed below). Reanalysis-based oceanic transports show generally good agreement with observations on the scale of single branches of the GSR, in terms of both mean and variability in volume and heat ﬂuxes. There is some indication that the higher- resolution products have a better representation of AW in- ﬂow in the I-F and F-S branches. All considered products underestimate net heat ﬂux into the Arctic Mediterranean. The magnitude of the low bias is correlated with the strength of AW volume ﬂux, but a warm bias in OW and cold bias in Davis Strait inﬂow further add to the found net heat ﬂux bias. We reiterate that reanalysis-based and observational trans- port estimates are obtained in different ways (closed line in- tegrations versus measurements from 11 branches with an in- verse model applied), but, as elaborated in Sect. 2, we deem this a fair and robust approach for an intercomparison. The energy-budget-based estimate from M. Mayer et al. (2022) suggests even higher net heat ﬂux than oceanic observations, which conﬁrms the underestimation of heat transports by the ocean reanalyses. A much smaller discrepancy was found in an analogous comparison of observed oceanic transports into the central Arctic and an energy-budget-based estimate (Mayer et al., 2019), potentially reﬂecting the different obser- vational strategies in Fram Strait and the Barents Sea Open- ing compared to the GSR (see, e.g., Dickson et al., 2008) or potential biases in the employed energy budget ﬁelds over the Nordic Seas. Actual SLA anomalies averaged over July 2017–June 2019 (Fig. 4c) indicate a weakened zonal SLA gradient at the GSR, albeit not very strongly pronounced, and anoma- lously low SLA in the SPG region. According to the correla- tion patterns discussed above, these two features suggest re- duced AW volume ﬂux (as suggested by patterns in Fig. 4a) and anomalously low AW volume-weighted temperatures (as suggested by patterns in Fig. 4b). We also note the positive SLA anomalies north of the GSR with a maximum in the central Nordic Seas. 4 Conclusions To put these results in context, we deﬁne two SLA-based indices (shown in Fig. 4d) from the correlation patterns found in Fig. 3a and b. The similarity of the correlations in Fig. 4a to the correlations between total OHT at the GSR and SLA shown in M. Mayer et al. (2022) reinforces use of their gradient-based index (i.e., standardized SLA difference be- tween 58–60◦N, 2–0◦W and 63–67◦N, 20–15◦W). This in- dex is correlated with AW volume ﬂux anomalies (r = 0.62 for observed transports, ranging from 0.47 to 0.70 for reanal- yses, which is statistically signiﬁcant in all cases). The sec- ond index uses spatial SLA averages in the North Atlantic re- gion (55–60◦N and 40–15◦W) as an inverse proxy for SPG strength. This index is correlated with anomalies of volume- weighted temperatures of AW transports. The two indices in Fig. 4d show different characteristics, with the SPG in- dex varying on decadal timescales, while the gradient index shows stronger interannual variations. The SPG index has been negative since ∼2014, which suggests a strong SPG and lower inﬂow temperatures in recent years, in agreement with results by Hátún and Chaﬁk (2018). This SPG index ex- hibits two minima during the July 2017–June 2019 period, although these are not extreme relative to the entire time se- ries. All reanalyses with data assimilation and observations show a pronounced reduction in OHT during the 2-year pe- riod July 2017–June 2019, with some recovery after that. Comparison of observed SLA patterns during this period with statistical relationships between SLA and oceanic trans- ports suggests that this reduction arose from a combination of interannual- (i.e., reduced zonal SLA gradient at the GSR) and decadal-scale changes (i.e., strong SPG in recent years). Another potential factor contributing to the OHT reduction during July 2017–June 2019 may have been the positive SLA anomalies centered in the Nordic Seas (Fig. 4c), which Chat- terjee et al. (2018) have related to a weakened gyre circu- lation in the Nordic Seas and may have contributed to the weakened AW inﬂow as well. Figure 4e shows volume-weighted temperatures of AW waters from different products and conﬁrms their overall de- crease in recent years. Comparison with the SPG index in Fig. 4d suggests a generally delayed response of volume- weighted temperature in Atlantic inﬂow water at the GSR to SPG strength. 3.3 Relationships between sea level and AW inﬂow For a better understanding of mechanisms contributing to the GSR transport anomaly around 2018, we ﬁrst consider ba- sic statistical relationships between SLA and oceanic quan- tities. We ﬁnd a statistically signiﬁcant temporal correlation of the zonal SLA gradient at GSR with observed AW vol- ume ﬂux anomalies (Fig. 4a), which is plausible in terms of geostrophic balance. The correlation pattern looks very sim- ilar when performed with the F-S branch volume ﬂux alone (not shown). The pattern of temporal correlation between the SLA ﬁeld and observed anomalies of volume-weighted tem- perature of AW transports (Fig. 4b) is distinct from the re- lationship with AW volume ﬂux (compare Fig. 4a). It em- phasizes SLA in the North Atlantic subpolar gyre (SPG), Observations and all reanalyses show large negative F-S volume inﬂow anomalies of −0.61, −0.56, −0.34, and −0.24 Sv in the observations, GREP_mean, GLO- RYS12, and GLOB16, respectively, during July 2017–June 2019 (Fig. 3e). In four out of seven datasets, this is the overall biennial minimum of the 1993–2021 record (not shown). The total AW volume ﬂux anomaly for that pe- riod was −0.24, −0.53, −0.37, and +0.08 Sv in the obser- vations, GREP_mean, GLORYS12, and GLOB16, respec- tively. Thus, F-S volume ﬂux anomalies were partly com- pensated by other AW branches, with no very clear signal in any of them (not shown). Only GLOB16 exhibits a positive AW volume ﬂux anomaly during that period, which appears State Planet, 1-osr7, 14, 2023 https://doi.org/10.5194/sp-1-osr7-14-2023 CHAPTER4.2 M. Mayer et al.: Recent variations in oceanic transports across the Greenland–Scotland Ridge M. Mayer et al.: Recent variations in oceanic transports across the Greenland–Scotland Ridge 9 M. Mayer et al.: Recent variations in oceanic transports across the Greenland–Scotland Ridge 10 Figure 4. Correlation of observation-based (a) AW volume ﬂux and (b) AW ﬂux-weighted temperature with SLA with a 12-monthly smoother applied. (c) SLA anomaly in July 2017–June 2019. (d) Temporal evolution (standardized) of two SLA-based indices based on the zonal SLA gradient at GSR and the SLA in the SPG region. (e) Temporal evolution of volume-weighted temperature (T _weight) anomalies of Atlantic waters at the GSR (red shading indicates ±1 standard error in the observational data, and temporal correlations of reanalyses with observations are provided in the legend). Stippling in (a) and (b) denotes statistically signiﬁcant correlations on the 95 % conﬁdence level. Figure 4. Correlation of observation-based (a) AW volume ﬂux and (b) AW ﬂux-weighted temperature with SLA with a 12-monthly smoother applied. (c) SLA anomaly in July 2017–June 2019. (d) Temporal evolution (standardized) of two SLA-based indices based on the zonal SLA gradient at GSR and the SLA in the SPG region. (e) Temporal evolution of volume-weighted temperature (T _weight) anomalies of Atlantic waters at the GSR (red shading indicates ±1 standard error in the observational data, and temporal correlations of reanalyses with observations are provided in the legend). Stippling in (a) and (b) denotes statistically signiﬁcant correlations on the 95 % conﬁdence level. The presented results indicate that decadal predictions of the SPG strength, which have been shown to exhibit skill (e.g., Robson et al., 2018; Borchert et al., 2021), may also allow us to infer near-term trends in OHT across the GSR. Another implication is that the strong interannual-to-decadal variability in OHT across the GSR hampers detection of longer-term (forced and unforced) trends in observed OHT, an aspect in which climate simulations show large spread (Burgard and Notz, 2017). Continued in situ monitoring of OHT, complemented with reanalysis efforts, is thus needed to provide observationally constrained time series of sufﬁ- cient length for climate model validation. Author contributions. MM and TT conceptualized the study. All co-authors were involved with data preparation and analysis, as well as interpretation of results. MM prepared the manuscript with con- tributions from all co-authors. Author contributions. MM and TT conceptualized the study. All co-authors were involved with data preparation and analysis, as well as interpretation of results. MM prepared the manuscript with con- tributions from all co-authors. Competing interests. The contact author has declared that none of the authors has any competing interests. Disclaimer. 4 Conclusions Lagged correlation analysis indeed suggests positive correlations peaking (at values around 0.3 to 0.5, de- pending on the dataset) when temperatures are lagging the SPG index ∼2–3 years (not shown), but the correlations are not signiﬁcant due to the high auto-correlation of time series. Our results also reveal a delayed response of AW inﬂow temperatures to SPG strength. This is consistent with ear- lier studies ﬁnding anti-correlation between SPG strength and GSR heat transport (Häkkinen et al., 2011; Hátún et al., 2005). Speciﬁcally, the generally weaker SPG during ∼1997 and ∼2014 (with more pronounced minima in 1997–1998, 2010–2011, and 2003–2006; see Hátún and Chaﬁk, 2018) was associated with warm inﬂow temperatures and stronger OHT after 2001 (Tsubouchi et al., 2021). 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Zarokanellos1 1SOCIB, Balearic Islands Coastal Observing and Forecasting System, 07122 Palma, Spain 2Puertos del Estado, 28042 Madrid, Spain Baptiste Mourre1, Emma Reyes1, Pablo Lorente2, Alex Santana1, Jaime Hernández-Lasheras1, Ismael Hernández-Carrasco1, Maximo García-Jove1, and Nikolaos D. Zarokanellos1 1SOCIB, Balearic Islands Coastal Observing and Forecasting System, 07122 Palma, Spain 2Puertos del Estado, 28042 Madrid, Spain Baptiste Mourre1, Emma Reyes1, Pablo Lorente2, Alex Santana1, Jaime Hernández-Lasheras1, Ismael Hernández-Carrasco1, Maximo García-Jove1, and Nikolaos D. Zarokanellos1 1SOCIB, Balearic Islands Coastal Observing and Forecasting System, 07122 Palma, Spain 2Puertos del Estado, 28042 Madrid, Spain rnicus Ocean State Report (OSR7) Correspondence: Baptiste Mourre (bmourre@socib.es) Correspondence: Baptiste Mourre (bmourre@socib.es) Received: 3 August 2022 – Discussion started: 30 September 2022 Revised: 10 March 2023 – Accepted: 31 March 2023 – Published: 27 September 2023 Received: 3 August 2022 – Discussion started: 30 September 2022 Revised: 10 March 2023 – Accepted: 31 March 2023 – Published: 27 September 2023 Abstract. This article analyses the Balearic Islands wind-driven coastal upwelling in response to the intense and long-lasting Storm Blas, which affected the western Mediterranean Sea in November 2021. The storm was associated with a pronounced pressure low, generating heavy rains and intense winds and showing some char- acteristics of a tropical cyclone. The Balearic Islands area was particularly affected since the core of the storm was moving over a 1-week-long period from the south-west of this area to just above the islands of Menorca and Mallorca. High-resolution regional forecast models indicated that the intense north-easterly winds blowing over the region during the ﬁrst days of the storm led to the development of intense upwellings along the north- western coasts of the islands of Mallorca and Ibiza together with a reversal of the surface current. While the clouds associated with the storm prevented the radiometers on board satellites to precisely observe the evolution of the sea surface signature of the upwelling, signals of enhanced chlorophyll concentration were still detected in the upwelling region. The high-resolution Western Mediterranean Operational Prediction System model, which downscales the Copernicus Marine Service Mediterranean predictions, is used to describe the characteristics of this intense coastal-upwelling event, as well as to analyse its singularity over the past-9-year time series through the comparison of different coastal-upwelling indices. 1https://www.aemet.es/en/conocermas/borrascas/2021-2022/ estudios_e_impactos/blas (last access: 14 April 2023) Intense wind-driven coastal upwelling in the Balearic Islands in response to Storm Blas (November 2021) The upwelling event is found to have a duration of 3 d (considering its effects on the sea surface temperature) with a spatial offshore extension of around 20 km. It was characterized by intense cold coastal sea surface anomalies of around 6 ◦C. While it was the most intense event over the past 9 years in terms of local cross-shore sea surface temperature gradients, it is ranked second in terms of the intensity of cross-shelf transports, behind the Storm Gloria upwelling event in January 2020. This study demonstrates the beneﬁts of operational oceanography for the characterization of extreme events through the provision of time series of high-resolution modelling results in the coastal area. an State Report (OSR7) Published by Copernicus Publications. M. Mayer et al.: Recent variations in oceanic transports across the Greenland–Scotland Ridge Climate, 32, 7915–7934, 2019. Mayer, M., Tsubouchi, T., von Schuckmann, K., Seitner, V., Winkelbauer, S., Haimberger, L.: Atmospheric and oceanic contributions to observed Nordic Seas and Arctic Ocean Heat Content variations 1993–2020, in: Copernicus Ocean State Report, Issue 6, J. Oper. Oceanogr., 15, s119–s126, https://doi.org/10.1080/1755876X.2022.2095169, 2022. Tsubouchi, T., Våge, K., Hansen, B., Larsen, K. M. H., Østerhus, S., Johnson, C., Jónsson, S., and Valdimarsson, H.: Increased ocean heat transport into the Nordic Seas and Arctic Ocean over the period 1993–2016, Nat. Clim. Change, 11, 21–26, 2021. Muilwijk, M., Smedsrud, L. H., Ilicak, M., and Drange, H.: Atlantic Water heat transport variability in the 20th century Arctic Ocean from a global ocean model and observations, J. Geophys. Res.- Oceans, 123, 8159–8179, 2018. Tsujino, H., Urakawa, S., Nakano, H., Small, R. J., Kim, W. M., Yeager, S. G., Danabasoglu, G., Suzuki, T., Bamber, J. L., State Planet, 1-osr7, 14, 2023 https://doi.org/10.5194/sp-1-osr7-14-2023 CHAPTER4.2 M. Mayer et al.: Recent variations in oceanic transports across the Greenland–Scotland Ridge 13 13 Woodgate, R. A.: Increases in the Paciﬁc inﬂow to the Arctic from 1990 to 2015, and insights into seasonal trends and driving mechanisms from year-round Bering Strait mooring data, Prog. Oceanogr., 160, 124–154, https://doi.org/10.1016/j.pocean.2017.12.007, 2018 (data avail- able at: http://psc.apl.washington.edu/HLD/Bstrait/bstrait.html, last access: 22 March 2023). Bentsen, M., Böning, C. W., Bozec, A., Chassignet, E. P., Cur- chitser, E., Boeira Dias, F., Durack, P. J., Grifﬁes, S. M., Harada, Y., Ilicak, M., Josey, S. A., Kobayashi, C., Kobayashi, S., Ko- muro, Y., Large, W. G., Le Sommer, J., Marsland, S. J., Masina, S., Scheinert, M., Tomita, H., Valdivieso, M., and Yamazaki, D.: JRA-55 based surface dataset for driving ocean–sea-ice models (JRA55-do), Ocean Model., 130, 79–139, 2018. https://doi.org/10.5194/sp-1-osr7-14-2023 State Planet, 1-osr7, 14, 2023 State Planet, 1-osr7, 14, 2023 CHAPTER4.2 Chapter 4.3 – 7th edition of the Copernicus Ocean State Report (OSR7) Chapter 4.3 – 7th edition of the Co Chapter 4.3 – 7th edition of the Copernicus Ocean State Planet, 1-osr7, 15, 2023 https://doi.org/10.5194/sp-1-osr7-15-2023 © Author(s) 2023. This work is distributed under the Creative Commons Attribution 4.0 License. ter 4.3 – 7th edition of the Copernicus Ocean State Report (O B. Mourre et al.: Intense coastal upwelling B. Mourre et al.: Intense coastal upwelling Table 1. Products from the Copernicus Marine Service and other complementary datasets used in this study, including the Product User Manual and quality information. For complementary datasets, the link to the product description, data access and scientiﬁc references are provided. Last access for all web pages cited in this table: 14 April 2023. Table 1. Products from the Copernicus Marine Service and other complementary datasets used in this study, including the Product User Manual and quality information. For complementary datasets, the link to the product description, data access and scientiﬁc references are provided. Last access for all web pages cited in this table: 14 April 2023. Product Product ID and type Data access Documentation ref. no. 1 Images from Copernicus Sentinel-3, Sentinel Hub services (EO Browser), Sentinel-3 mission description: satellite observations https://www.sentinel-hub.com/ https://sentinels.copernicus.eu/web/sentinel/ missions/sentinel-3; data acquired by the Ocean and Land Colour Instrument (OLCI) 2 INSITU_MED_PHYBGCWAV EU Copernicus Marine Quality Information Document _DISCRETE_MYNRT_013_035, Service Product (2022a) (QUID): Wehde et al. (2022); in situ observations Product User Manual (PUM): Pouliquen et al. (2022) 3 HARMONIE-AROME, AEMET, Ministerio para System description: https://www.aemet.es/ numerical models la Transición Ecológica en/noticias/2017/07/modelo_harmonie-arome y el Reto Demográﬁco. (Bengtsson et al., 2017) 4 SOCIB-WMOP, SOCIB: http://thredds.socib.es/ Product description: http://socib.es/?seccion= numerical models thredds/catalog/operational_models/ modelling&facility=forecast_system_description oceanographical/hydrodynamics/ (Juza et al., 2016; Mourre et al., 2018; wmop/catalog.html Hernandez-Lasheras and Mourre, 2018; Hernandez-Lasheras et al., 2021) 5 MEDSEA_ANALYSIS EU Copernicus Marine QUID: Goglio et al. (2022); _FORECAST_PHY_006_013, Service Product (2022b) PUM: Lecci et al. (2022) numerical models 6 SST_MED_SST_L4_NRT EU Copernicus Marine QUID: Pisano et al. (2022b); _OBSERVATIONS_010_004, Service Product (2022c) PUM: Pisano et al. (2022a) satellite observations 7 SEALEVEL_EUR_PHY_L3_NRT EU Copernicus Marine QUID: Pujol et al. (2023); _OBSERVATIONS_008_059, Service Product (2023) PUM: Pujol (2022) satellite observations 8 INSITU_GLO_PHY_UV EU Copernicus Marine QUID: Verbrugge et al. (2022b); _DISCRETE_NRT_013_048, Service Product (2022d) PUM: Verbrugge et al. (2022a) in situ observations 9 OCEANCOLOUR_MED_BGC EU Copernicus Marine QUID: Colella et al. (2022b); _L3_NRT_009_141, Service Product (2022e) PUM: Colella et al. (2022a) satellite observations This situation created extreme conditions of intense winds and high waves as well as heavy rainfall in the Balearic archipelago. In particular, intense north-easterly winds were blowing over the area during the ﬁrst phase of the storm from 5 to 7 November (Fig. 1c, d), creating favourable conditions for coastal upwelling along the north-western coasts of the islands of Mallorca and Ibiza. 1 Introduction ing a well-deﬁned, circular, deep (∼10 hPa) low-pressure centre, it then moved eastwards while developing a spiral structure resembling that of tropical cyclones. The storm then moved over the islands of Sardinia and Corsica before weak- ening and dissipating in the Tyrrhenian Sea on 18 November. Figure 1a and b show the distribution of clouds over the af- fected area on 7 and 13 November, as observed by Sentinel- 3 Ocean and Land Colour Instrument (OLCI) satellite true- colour images. Storm Blas1 was an intense Mediterranean cyclone which af- fected the western Mediterranean Sea from 6 to 18 Novem- ber 2021. It was ﬁrst identiﬁed on 6 November between the Balearic Islands and Sardinia before moving westwards to- wards the Balearic archipelago. After the core of the storm looped over the island of Mallorca on 11 November, exhibit- Published by Copernicus Publications. CHAPTER4.3 CHAPTER4.3 2 B. Mourre et al.: Intense coastal upwelling The peak of this wind event occurred on 6 November around 10:00 UTC, with hourly mean values over 17.5 m s−1 off the island of Mallorca, as represented by the HARMONIE-AROME model (Bengts- son et al., 2017) from the Spanish Meteorological Agency (AEMET). shore transport of surface waters produced under the action of an intense or sustained alongshore wind stress. In the Northern Hemisphere, the wind has to blow with the coast on its left-hand side to be favourable to upwelling. By bring- ing cold and nutrient-rich deep waters close to the surface, the upwelling has signiﬁcant effects on the physical and bio- geochemical characteristics of coastal waters, generally en- hancing the local primary productivity and then playing an important role in marine ecosystems (Pauly and Christensen, 1995). In the western Mediterranean Sea, wind-induced up- welling is known to occur along the French coast (Millot et al., 1979; Rey et al., 2000; Bakun and Agostini, 2001), the western and eastern coasts of Sardinia (Olita et al., 2013; Salusti, 1998), and the north-western Alboran Sea (Sarhan Coastal upwelling is an oceanic process which generates upward vertical currents at the coast in response to an off- State Planet, 1-osr7, 15, 2023 https://doi.org/10.5194/sp-1-osr7-15-2023 CHAPTER4.3 CHAPTER4.3 3 B. Mourre et al.: Intense coastal upwelling et al., 2000; Macias et al., 2008). Eddy-induced coastal up- welling has also been evidenced in the Ligurian Sea (Casella et al., 2011) and along the Algerian coast (Millot et al., 1985). To our knowledge, no upwelling has been reported yet in the Balearic Islands. While the frequent north-north-westerly mistral wind events do not present the appropriate orientation to generate upwelling along the north-western coast of the Balearic Islands, favourable conditions with intense north- easterly winds are occasionally met during speciﬁc storms such as Gloria in January 2020 (Amores et al., 2020; Sotillo et al., 2021; Álvarez-Fanjul et al., 2022) or Blas in Novem- ber 2021. Notice that these winds blow in the opposite direc- tion of the established Balearic Current, which ﬂows north- eastwards along the north-western coast of the Balearic Is- lands (Lopez-García et al., 1994). as well as surface currents in the Ibiza channel, applying a local multimodel ensemble optimal interpolation approach as described in Hernandez-Lasheras and Mourre (2018) and Hernandez-Lasheras et al. (2021), with a 3 d cycle. We also consider the outputs of the CMEMS-MED model, which provides a spatial resolution of around 4 km. 3https://www.ecmwf.int (last access: 14 April 2023) 2https://www.socib.es/?seccion=modellingfacility=forecast (last access: 14 April 2023) 2.2 Ocean colour satellite observations This study was motivated by the intense sea surface tem- perature (SST) gradients along the north-western coast of the Balearic Islands seen in high-resolution regional prediction models during the ﬁrst phase of Storm Blas, together with observations of surface current reversal from high-frequency (HF) radar measurements along the north-western coast of the island of Ibiza. The objective of this study is to character- ize this unusually intense upwelling in terms of both its sur- face signature and its vertical structure, as well as to evaluate its singularity within the time series of different upwelling indices computed over the past decade. The main source of information is a high-resolution numerical prediction model given the very limited number of in situ and satellite obser- vations available during this event. High-resolution ocean colour imagery was analysed to detect the enhancement of the surface chlorophyll a (Chl a) con- centration during the upwelling. The Level 3 ocean colour product distributed by the Copernicus Marine Service (Volpe et al., 2019) was used. It provides surface Chl a concentra- tion from the OLCI on board the Sentinel-3 satellite with a 300 m resolution. Notice that clouds were present over the study area during the storm period, which limited the avail- ability of exploitable high-resolution satellite observations. The Sentinel-3 OLCI observations were the only available satellite data that were found to give relevant information for the detection of the upwelling signature during the study pe- riod. B. Mourre et al.: Intense coastal upwelling This sys- tem includes variational data assimilation of temperature and salinity vertical proﬁles and along-track satellite sea level anomaly observations through a 3D-VAR scheme (Dobricic and Pinardi, 2008). The atmospheric forcing is provided by the predictions from the European Centre for Medium-Range Weather Forecasts3 with a spatial resolution close to 10 km and a temporal resolution of 1 h. 2.1 Numerical models Several upwelling indices (UIs) have been used in the litera- ture with the objective of estimating the intensity of coastal upwelling. These indices are based on either the cross-shore SST differences (Demarcq and Faure, 2000), the alongshore surface current velocities (Lorente et al., 2020) or the cross- shore Ekman transport related to the forcing winds (Bakun, 1973), sometimes also incorporating the effect of cross-shore geostrophic ﬂows (Marchesiello and Estrade, 2010; Rossi et al., 2013; Jacox et al., 2018). This study compares four UIs computed over the 9-year time series of the WMOP opera- tional model outputs: We analyse the outputs of the Western Mediterranean Op- erational Prediction System (WMOP2; Juza et al., 2016; Mourre et al., 2018) developed at the Balearic Islands Coastal Observing and Forecasting System (SOCIB; Tintoré et al., 2013). The WMOP is a 2 km regional conﬁguration of the Regional Ocean Modeling System (ROMS; Shchep- etkin and McWilliams, 2005) implemented over the west- ern Mediterranean Sea. It uses high-resolution (1 h, 2.5 km) atmospheric forcing from the HARMONIE-AROME model (Bengtsson et al., 2017) provided by AEMET. Notice that before February 2019, the HIRLAM model was used as at- mospheric forcing with a resolution of 5 km and 1 h (3 h be- fore March 2017). The WMOP downscales the conditions of the Copernicus Marine Service Mediterranean analysis and forecast model (CMEMS-MED; Clementi et al., 2021), which are used as open boundary conditions. It also includes assimilation of observations from satellite SST, along-track sea level anomaly, Argo temperature and salinity proﬁles i. UISST ﬁrst quantiﬁes the temperature differences be- tween the coast (SSTcoast) and a location 25 km off- shore (SSToffshore) off the north-western coast of the island of Mallorca (edges of the magenta cross-shore section illustrated in Fig. 2a). As in Marchesiello and Estrade (2010), the observed differences are normalized by the vertical temperature differences between the sur- face and 200 m depth (T 200 m) at the offshore end of the https://doi.org/10.5194/sp-1-osr7-15-2023 State Planet, 1-osr7, 15, 2023 CHAPTER4.3 B. Mourre et al.: Intense coastal upwelling Figure 1. Upper panels: true-colour image from Sentinel-3 OLCI captured over the western Mediterranean Sea on (a) 6 November 2021 and (b) 13 November 2021. 2.1 Numerical models Lower panels: (c) 10 m wind map on 6 November 2021 from the AEMET HARMONIE-AROME prediction model over the area delimited by the black rectangles in panels (a) and (b), (d) time series of alongshore 10 m wind speed from the model and observations at the three locations (1: automatic weather station of Puig des Galfí in Ibiza; 2: oceanographic buoy of Palma Bay in Mallorca; 3: virtual station off the north-western coast of Mallorca) represented in panel (c). “Alongshore” is deﬁned here considering the direction of the north-western coast of the island of Mallorca making an angle of 123◦with respect to the north, with positive values south-westwards. Figure 1. Upper panels: true-colour image from Sentinel-3 OLCI captured over the western Mediterranea Figure 1. Upper panels: true-colour image from Sentinel-3 OLCI captured over the western Mediterranean Sea on (a) 6 November 2021 and (b) 13 November 2021. Lower panels: (c) 10 m wind map on 6 November 2021 from the AEMET HARMONIE-AROME prediction model over the area delimited by the black rectangles in panels (a) and (b), (d) time series of alongshore 10 m wind speed from the model and observations at the three locations (1: automatic weather station of Puig des Galfí in Ibiza; 2: oceanographic buoy of Palma Bay in Mallorca; 3: virtual station off the north-western coast of Mallorca) represented in panel (c). “Alongshore” is deﬁned here considering the direction of the north-western coast of the island of Mallorca making an angle of 123◦with respect to the north, with positive values south-westwards. g pp p g p ( ) (b) 13 November 2021. Lower panels: (c) 10 m wind map on 6 November 2021 from the AEMET HARMONIE-AROME prediction model over the area delimited by the black rectangles in panels (a) and (b), (d) time series of alongshore 10 m wind speed from the model and observations at the three locations (1: automatic weather station of Puig des Galfí in Ibiza; 2: oceanographic buoy of Palma Bay in Mallorca; 3: virtual station off the north-western coast of Mallorca) represented in panel (c). “Alongshore” is deﬁned here considering the direction of the north-western coast of the island of Mallorca making an angle of 123◦with respect to the north, with positive values south-westwards. off the north-western coast of the island of Mallorca, following Eq. (2). 2.1 Numerical models section according to the following formula: UISST = SSToffshore −SSTcoast SSToffshore −T 200 m offshore . (1) (1) UIalongshore velocity = V surf alongshore (2) (2) This normalized formulation allows scaling of the hor- izontal temperature differences by the vertical ones. A value close to 1 indicates a fully developed upwelling with SST values at the coast equal to the temperatures at 200 m depth. Since this formulation of UISST gener- ates very high values in situations when the upper ocean is vertically mixed during winter (denominator close to 0), the index is only computed here when the vertical temperature difference is larger than 1 ◦C. The overbar denotes the average over the rectangular box containing the two magenta sections represented in Fig. 2. The alongshore direction is the direction of the corresponding magenta section. It makes an angle of 123◦with respect to the north. Positive values de- note south-westward ﬂows. Further details about the methodology and application of this index can be found in Lorente et al. (2023; present issue of the Ocean State Report). ii. The second index is based on alongshore surface current velocities, as ﬁrst proposed in Lorente et al. (2020) as a proof-of-concept investigation. This index was speciﬁ- cally designed to be applied in any coastal area where surface velocities are available from HF radar instru- ments. It assumes that the alongshore wind stress is the primary driver of upwelling circulation and that the sur- face currents are highly responsive to local winds (e.g. Paduan and Ronsenfeld, 1996; Kohut et al., 2006). This index is deﬁned here as the average alongshore surface velocity (V surf alongshore) in the 25 km wide coastal region iii. The third index is the classical cross-shore Ekman trans- port index, computed from the alongshore wind stress. It is deﬁned as follows: UIEkman transport = x=L x=0 τalongshore ρ f dx , (3) (3) where τalongshore is the alongshore wind stress, ρ is the water density, and f is the Coriolis frequency. The inte- gral is computed along the magenta alongshore section over a distance L of 60 km. UIEkman transport represents where τalongshore is the alongshore wind stress, ρ is the water density, and f is the Coriolis frequency. The inte- gral is computed along the magenta alongshore section over a distance L of 60 km. UIEkman transport represents State Planet, 1-osr7, 15, 2023 https://doi.org/10.5194/sp-1-osr7-15-2023 https://doi.org/10.5194/sp-1-osr7-15-2023 State Planet, 1-osr7, 15, 2023 CHAPTER4.3 CHAPTER4.3 B. Mourre et al.: Intense coastal upwelling 5 the total transport across this section. Positive values in- dicate an offshore transport. storm, a signiﬁcant increase was observed on 7 November. The Chl a concentration reached 0.1 mg m−3, a magnitude more than twice as large as that observed 3 d before. While the satellite-derived Chl a in coastal areas should be inter- preted carefully, particularly under cloudy conditions, no ev- ident errors have been identiﬁed in the associated quality in- dex (QI). The QI measures for every pixel the difference be- tween the observation and the climatological average, nor- malized by the climatological standard deviation. The QI maps for 4 and 7 November are included in the Supplement (Fig. S1), showing values ranging from 1 to 2 in the area of enhanced Chl a, with a peak above 4 in the near-coastal zone. Moreover, sediment resuspension is unlikely to affect the quality of the data due to the steep local topography. Also, the Lagrangian analysis of surface water trajectories in the northern part of the island of Mallorca (Fig. S2) allows the hypothesis of a lateral advection of Chl a from the northern bay (Alcúdia Bay) towards the north-western coast of the is- land to be rejected. As a consequence, and within the limita- tions of both the model and observations, we believe that the spatial consistency between the observed patch of enhanced Chl a and the model upwelling area is probably indicative of an upwelling-induced local enhancement of Chl a. Finally, the last index computes the total cross-shore model transport in the upper 50 m across the same sec- tion. This depth approximately corresponds to the depth of the Ekman layer as illustrated in Fig. 3. It includes not only the Ekman transport, but also the cross-shore geostrophic transport and other contributions due to the cross-shore wind component and potential effects asso- ciated with the spatial variability in the wind stress curl. It is calculated following Eq. (4): UItotal transport = z=50 m z=0 x=L x=0 Vcross-shoredxdz, (4) (4) where Vcross-shore is the model horizontal velocity in the cross-shore direction. where Vcross-shore is the model horizontal velocity in the cross-shore direction. 3 Surface expression of the upwelling Figure 2 illustrates the surface circulation patterns and as- sociated SST in the WMOP and CMEMS-MED predic- tions models on 7 November 2022 at 06:00 UTC (the time with the most signiﬁcant impact of the upwelling on the SST). The SST exhibits marked upwelling signatures in the WMOP model along the north-western coast of the islands of Mallorca and Ibiza, with the temperature of surface waters around 6 ◦C colder at the coast compared to 20 km offshore. The surface coastal current ﬂows south-westwards along the wind direction, which is reversed with respect to the direc- tion of the Balearic Current under normal conditions (Lopez- García et al., 1994). The offshore width of the upwelled- water region ranges from 10 to 20 km, which slightly extends off the 200 m isobath. The upwelling is also represented in the CMEMS-MED model, but with a less pronounced sig- nature. It is out of the scope of this short article to inves- tigate these differences between models, but the lower spa- tial resolution of both the model grid and atmospheric forc- ing may have an important role in producing these discrep- ancies. The upwelled water only reaches the surface in the northern half of the north-western coast of the island of Mal- lorca in CMEMS-MED, yet with a larger offshore extension compared to the WMOP. The event duration based on its effect on the SST was around 3 d, i.e. from 5 November at 15:00 UTC, when the ﬁrst cross-shore coastal SST gradients were observed, until 8 November at 12:00 UTC, when they vanished. Figure 2 illustrates the surface circulation patterns and as- sociated SST in the WMOP and CMEMS-MED predic- tions models on 7 November 2022 at 06:00 UTC (the time with the most signiﬁcant impact of the upwelling on the SST). The SST exhibits marked upwelling signatures in the WMOP model along the north-western coast of the islands of Mallorca and Ibiza, with the temperature of surface waters around 6 ◦C colder at the coast compared to 20 km offshore. https://doi.org/10.5194/sp-1-osr7-15-2023 4 Vertical structure Figure 3 illustrates the vertical structure of the temperature and velocity ﬁelds along the cross-shore section off the north- western coast of the island of Mallorca, as represented by the WMOP model. While velocities are illustrated on 6 Novem- ber at 12:00 UTC, corresponding to the maximum wind in- tensity in this area (Fig. 1), the temperature is displayed 18 h later to illustrate the time with the maximum signature in SST. The temperature section (Fig. 3a) shows a typical pat- tern of upwelling with an upward tilting of the isotherms to- wards the coast, which is associated with an upward trans- port of colder deep waters along the topographic slope. The surface temperature decreases by 6 ◦C across the upwelling front, varying from 20.6 ◦C 20 km offshore, where the mixed layer depth is around 50 m, to 14.6 ◦C at the coast. The cross- shore velocity section (Fig. 3b) shows an offshore displace- ment of surface waters in the upper 50 m, consistent with the Ekman theory in the presence of alongshore winds. Cross- shore velocities (Fig. 3c) reach 0.3 m s−1. The alongshore velocity along this cross-shelf section is characterized by the intense south-westward coastal jet with surface velocities ex- ceeding 1 m s−1. Upward vertical velocities (Fig. 3d) are en- hanced along the topographic slope, with values between 40 and 60 m d−1 in the upper 100 m and in the ﬁrst 12 km from the coast. Overall, these cross-shelf sections illustrate a rela- tively thin (cross-shore extension around 20 km) but intense upwelling with a signiﬁcant coastal jet. As illustrated in Fig. 1, the storm was associated with a dense cloud coverage, which only allowed very partial satel- lite remote sensing information on SST and Chl a concen- tration. Despite this important limitation, the two most rel- evant ocean colour images illustrated in Fig. 2 hint at the enhancement of the Chl a concentration along the north- western coast of the island of Mallorca. While a low con- centration of Chl a was detected on 4 November before the State Planet, 1-osr7, 15, 2023 State Planet, 1-osr7, 15, 2023 https://doi.org/10.5194/sp-1-osr7-15-2023 CHAPTER4.3 CHAPTER4.3 CHAPTER4.3 B. Mourre et al.: Intense coastal upwelling 6 Figure 2. Upper panels: SST and surface currents around the islands of Mallorca and Ibiza on 7 November 2021 at 06:00 UTC as represented by the (a) WMOP and (b) CMEMS-MED models. 5 Interannual perspective tal transport compared to Blas. The effects on the SST and coastal jet are particularly marked during the Blas upwelling event due to its occurrence in early November, when the sur- face ocean stratiﬁcation was still signiﬁcant. In contrast and despite the intense offshore transport during the upwelling event associated with Storm Gloria, the effects on the SST were hardly noticeable due to the presence of mixed sur- face waters. Note that the SST index is only computed when the vertical temperature difference between the surface and 200 m depth is larger than 1 ◦C, which signiﬁcantly limits the use of the SST-based index during the winter season. How- ever, this was not the case during the Gloria event, which showed values just above 1 ◦C. How singular was this intense wind-driven coastal-upwelling event? To answer this question, we compute here several upwelling indices (described in Sect. 2.3) over the whole WMOP model time series covering the 9-year period be- tween August 2013 and June 2022. The ﬁrst index, repre- senting the normalized cross-shelf SST gradients, ranks the November 2021 event (i.e. Storm Blas) as the most intense event of the whole time series. It is the only event with a nor- malized cross-shelf SST gradient exceeding 0.5. The event is also the most intense according to the second index, which measures the intensity of the alongshore surface velocity. However, the Ekman and total cross-shelf transport indices indicate a larger transport during the other extreme event as- sociated with Storm Gloria in January 2020. This is espe- cially true for the total transport, which also accounts for the effects of the wind stress curl and cross-shore geostrophic transport. During both the Gloria and Blas upwelling events, the offshore total transport was enhanced with respect to the Ekman estimate. A third very intense storm in January 2017 (also documented in García-León et al., 2018) led to a slightly larger cross-shore Ekman transport but a lower to- 4 Vertical structure In panel (a), the lines in magenta represent the alongshore and cross-shore sections used in Sects. 3 and 4. Lower panels: Chl a concentration as observed by Sentinel-3 OLCI on (c) 4 November and (d) 7 November (i.e. before and during Storm Blas, respectively). Figure 2. Upper panels: SST and surface currents around the islands of Mallorca and Ibiza on 7 November 2021 at 06:00 UTC as represented by the (a) WMOP and (b) CMEMS-MED models. In panel (a), the lines in magenta represent the alongshore and cross-shore sections used in Sects. 3 and 4. Lower panels: Chl a concentration as observed by Sentinel-3 OLCI on (c) 4 November and (d) 7 November (i.e. before and during Storm Blas, respectively). State Planet, 1-osr7, 15, 2023 6 Conclusions This study describes some of the characteristics of a short (5–8 November 2021) but intense wind-driven coastal- upwelling event along the north-western coast of the Balearic Islands, as represented by a high-resolution forecast model during Storm Blas in November 2021. The time series of sev- eral upwelling indices illustrate the episodicity of upwelling State Planet, 1-osr7, 15, 2023 https://doi.org/10.5194/sp-1-osr7-15-2023 CHAPTER4.3 7 B. Mourre et al.: Intense coastal upwelling Figure 3. Sections of WMOP model ﬁelds along the cross-shore magenta line represented in Fig. 2: (a) temperature on 7 November 2021 at 06:00 UTC, (b) cross-shore velocity, (c) alongshore velocity and (d) vertical velocity on 6 November 2021 at 12:00 UTC. Figure 3. Sections of WMOP model ﬁelds along the cross-shore magenta line represented in Fig. 2: (a) temperature on 7 November 2021 at 06:00 UTC, (b) cross-shore velocity, (c) alongshore velocity and (d) vertical velocity on 6 November 2021 at 12:00 UTC. Figure 4. Time series of upwelling indices deﬁned in Sect. 2.3 between August 2013 and June 2022 (3-hourly values are shown after applying a 24 h running average): (a) cross-shore normalized SST gradient index, (b) alongshore velocity index, (c) cross-shore Ekman transport index, (d) cross-shore total transport index. The red, blue and green triangles on the x axis mark the time of the Blas, Gloria and January 2017 storms, respectively. Figure 4. Time series of upwelling indices deﬁned in Sect. 2.3 between August 2013 and June 2022 (3-hourly values are shown after applying a 24 h running average): (a) cross-shore normalized SST gradient index, (b) alongshore velocity index, (c) cross-shore Ekman transport index, (d) cross-shore total transport index. The red, blue and green triangles on the x axis mark the time of the Blas, Gloria and January 2017 storms, respectively. CHAPTER4.3 https://doi.org/10.5194/sp-1-osr7-15-2023 State Planet, 1-osr7, 15, 2023 State Planet, 1-osr7, 15, 2023 https://doi.org/10.5194/sp-1-osr7-15-2023 CHAPTER4.3 CHAPTER4.3 B. Mourre et al.: Intense coastal upwelling 8 events in this area, mainly related to their nature related to the occurrence of storms with intense north-easterly winds. pacity to operate under any weather conditions. In the fu- ture, the possible increase in the intensity and duration of the storms affecting the western Mediterranean area (Gaertner et al., 2007; Romero and Emanuel, 2013; González-Alemán et al., 2019) could lead to enhanced impacts of such storm- related upwelling events. 6 Conclusions While this study presents a ﬁrst analysis over the last 9 years, it will be important to anal- yse these events in the longer term with a climate perspective when multi-decadal, kilometre-scale reanalysis simulations become available in the study area. While the November 2021 Blas-related event was the most intense in terms of the effects on the SST and alongshore velocities over the 9-year-long time series analysed, the in- duced cross-shelf surface transport was lower than that mod- elled during Storm Gloria in January 2020, whose effects on the wind, waves, sea level and currents were already reported as a record-breaking event in the literature (Álvarez-Fanjul et al., 2022). This comparison illustrates the difference between upwelling indices for the characterization of the intensity of these events. In particular, the magnitude of SST gradients or alongshore velocity does not necessarily reveal the whole intensity of the underlying offshore transport. A careful anal- ysis of the time series would allow appropriate thresholds to be determined for the different indices so as to deﬁne suitable criteria for the identiﬁcation of the upwelling. The duration would certainly be an additional relevant parameter for the operational deﬁnition of these events. Here, for instance, the event duration corresponding to a threshold of 0.25 Sv for the total offshore transport was 42 h for the Blas-related up- welling, 51 h for Gloria and 21 h for the January 2017 event. In the particular case of Blas, this duration is shorter than 3 d, i.e. the value obtained when considering the effect on the SST, highlighting the sensitivity of these metrics to the index and threshold under consideration. Data availability. The model and observation products used in this study from both the Copernicus Marine Service and other sources are listed in Table 1. Supplement. The supplement related to this article is available online at: https://doi.org/10.5194/sp-1-osr7-15-2023-supplement. Author contributions. BM, ER, AS, JHL, IHC, MGJ and NDZ designed the study through interactive discussions in the frame- work of working team meetings. BM and ER created the ﬁgures. BM prepared the article with contributions from all co-authors. ER analysed HF radar and in situ observations. PL provided exper- tise in the deﬁnition of upwelling indices. AS analysed the atmo- spheric ﬁelds and computed the Ekman transport. NDZ and IHC contributed to the analysis of ocean colour images. JHL and MGJ analysed WMOP and CMEMS-MED model outputs. 6 Conclusions All authors participated in the iterations and revision of the manuscript. Such storm-driven coastal upwelling is especially difﬁcult to monitor given their short timescale and the absence of in situ observations in this area. The dense cloud coverage asso- ciated with the storm also signiﬁcantly limits the availability of high-resolution satellite observations to characterize the surface signature of the phenomenon. In this context, high- resolution operational regional ocean models help overcome these limitations, providing timely and accurate information to identify and characterize these events. The only available ocean colour image from satellites suggests an increase in primary productivity in a coastal area which corresponds to the area of upwelled water in the model. While the lack of complementary observations prevents the realism of the model ﬁelds from being properly evaluated, the consistency between the spatial structure of observed Chl a enhancement and model upwelled water seems to indicate a reasonable representation of the effect of the upwelling process in the model. High spatial resolution (close to kilometric) is needed in both the model grid and the atmospheric forcing to de- scribe such coastal phenomena whose cross-shore extension does not exceed 20 km. Competing interests. The contact author has declared that none of the authors has any competing interests. Disclaimer. Publisher’s note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Acknowledgements. This study was conducted using informa- tion from the European Union’s Copernicus Marine Service. The re- sults of this article were obtained using the HARMONIE-AROME atmospheric ﬁelds provided by the Spanish Meteorological Agency (AEMET, Ministerio para la Transición Ecológica y el Reto De- mográﬁco). The authors are very grateful to AEMET for provid- ing these ﬁelds. The authors also thank SOCIB and Puertos del Estado for collecting oceanic and atmospheric observations around the Balearic Islands and distributing them through both their insti- tutional websites and the Copernicus database. Despite its short duration, this phenomenon was found to be sufﬁcient to enhance the surface Chl a concentra- tion. A more comprehensive understanding of the impacts of these storm-related upwelling events on local ecosystems would need further investigation requiring dedicated mon- itoring systems. Underwater gliders equipped with sensors of both physical and biogeochemical parameters can be suit- able observation platforms for these studies given their ca- Review statement. 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K. von Schuckmann et al. 1.2 Evaluation of operational ocean forecasting systems from the perspective of the users and the experts. S. A. Ciliberti et al. Chapter 2: Updated and new pathways in ocean science 2.1 Comparing global trends in marine cold spells and marine heatwaves using reprocessed satellite data. R. Peal et al. 2.2 South Atlantic overturning and heat transport variations in ocean reanalyses and observation-based estimates. J. A. Baker et al. 2.3 Satellite monitoring of surface phytoplankton functional types in the Atlantic Ocean over 20 years (2002–2021). H. Xi et al. 2.4 The dynamical role of upper layer salinity in the Mediterranean Sea. A. Aydogdu et al. 2.5 Baltic Sea freshwater content. U. Raudsepp et al. 2.6 High-frequency radar-derived coastal upwelling index. P. Lorente et al. 2.7 Ocean heat content in the Iberian–Biscay–Ireland regional seas. Á. de Pascual-Collar et al. Chapter 3: Ocean state and change for relevance to society 3.1 Dissolved oxygen as an indicator of multiple drivers of the marine ecosystem: the southern Adriatic Sea case study. V. Di Biagio et al. 3.2 Characterization of the organic vs. inorganic fraction of suspended particulate matter in coastal waters based on ocean color radiometry remote sensing. H. Loisel et al. 3.3 Recent changes in extreme wave events in the south-western South Atlantic. C. B. Gramcianinov et al. Chapter 4: Specific events in the ocean in 2021 4.1 Unusual coccolithophore blooms in Scottish waters. R. Renshaw et al. 4.2 Recent variations in oceanic transports across the Greenland–Scotland Ridge. M. Mayer et al. 4.3 Intense wind-driven coastal upwelling in the Balearic Islands in response to Storm Blas (November 2021). B. Mourre et al. 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J., 47, 17 pp., 2013. Wehde, H., von Schuckmann, K., Pouliquen, S., Grouazel, A., Bartolome, T., Tintore, J., De Alfonso Alonso-Munoyerro, M., Carval, T., Racapé, V., and the INSTAC team: EU Coper- nicus Marine Service Quality Information Document for the Mediterranean Sea- In-Situ Near Real Time Observations, IN- SITU_MED_PHYBGCWAV_DISCRETE_MYNRT_013_035, Issue 2.2, Mercator Ocean International, https: //catalogue.marine.copernicus.eu/documents/QUID/ CMEMS-INS-QUID-013-030-036.pdf (last access: 14 April 2023), 2022. Mason, E., Escudier, R., Conti, D., Sayol, J. M., Barceló, B., Oguz, T., Gómez, M., Álvarez, E., Ansorena, L., and Manriquez, M.: SOCIB: the Balearic Islands Observing and Forecasting Sys- tem responding to science, technology and society needs, Mar. Technol. Soc. J., 47, 17 pp., 2013. Table of contents Copernicus Ocean State Report \| OSR7 \| 2023 https://sp.copernicus.org/articles/1-osr7 https://sp.copernicus.org/articles/1-osr7
https://openalex.org/W2474156429	http://www.revistas2.uepg.br/index.php/pauta/article/download/8697/5126	Portuguese	null	A prática da Docência no Curso de Jornalismo da Unicentro/Guarapuava-PR	Pauta Geral	2,016	cc-by-sa	4,934	1 Mestre e Doutora em Educação pela Universidade Estadual de Campinas e Pós-doutorado pelo IG/UNICAMP em Geociências Aplicadas ao Ensino. É professora associada da Universidade Estadual do Centro-Oeste do Campus de Irati- PR. Participa como membro do Projeto Prodocência/CAPES/UNICENTRO do curso de Geografia do Campus de Irati - PR. Coordenadora Geral na Unicentro do Programa de Desenvolvimento Educacional - PDE/SEED - PR. Coordenadora no Campus de Irati - PR do Projeto Laboratórios Interdisciplinares das Licenciaturas - LIFE/CAPES. Coordenadora de área - Geografia/Campus de Irati-PR - PIBID/CAPES/UNICENTRO. 2 Formada em Comunicação Social com habilitação em Jornalismo pelo Centro Universitário de União da Vitória (Uniuv) em 2012. Especialização em Docência Universitária concluída em 2015 pela Universidade Estadual do Centro-Oeste (Unicentro). A prática da Docência no Curso de Jornalismo da Unicentro/Guarapuava-PR Wanda Terezinha Pacheco dos SANTOS1 Fernanda Pereira SANTOS2 Revista Pauta Geral-Estudos em Jornalismo, Ponta Grossa, vol.3, n.1 p. 110-121, Jan/Jun, 2016. Abstract This article is the result of a research made with Journalism bachelor’s degree course teachers of the State University of the Midwest, also called Unicentro - Guarapuava- PR, whose objective was to investigate the importance of pedagogical training, considering that the course trains bachelors professional which don’t have pedagogical content in their course curriculum. In the grounds of this work were studied authors that research about journalism and university teaching as Andrade and Linhares, Masetto, Anastasiou, Nóvoa, Marcelo, among others. To gather information, was sent by e-mail a questionnaire to the Journalism teachers with six open questions. The analysis took into account four questionnaires. Considering the answers, for the respondents, the pedagogical training makes the differential in the classroom, though not all, while recognizing the importance of teacher training, are interested in improving their practice. In this sense, for this training be consolidated it’s important to develop institutional actions, from an organized continuing education project, systematic and intentional and that involves all university professors. Keywords: journalism; university teaching; teacher training. 112 Resumo O presente artigo é resultado de uma pesquisa realizada com professores do curso de Jornalismo da Universidade Estadual do Centro-Oeste – Unicentro – Guarapuava- PR em que o objetivo foi investigar a importância da formação pedagógica do docente, uma vez que o curso forma profissionais bacharéis e que não possuem conteúdos pedagógicos em sua estrutura curricular. Na fundamentação deste trabalho, foram estudados autores que pesquisam tanto sobre jornalismo quanto docência universitária como Andrade e Linhares, Masetto, Anastasiou, Nóvoa, Marcelo, entre outros. Para coleta dos dados, foi encaminhado via e-mail um questionário aos professores do curso de Jornalismo da Unicentro com seis perguntas abertas. A análise levou em conta quatro questionários respondidos. Diante dos relatos, para os entrevistados a formação pedagógica faz todo diferencial em sala de aula, no entanto nem todos, mesmo reconhecendo a importância da formação docente, se interessam em melhorar sua prática. Nesse sentido, para que essa formação seja consolidada é importante o desenvolvimento de ações institucionais, a partir de um projeto de formação continuada organizado, sistemático e intencional e que envolva todos os professores universitários. Palavras-chave: jornalismo; docência universitária; formação pedagógica 2 Formada em Comunicação Social com habilitação em Jornalismo pelo Centro Universitário de União da Vitória (Uniuv) em 2012. Especialização em Docência Universitária concluída em 2015 pela Universidade Estadual do Centro-Oeste (Unicentro). Revista Pauta Geral-Estudos em Jornalismo, Ponta Grossa, vol.3, n.1 p. 110-121, Jan/Jun, 2016. Revista Pauta Geral-Estudos em Jornalismo, Ponta Grossa, vol.3, n.1 p. 110-121, Jan/Jun, 2016. REVISTA PAUTA GERAL ESTUDOS EM JORNALISMO Doi 10.5212/RevistaPautaGeral.v.3.i1.0007 Para a fundamentação teórica diversos autores foram estudados como Masetto (2005) Anastasiou (2006) Libâneo (2003) Isaia (2006) Pimenta (2002) Marcelo (2009). A chefia do departamento de Comunicação Social foi contatada e repassou os e-mails dos professores de Jornalismo. Em seguida, o questionário foi enviado. Infelizmente, mesmo insistindo somente quatro professores retornaram com o questionário respondido. Dessa forma, análise levou em consideração os dados recebidos via e-mail, através do questionário com 06 perguntas abertas que tratavam do ser professor universitário, da descrição de como acontece uma aula da sua disciplina, das dificuldades ou não de ensinar, se durante sua formação teve alguma disciplina pedagógica e dos temas que ela tratou, sobre a importância da formação pedagógica do professor universitário e por último, se faria algum curso de capacitação ofertado pela universidade visando a melhoria da sua formação como professor universitário e que tema poderia ser trabalhado. Revista Pauta Geral-Estudos em Jornalismo, Ponta Grossa, vol.3, n.1 p. 110-121, Jan/Jun, 2016. Introdução O curso de Comunicação Social com habilitação em Jornalismo dá ao acadêmico a formação de bacharel, porém como todas as áreas, o Jornalismo também necessita de professores para preparar novos jornalistas ao mercado de trabalho. No entanto, esse professor não possui embasamento pedagógico, em razão do seu curso de bacharelado não ofertar nenhuma disciplina que trate da formação do professor de Jornalismo, isto é, da formação pedagógica desse professor. Assim sendo, houve uma preocupação de como esse profissional se prepara para ensinar tanto as teorias quanto as práticas jornalísticas aos seus alunos. Em quê ou quem se baseia para planejar e desenvolver suas aulas? O objetivo foi averiguar com os professores do curso de Jornalismo da Unicentro, Campus Santa Cruz, em Guarapuava - PR qual a importância do conhecimento pedagógico para sua formação enquanto professor universitário, bem como verificar como esse professor desenvolve sua prática pedagógica no curso com seus alunos. REVISTA PAUTA GERAL ESTUDOS EM JORNALISMO Doi 10.5212/RevistaPautaGeral.v.3.i1.0007 Desenvolvimento O primeiro passo da pesquisa foi relembrar quais dos meus professores, no período de graduação, no Centro Universitário de União da Vitória (UNIUV) possuíam alguma formação pedagógica. Para minha surpresa, encontrei oito deles com especialização na prática docente – número que acredito ser relevante. 113 O passo seguinte foi encontrar materiais sobre a docência universitária, também relacionada à formação bacharel, e se havia algum estudo específico sobre o curso de Jornalismo. Foram encontrados dois artigos na internet, um sobre a Comunicação e sobre o Jornalismo e outro relacionado à prática da docência de maneira ampla para embasar o início das pesquisas. Além da procura pelo conhecimento teórico inicial, a chefia do Departamento de Comunicação Social na UNICENTRO – Campus Santa Cruz – Guarapuava – PR, oi procurada no sentido de autorizar a pesquisa e a coleta de dados junto aos professores do curso. Os dados solicitados inicialmente foram o projeto político-pedagógico do curso - PPP, número de professores atuantes e quais deles possuíam algum tipo de formação ou capacitação relacionada à docência. Com os dados avaliados, a etapa seguinte foi enviar um e-mail informando os professores de que receberiam um questionário para coleta de dados da pesquisa. Dois REVISTA PAUTA GERAL ESTUDOS EM JORNALISMO Doi 10.5212/RevistaPautaGeral.v.3.i1.0007 dias depois, as perguntas foram repassadas com prazo para envio das respostas. Dos 12 professores do corpo docente, apenas quatro deles enviaram o questionário respondido. dias depois, as perguntas foram repassadas com prazo para envio das respostas. Dos 12 professores do corpo docente, apenas quatro deles enviaram o questionário respondido. Questionados sobre quais os motivos de terem escolhido a profissão, os professores assim se manifestaram: P1 – Minha afinidade com a pesquisa. P2- Para contribuir com a formação de profissionais melhores. P3- Desde o tempo de graduação, desejava ser professora universitária. Sempre gostei da leitura, da escrita e da pesquisa acadêmica. P4- Para poder repassar um pouco do meu conhecimento adquirido nos últimos 20 anos na carreira. Autores como Nóvoa (2004), Marcelo (2009) Pimenta e Anastasiou (2002) discutem a identidade do professor universitário. Afinal ser professor é questão de aprendizado, escolha ou dom? De qualquer maneira, “a identidade não é um dado adquirido, não é uma propriedade, não é um produto. A identidade é um lugar de lutas e de conflitos, é um espaço de construção de maneiras de ser e de estar na profissão” (NÓVOA, 1997, p.34). Revista Pauta Geral-Estudos em Jornalismo, Ponta Grossa, vol.3, n.1 p. 110-121, Jan/Jun, 2016. Desenvolvimento Para os quatro professores a escolha teve objetivos bem definidos, dois deles relacionam a produção com a pesquisa e dois têm a preocupação com a formação de melhores profissionais. Como funciona sua aula? Para P1 “há disciplinas teóricas e práticas. No geral, como estratégia didática, busco transformar as aulas em uma conversa”. Entende-se que “transformar as aulas em uma conversa” pode ser que seja uma aula expositiva dialogada, como destaca Anastasiou (2005, p.161) “A aula, no que se refere à exposição do conteúdo, deve ser vivenciada o tempo todo com a participação do estudante: a isto chamamos de aula expositiva dialogada.” Também P4 diz que “basicamente as aulas são expositivas, dialogadas, com textos, slides e avaliações”. O P2 trabalhando com “disciplinas que mesclam teoria e prática na própria ementa”, procura “transportar essa mesma mescla para os planos de aula, tentando mostrar de que forma o universo teórico e universo mercadológico dialogam ou se repelem. Nesse contexto, busco trazer exemplos, situações, problemas, etc. que façam os alunos refletirem sobre determinado ponto”. contexto, busco trazer exemplos, situações, problemas, etc. que façam os alunos refletirem sobre determinado ponto”. Na mesma linha, P3 nos informa que “nas teóricas geralmente há um texto para discussão e cada grupo apresenta. Após faço uma exposição do texto, utilizando-me de vídeos, músicas, trechos de filmes, fotografias, etc”. Acrescenta que “nas aulas teórico- práticas, os alunos realizam um projeto sobre determinado tema e ao final há uma apresentação com banca com a participação de professor ou profissional do mercado para avaliar o produto final”. É isso que ressalta Libâneo (2003, p.8) quando apresenta a aula para um curso universitário como a teoria e a prática sempre se complementando. A sala de aula implica uma aproximação entre a teoria e a prática. A aprendizagem se realiza mais facilmente e com maior compreensão e retenção quando acontece nos vários ambientes profissionais fora da sala de aula porque coloca o aprendiz mais em contato com a realidade. O conhecimento da realidade parte da leitura da prática referente à disciplina estudada, de forma a se superar uma prática sem reflexão e uma teoria que não consegue atingir a prática. 115 Para esse autor a aula e o professor de métodos tradicionais estão, de certa forma, perdendo seu campo no processo só de repassar o conhecimento. Desenvolvimento Ele defende a aula universitária alternativa, como “lugar onde professores e alunos buscam juntos o conhecimento, estabelecem interações, diálogos e trocas” (LIBÂNEO, 2003, p.7). Isso vai ao encontro ao que pensa Isaia (2005, p.70-71): a aula universitária precisa deixar de ser “um espaço apenas de transmissão mecânica e fragmentada de conhecimentos científicos e profissionais para se instaurar como um lugar que possibilita ao aluno uma compreensão genuína em seu campo de atuação, que o torna capaz de aplicar conhecimentos, destrezas e saberes a situações novas e imprevisíveis, ao longo de sua carreira profissional”. Também Masetto (2005, p. 6) sugere e acredita que a aula precisa ser diversificada, transformando a universidade em mais um espaço de aprendizado, mas não o único. biblioteca, redigir trabalhos, participar de conferências de especialistas, entrevistá-los, fazer perguntas, solucionar dúvidas, orientar trabalhos de investigação e pesquisa, desenvolver diferentes formas de expressão e comunicação, realizar oficinas e trabalhos de campo. Para ele, este conceito faz com que a aula universitária passe a ser um espaço de discussões, diálogo, sugestões, tornando as aulas ‘vivas’. A mudança de perfil dos alunos hoje também é um tema que merece atenção. Em consequência disso, é que a universidade necessita de inovação, ou seja, encarar a nova realidade e os desafios. Por isso, Maraschin (2009, p.8273,) defende que o professor e seus métodos de ensinar também devem acompanhar esse processo contínuo. A autora acredita que a aula universitária é “um exercício de interlocução humana, instituída e instituinte de ações consentidas, permeadas por contradições inerentes a tempos e espaços diferenciados, amparadas num movimento epistemológico de teorias e práticas”(p. 8273). Entretanto, dar aula não é simplesmente estar munido de conhecimento e nem de diversidades de como repassar esse conhecimento. Para Neto (2011, p.119) dar aula é muito mais que isso. É uma dedicação e, acima de tudo, é transpor aos alunos o seu amor. 116 A aula, toda ela, todas elas, deve ser um ato de amor, uma dança, um orgasmo múltiplo, um gozo ensurdecedor, uma festa, um ato político, uma manifestação de indignação contra as injustiças. Aqueles que não veem isso em uma aula, aqueles que jamais se arrepiaram com a descoberta de um dos seus alunos, aqueles que jamais souberam o que é velar à noite as palavras do dia seguinte, jamais saberão, jamais sentirão o prazer que a profissão de professor pode proporcional (NETO, 2011, p.119). Encontram dificuldades em ensinar? Sim? Não? Revista Pauta Geral-Estudos em Jornalismo, Ponta Grossa, vol.3, n.1 p. 110-121, Jan/Jun, 2016. A aula, toda ela, todas elas, deve ser um ato de amor, uma dança, um orgasmo múltiplo, um gozo ensurdecedor, uma festa, um ato político, uma manifestação de indignação contra as injustiças. Aqueles que não veem isso em uma aula, aqueles que jamais se arrepiaram com a descoberta de um dos seus alunos, aqueles que jamais souberam o que é velar à noite as palavras do dia seguinte, jamais saberão, jamais sentirão o prazer que a profissão de professor pode proporcional (NETO, 2011, p.119). Desenvolvimento Quais? Foi outra questão apresentada aos professores. Para o P1, “a falta de leitura prévia dos conteúdos atrapalha muito”. P3 diz que como trabalha com disciplinas teóricas, percebe que, cada vez mais, há um crescente desinteresse dos acadêmicos pela leitura e até mesmo pela escrita (embora seja um curso de Comunicação), o que dificulta o processo ensino-aprendizado. Destaca que o perfil do aluno de Comunicação mudou muito nestes últimos anos, cada vez mais jovens – o que certamente reflete na maturidade necessária para cursar um curso superior, sendo responsável pela sua formação profissional. Já o P2 assim se manifestou: “O mercado jornalístico, talvez justamente por estar em crise, se movimenta muito rápido, no sentido de tentar reencontrar seu espaço. Absorver essas tendências, estar antenado com novas iniciativas, e ao mesmo tempo relacioná-las com os velhos paradigmas e problemas da comunicação geram alguma dificuldade, de fato, mas creio que seja uma dificuldade fundamental. Então, sim, existe uma dificuldade nessa tarefa de filtrar e transformar a situação em reflexão; e não, porque esse trabalho é essencial”. Sobre o desinteresse dos alunos, Cardeal et al (2012, p.2) diz que essa é a realidade enfrentada por todos os docentes – o que pode ser a maior dificuldade encontrada para dar aula. “A maior parte dos alunos quase sempre não mostra interesse em aprender o que o professor ensina, há diversas outras coisas que chamam mais a atenção deles do que seu professor e o que ele está lecionando”. Os pesquisadores fazem essa observação não só na Sociologia, disciplina foco de seus estudos, mas em todas as outras que acabam desmotivando o professor, afetando sua relação com o aluno. Aliado a essa falta de interesse, há a indisciplina. O professor pode preparar seu conteúdo, mas acaba se decepcionando ao chegar à sala de aula, quando seus alunos não prestam atenção, tornando impossível o desenvolvimento da aula. Como Jornalismo é um curso de bacharelado, os professores foram indagados se tiveram algum estudo focado na Didática ou alguma disciplina pedagógica durante seus estudos de formação. P2 e P4 disseram que não tiveram nenhuma disciplina pedagógica. P3 relatou que também não teve essa formação, no entanto para poder suprir essa necessidade, procurou curso de especialização na área. Mesmo assim, suas expectativas não foram supridas. Revista Pauta Geral-Estudos em Jornalismo, Ponta Grossa, vol.3, n.1 p. 110-121, Jan/Jun, 2016. Desenvolvimento P1 disse ter tido Didática e Metodologia de Ensino em seus cursos de especialização e mestrado, junto aos Fundamentos da Educação, Metodologias de Ensino e Práticas Pedagógicas. Mesmo não tendo essa formação durante sua graduação ou na pós-graduação, todos consideraram importante a formação pedagógica para o professor universitário. De acordo com o P3 “é essencial para o desenvolvimento de aulas dinâmicas, para a compreensão do processo ensino-aprendizado e para sabermos lidar, de uma forma mais individualizada, com os mais diversos perfis de alunos que recebemos anualmente”. Para o P1, “trata-se de docência, seja qual área for. Conhecimento pedagógico ajuda num ensino mais com mais êxito”. P4 comentou que “a formação ajuda no contato com os alunos e na didática das aulas”. Acreditando que o conhecimento de práticas pedagógicas é fundamental, P2 ressalta que “é importante o docente entender que ele precisa ser entendido, ou seja, não adianta ele dominar o conteúdo se ele não consegue transmiti-lo aos seus alunos”. entendido, ou seja, não adianta ele dominar o conteúdo se ele não consegue transmiti-lo aos seus alunos”. Como em todo o processo de aprendizado, no Jornalismo também há a necessidade de professores capacitados para lecionar o conteúdo teórico e prático. Porém, a questão está na formação desse docente, uma vez que o curso dá o diploma de bacharel. Andrade e Linhares (2011) dizem que o profissional com mais experiência e conhecimento em áreas específicas, ainda tem a preferência para atuar na docência, mesmo não tendo passado por algum curso ou especialização educacional. [...] Num primeiro olhar, vale mais a experiência no mercado e a competência técnico-profissional como fundamento do domínio do conteúdo do que propriamente a prática docente, a forma como apresenta este conteúdo ao aluno, o domínio de saberes necessários para a construção de um ambiente propício a aprendizagem e a formação profissional, considerando aqui a concepção da didática como sistematizadora da prática de ensinar/aprender ( ANDRADE; LINHARES, 2011, p.3). Independente da formação bacharel ou licenciatura é necessária que a formação pedagógica seja realizada. Tendo esses princípios, ele pode, com toda a certeza, ter uma linha de trabalho eficaz, porque possui uma metodologia planejada – um dos motivos fundamentais e claros para se procurar se especializar no ‘ser professor’. Desenvolvimento 118 “O docente desenvolve então, habilidades pessoais, tais como capacidade de improvisação, macetes, gestos, atitudes e estilos que possibilitam vencer as barreiras e construir uma maneira própria de ensinar” (CUNHA, BRITO E, CICILLINI, 2006, p. 5). Muitos são os pesquisadores da área de formação de professores que enfatizam a questão da formação pedagógica do professor universitário. Entre eles, Cunha e Lima (2009), afirmam que o professor necessita de uma atenção continuada para a sua formação. Revista Pauta Geral-Estudos em Jornalismo, Ponta Grossa, vol.3, n.1 p. 110-121, Jan/Jun, 2016. ç Levando-se em conta a importância do professor e do seu papel no processo educativo urge a necessidade de investir nele, como profissional e como pessoa no contexto educacional, na contemporaneidade. É preciso dar voz e vez ao professor tendo em vista sua própria reflexão sobre o seu saber e o seu saber fazer, acreditando que da sua fala e da sua história possa surgir grandes e ricas experiências que apontam alternativas de formação, dos seus saberes e de sua prática pedagógica (CUNHA; LIMA, 2009, p.8). Levando-se em conta a importância do professor e do seu papel no processo educativo urge a necessidade de investir nele, como profissional e como pessoa no contexto educacional, na contemporaneidade. É preciso dar voz e vez ao professor tendo em vista sua própria reflexão sobre o seu saber e o seu saber fazer, acreditando que da sua fala e da sua história possa surgir grandes e ricas experiências que apontam alternativas de formação, dos seus saberes e de sua prática pedagógica (CUNHA; LIMA, 2009, p.8). Revista Pauta Geral-Estudos em Jornalismo, Ponta Grossa, vol.3, n.1 p. 110-121, Jan/Jun, 2016. Revista Pauta Geral-Estudos em Jornalismo, Ponta Grossa, vol.3, n.1 p. 110-121, Jan/Jun, 2016. REVISTA PAUTA GERAL ESTUDOS EM JORNALISMO Doi 10.5212/RevistaPautaGeral.v.3.i1.0007 A tarefa de educar, além de incluir a formação técnica e teórica, exige muito dinamismo e entrega à profissão, pois o professor encontra diversidades envolvidas no complexo sistema educacional. Em seu trabalho muitos contextos sociais afetam seu desempenho, como sugere Neuenfeldt (2014, p.2) Dessa forma, para que a atitude reflexiva se estabeleça, o professor precisa desenvolver algumas características, tais como: não se ver como detentor de um saber acabado, permitindo-se aprender constantemente; compreender que os modelos são construídos em conjunto; lidar com as dúvidas e incertezas como parte do processo de aprendizagem, compartilhando os saberes e fazeres da prática pedagógica. 119 Para que essa formação ocorra, concordamos com Isaia (2005, p. 67) que “a formação permanente, para se consolidar, precisa ser entendida como um processo organizado, sistemático e intencional, a partir do grupo de professores, das instituições e das políticas educativas de nível superior”. Importante destacar O Programa Entredocentes que é o Programa Institucional de Formação de Professores da Unicentro e que tem por objetivo, segundo a coordenadora, a formação continuada dos professores da universidade, inicialmente com o foco nos professores em início de carreira e que se encontram em estágio probatório. Revista Pauta Geral-Estudos em Jornalismo, Ponta Grossa, vol.3, n.1 p. 110-121, Jan/Jun, 2016. Desenvolvimento Questionados sobre a possibilidade de participarem de um curso de formação docente oferecido pela sua instituição, assim se manifestaram: P1 respondeu que não participaria e não comentou os motivos. P3 faria, sim e sua sugestão de tema refere-se “às mudanças do perfil de aluno que os cursos de graduação vêm recebendo: mais jovens, conectados, maduros em alguns sentidos e imaturos em outros, como para a tomada de decisão de uma profissão, extremamente dinâmicos e criativos, mas dispersos e, muitas vezes, desinteressados em aprender. Como conquistá-los? Como evitarmos a crescente evasão que nossos cursos de graduação vêm apresentando?”. P2 manifestou que provavelmente sim, mas dependeria muito do programa do curso. “Mesmo que nesses quatro anos tenha conseguido alguma experiência é sempre importante continuar se aprimorando”. P4 também participaria e sua sugestão seria de algo voltado para a didática em sala de aula. REVISTA PAUTA GERAL ESTUDOS EM JORNALISMO Doi 10.5212/RevistaPautaGeral.v.3.i1.0007 Este trabalho tinha como finalidade investigar a importância da formação pedagógica dos professores do curso de Jornalismo da Unicentro – Campus Santa Cruz- Guarapuava- PR. Dos 12 questionários enviados, apenas quatro foram respondidos. Diante dos relatos sobre a prática docente, os entrevistados apontaram que, mesmo tendo ou não um estudo específico acreditam que a formação pedagógica faz todo o diferencial em sala de aula, ou seja, no ‘ser professor’. No entanto, não são todos que, mesmo reconhecendo a importância da formação docente, se interessam em melhorar sua prática – o que é contraditório: não se preocupar por fazer cursos de formação continuada. Com a capacitação do professor universitário, aliada ao conhecimento teórico e prático, é possível transformar positivamente suas aulas, qualificando o ensino e contribuindo para a formação de seus alunos. Por esse motivo, é necessário que a ação docente seja desenvolvida nas universidades com o objetivo de intensificar, melhorar e estimular o aprendizado. Também foi possível perceber que os professores buscam o diálogo como a melhor maneira de se aplicar e receber o conhecimento. A aula expositiva, de certa forma, é o melhor método para que, além do conhecimento científico, possa haver a interação entre professor e aluno. Esse processo, pela forma semelhante abordada pelos entrevistados, é o mais eficaz, até então. Mesmo utilizando essa metodologia, que proporciona a diversidade e inovação, os professores sentem muito que os alunos não correspondem às suas expectativas, deixando de prestar atenção e demonstrando desinteresse pelo material que é apresentado para estudo. Esta situação é preocupante porque a universidade está formando profissionais que atuarão no mercado de trabalho e que, em pouco tempo, prestarão serviços à sociedade. Considerações Finais Revista Pauta Geral-Estudos em Jornalismo, Ponta Grossa, vol.3, n.1 p. 110-121, Jan/Jun, 2016. REVISTA PAUTA GERAL ESTUDOS EM JORNALISMO Doi 10.5212/RevistaPautaGeral.v.3.i1.0007 smo_frente_%C3%A0s_demandas_da_sociedade_contempor%C3%A2nea Acesso em: 25 abr. 2015. CARDEAL, D. M.; MARIANO, M. L. S.’; LUCATELLI, N. G. O trabalho docente: desafios e dificuldades. II Seminário de Socialização do PIBID – UNIFAL – MG. Minas Gerais, 2012. Disponível em: <www.unifal-mg.edu.br/sspibid/sites/default/files/file/S02771.pdf>. Acesso em: 15 ago. 2015. CARDEAL, D. M.; MARIANO, M. L. S.’; LUCATELLI, N. G. O trabalho docente: desafios e dificuldades. II Seminário de Socialização do PIBID – UNIFAL – MG. Minas Gerais, 2012. Disponível em: <www.unifal-mg.edu.br/sspibid/sites/default/files/file/S02771.pdf>. Acesso em: 15 ago. 2015. g CUNHA, A. de F.; LIMA, M. da G. S. Formação continuada de professores bacharéis: saberes e práticas. Disponível em p p www.ufpi.br/.../2_Aldina%20de%20Figueredo%20Cunha%20e%20Mari... Acesso em: 07 out. 2014. CUNHA, A. M. de O.; BRITO, T. T. R.; CICILLINI, G. A. Dormi aluno(a)... acordei professor(a) – interfaces da formação para o exercício do ensino superior. In: SILVA JÚNIOR, João dos Reis; OLIVEIRA, João Ferreira de; MANCEBO,Deise. (orgs). Reforma universitária: dimensões e perspectivas. Campinas, SP: Alínea, 2006. ISAIA S M de A Desafios à docência superior: pressupostos a considerar In: In: CUNHA, A. M. de O.; BRITO, T. T. R.; CICILLINI, G. A. Dormi aluno(a)... acordei professor(a) – interfaces da formação para o exercício do ensino superior. In: SILVA JÚNIOR, João dos Reis; OLIVEIRA, João Ferreira de; MANCEBO,Deise. (orgs). Reforma universitária: dimensões e perspectivas. Campinas, SP: Alínea, 2006. ISAIA, S. M. de A. Desafios à docência superior: pressupostos a considerar. In: In: Docência na educação superior. Instituto Nacional de Estudos e Pesquisas Educacionais Anísio Teixeira, 2006. XX p. – (Coleção Educação Superior em Debate). v. p p p , , ISAIA, S. M. de A. Desafios à docência superior: pressupostos a considerar. In: In: Docência na educação superior. Instituto Nacional de Estudos e Pesquisas Educacionais Anísio Teixeira, 2006. XX p. – (Coleção Educação Superior em Debate). v. 5. LIBÂNEO, J. C. O ensino de graduação na universidade: a aula universitária. Goiânia: UCG, 2003. MARASCHIN, M. L. C. A aula universitária em debate: entre expectativas e perspectivas. Curitiba, 2009. Disponível em: MARASCHIN, M. L. C. A aula universitária em debate: entre expectativas e perspectivas. Curitiba, 2009. Disponível em: p p p <www.pucpr.br/eventos/educere/educere2009/anais/pdf/1876_1014.pdf>. Acesso em: 22 ago. 2015. <www.pucpr.br/eventos/educere/educere2009/anais/pdf/1876_1014.pdf>. Acesso em: 22 ago. 2015. 121 ago. 2015. MARCELO, C. A identidade docente: constantes e desafios. Revista Formação Docente. Belo Horizonte, MG, v.01, n.01, p-109-131, ago/dez 2009. g MARCELO, C. A identidade docente: constantes e desafios. Revista Formação Docente. Belo Horizonte, MG, v.01, n.01, p-109-131, ago/dez 2009. p g MASETTO, M. Referências Bibliográficas Revista Pauta Geral-Estudos em Jornalismo, Ponta Grossa, vol.3, n.1 p. 110-121, Jan/Jun, 2016. ANASTASIOU, L. das G. C. Docência na educação superior. In: Docência na educação superior. Instituto Nacional de Estudos e Pesquisas Educacionais Anísio Teixeira, 2006. XX p. – (Coleção Educação Superior em Debate. v. 5) ANDRADE, P. B.; LINHARES, R. N. A formação do professor de Jornalismo frente às demandas da sociedade contemporânea. Disponível em: http://www.academia.edu/1974434/A_forma%C3%A7%C3%A3o_do_professor_de_Jornali ANASTASIOU, L. das G. C. Docência na educação superior. In: Docência na educação superior. Instituto Nacional de Estudos e Pesquisas Educacionais Anísio Teixeira, 2006. XX p. – (Coleção Educação Superior em Debate. v. 5) ANDRADE, P. B.; LINHARES, R. N. A formação do professor de Jornalismo frente às demandas da sociedade contemporânea. Disponível em: http://www.academia.edu/1974434/A_forma%C3%A7%C3%A3o_do_professor_de_Jornali ANASTASIOU, L. das G. C. Docência na educação superior. In: Docência na educação superior. Instituto Nacional de Estudos e Pesquisas Educacionais Anísio Teixeira, 2006. XX p. – (Coleção Educação Superior em Debate. v. 5) ANDRADE, P. B.; LINHARES, R. N. A formação do professor de Jornalismo frente às demandas da sociedade contemporânea. Disponível em: http://www.academia.edu/1974434/A_forma%C3%A7%C3%A3o_do_professor_de_Jornali Revista Pauta Geral-Estudos em Jornalismo, Ponta Grossa, vol.3, n.1 p. 110-121, Jan/Jun, 2016. smo_frente_%C3%A0s_demandas_da_sociedade_contempor%C3%A2nea Acesso em: 25 abr. 2015. T. Docência universitária: repensando a aula. Ensinar e aprender no ensino superior: por uma epistemologia da curiosidade na formação universitária, v. 2, p. 79-108, 2005. p g MASETTO, M. T. Docência universitária: repensando a aula. Ensinar e aprender no ensino superior: por uma epistemologia da curiosidade na formação universitária, v. 2, p. 79-108, 2005. NETO, M. de S. F.. A aula. Geografares, Vitória, n. 2, jun. 2001. Disponível em: <periodicos.ufes.br/geografares/article/view/1146/859>. Acesso em: 24 ago. 2015. NETO, M. de S. F.. A aula. Geografares, Vitória, n. 2, jun. 2001. Disponível em: <periodicos.ufes.br/geografares/article/view/1146/859>. Acesso em: 24 ago. 2015. NETO, M. de S. F.. A aula. Geografares, Vitória, n. 2, jun. 2001. Disponível em: <periodicos.ufes.br/geografares/article/view/1146/859>. Acesso em: 24 ago. 2015. NEUENFELDT, M. C. Formação de professores para o ensino superior: reflexões sobre a docência orientada. Santa Maria, [200-]. Disponível em: <http://www.ufsm.br/gpforma/2senafe/PDF/019e5.pdf>. Acesso em: 12 fev. 2010. NÓVOA, A., Diz-me como ensinas, dir-te-ei quem és e vice-versa. In: FAZENDA, Ivani Catarina Arantes. (Org.). A pesquisa em educação e as transformações do conhecimento. 6. ed. Campinas, SP: Papirus, p. 29-41, 2004. PIMENTA, S. G.; ANASTASIOU, L. das G. C. Docência no ensino superior. São Paulo: C 2002 <periodicos.ufes.br/geografares/article/view/1146/859>. Acesso em: 24 ago. 2015. NEUENFELDT, M. C. Formação de professores para o ensino superior: reflexões sobre a docência orientada. Santa Maria, [200-]. Disponível em: <http://www.ufsm.br/gpforma/2senafe/PDF/019e5.pdf>. Acesso em: 12 fev. 2010. Ó p g g g NEUENFELDT, M. C. Formação de professores para o ensino superior: reflexões sobre a docência orientada. Santa Maria, [200-]. Disponível em: <http://www.ufsm.br/gpforma/2senafe/PDF/019e5.pdf>. Acesso em: 12 fev. 2010. Ó <http://www.ufsm.br/gpforma/2senafe/PDF/019e5.pdf>. Acesso em: 12 fev. 2010. NÓVOA, A., Diz-me como ensinas, dir-te-ei quem és e vice-versa. In: FAZENDA, Ivani Catarina Arantes. (Org.). A pesquisa em educação e as transformações do conhecimento. 6. ed. Campinas, SP: Papirus, p. 29-41, 2004. PIMENTA, S. G.; ANASTASIOU, L. das G. C. Docência no ensino superior. São Paulo: p gp p NÓVOA, A., Diz-me como ensinas, dir-te-ei quem és e vice-versa. In: FAZENDA, Ivani Catarina Arantes. (Org.). A pesquisa em educação e as transformações do conhecimento. 6. ed. Campinas, SP: Papirus, p. 29-41, 2004. p gp p NÓVOA, A., Diz-me como ensinas, dir-te-ei quem és e vice-versa. In: FAZENDA, Ivani Catarina Arantes. (Org.). A pesquisa em educação e as transformações do conhecimento. 6. ed. Campinas, SP: Papirus, p. 29-41, 2004. p p p PIMENTA, S. G.; ANASTASIOU, L. das G. C. Docência no ensino superior. São Paulo: Cortez, 2002. smo_frente_%C3%A0s_demandas_da_sociedade_contempor%C3%A2nea Acesso em: 25 abr. 2015. Enviado em 22/02/2016 Publicado em 27/04/2016 Enviado em 22/02/2016 Publicado em 27/04/2016 Revista Pauta Geral-Estudos em Jornalismo, Ponta Grossa, vol.3, n.1 p. 110-121, Jan/Jun, 2016. Revista Pauta Geral-Estudos em Jornalismo, Ponta Grossa, vol.3, n.1 p. 110-121, Jan/Jun, 2016.
https://openalex.org/W4287671619	https://zenodo.org/records/4016877/files/133.252545%20BBDSSS-converted.pdf	English	null	DEMOGRAPHIC ANALYSIS OF MAXILLOFACIAL FRACTURES IN MAYO HOSPITAL LAHORE	Zenodo (CERN European Organization for Nuclear Research)	2,020	cc-by	2,475	Corresponding author: Dr Sabina Alim, Article Received: June 2020 Accepted: July 2020 Article Received: June 2020 Accepted: July 2020 Abstract: Aim: A descriptive study was undertaken to analyze the demographic distribution of maxillofacial fractures in 320 patients reported to the Department of Oral and Maxillofacial Surgery, Mayo Hospital Lahore. Duration: For one-year duration from May 2019 to May 2020. Aim: A descriptive study was undertaken to analyze the demographic distribution of maxillofacial fractures in 320 patients reported to the Department of Oral and Maxillofacial Surgery, Mayo Hospital Lahore. atients reported to the Department of Oral and Maxillofacial Surgery, Mayo Hospital Lahore. tion: For one-year duration from May 2019 to May 2020. f Methods: Patient records and radiographs were taken. Data on age, gender, and cause of fractures were reviewed. The age range was 2-76 years (mean 25 years) and the peak incidence was in the age group 21-30 years. The male-to-female ratio was 5.4: 1. y f Results and Conclusion: The study found that 64.7% (n = 206) were due to road traffic accidents (RTAs) followed by a fall (n = 60; 18.8%), seizure (n = 26; 8.1%), sports (n = 17; 5.3%), firearms injuries [FM (n = 3; 0.9%)], occupational injuries (n = 2; 0.6%), while 5 cases (1.65%) were related to other causes such as bomb blasts, animal injuries etc. This study could provide a guide for creating prevention and treatment programs. Keywords: injuries, facial fractures, etiology, road accidents. Sabina Alim et al Sabina Alim et al Sabina Alim et al IAJPS 2020, 07 (08), 766-769 ISSN 2349-7750 Research Article Available online at: http://www.iajps.com Dr Sabina Alim, Senior Lecturer, Rawal Institute of Health Sciences, Islamabad Please cite this article in press Sabina Alim et al, Demographic Analysis Of Maxillofacial Fractures In Mayo Hospital Lahore., Indo Am. J. P. Sci, 2020; 07(08). Page 766 w w w . i a j p s . c o m Sabina Alim et al ISSN 2349-7750 ISSN 2349-7750 IAJPS 2020, 07 (08), 766-769 INTRODUCTION: trauma victims over the last 100 years, such as external skeleton stiffening, open reduction, craniofacial exposure, internal wire fixation, primary bone grafting, mini-plates and orbital reconstruction. Consequently, such injuries today adversely affect quality of life less often than before, due to the progress made by countless people from a variety of disciplinary backgrounds. Taken together, these advances have had tremendous benefits in improving the primary and secondary correction of posttraumatic maxillofacial deformities. The face, as the most exposed part of the body, is particularly vulnerable to injury. The main causes around the world are road accidents, falls, assaults, sports, injuries to the hands of firefighters and work injuries. Of course, one would expect the etiology to influence the severity and type of injuries suffered. " Data collected in the 1960s and early 1970s show that 20% -60% of all people injured in R.T.A have some degree of maxillofacial injuries. A high incidence of R.T.As maxillofacial injuries is reported in developing countries, while the incidence of personal violence is higher in developed countries. The introduction of mandatory seat belts and drunk driving regulations significantly reduced both the number and severity of road traffic injuries by 25%, and more serious facial injuries were reduced by two-thirds. It turns out that falls were the second most common cause after being assaulted, although it is known that many patients who committed the assault reported that their injuries were due to falls. The constant improvement in the quality of life of the individual and the growing interest in sports activities resulted in the increasing use of sports in free time at the amateur level. As a result, the number of sports injuries has steadily increased since the late 1980s. Maxillofacial injuries from fire arm injuries have been increasing over the past decades, presenting one of the greatest challenges for maxillofacial surgeons. More industrial trauma is reported in industrialized cities. Age and gender are mentioned as important factors influencing the occurrence of maxillofacial injuries. Dr Sabina Alim, “The highest incidence is observed in the 21-30 age group. The lowest incidence is observed in the age group over 60 and under 5. patients are male with a male-to-female ratio of approximately 3: 1.1 'Considerable progress has been made in the care of maxillofacial MATERIALS AND METHODS: MATERIALS AND METHODS: The information obtained was based on an analysis of maxillofacial injuries recorded at Maxillofacial Surgery Department, Mayo Hospital, Lahore. The study was conducted from May 2019 to May 2020. This randomized study was conducted in 320 consecutive maxillofacial trauma patients. The study included all patients of all ages and both sexes who suffered from maxillofacial trauma in the ward. A detailed patient history was collected and a detailed clinical examination was performed. Basic and detailed examinations, such as a radiograph, were performed to confirm the bone injury. Maxillofacial fractures were assessed on the basis of etiology, age and gender. Maxillofacial injuries from fire arm injuries have been increasing over the past decades, presenting one of the greatest challenges for maxillofacial surgeons. More industrial trauma is reported in industrialized cities. Age and gender are mentioned as important factors influencing the occurrence of maxillofacial injuries. “The highest incidence is observed in the 21-30 age group. The lowest incidence is observed in the age group over 60 and under 5. patients are male with a male-to-female ratio of approximately 3: 1.1 'Considerable progress has been made in the care of maxillofacial DISCUSSION: reducing facial injuries. In Pakistan, the law mandating the use of seat belts has not been properly implemented. In interviews with victims of facial injuries due to RTA, it was observed that the carelessness of many drivers, failure to give priority, excessive speed on highways in the competition of addicted drivers, contributed to the increase in the number of jaw fractures caused by RTA. Hill et al. And Voss reported seizure as the leading cause of maxillofacial fractures in England and Norway, respectively. The 8.1% incidence in this study is due to a strong control of contrast with the 55% reported in Scotland, which may be related to differences in social alcohol consumption. Due to their religious background, Pakistanis do not drink alcohol. Epidemiological studies of the causes and incidence of maxillofacial fractures vary by geographic region, socioeconomic status, culture, religion and age. The prevalence of maxillofacial injuries in the 21-30 age group is consistent with the results of previous published studies. But it contrasts with the Karyouti report, which identified the 0-5 year age group as the group with the highest incidence. A possible explanation for the high frequency in the 21-30 age group is that people in this age group engage in hazardous exercise and sports, drive motor vehicles carelessly and are most likely violent. The lowest incidence was observed in the age group over 60 years (1.3%) compared to the Kapoor and Srivastava study of 0-5 years. A possible reason would be limited outdoor activities in old age. Previous studies have found a lower incidence of maxillofacial fractures in women with a male to female ratio ranging from 5.2: 1 to 5.4: 1. In this study, there was 5.4: 1.3 left. The higher age of a person may be due to the fact that men are mainly engaged in outdoor activities and are also at risk of violent interactions. Men are more than women. In most previous epidemiological studies, road traffic accidents were the most common cause of jaw fractures, and this study supports these findings. In England, it has been reported that the introduction of compulsory seat belts has a significant impact in RESULTS: 320 patients with various types of maxillofacial fractures were treated. The most common causes of maxillofacial injuries were RTA (n = 207; 64.7%), followed by accidental falls (n = 60; 18.8) and combat injuries (n = 26; 8.1%); sports injuries in 17 cases (5.3%). FAI (n = 3: 0.9%) Industrial (n = 2: 0.6%). The causes of the injuries are summarized in Table 1. a j p s . c o m Page 767 TABLE 1: DISTRIBUTION OF MAXILLOFACIAL FRACTURES ACCORDING TO ETIOLOGY Number of cases Percent RTA 207 64.7 Fall 60 18.8 Assault 26 8.1 Sports 17 5.3 FAI 3 0.9 Industrial 2 0.6 Other Causes 5 1.6 Total 320 100.0 The remaining fractures resulted from various causes (n = 5: 1.6%), such as bomb explosion, animal injuries, etc. 236 The patient's age at the time of injury ranged from 2 years. -76 years, with an average age of 25 + 1-13 years. In virtually all age groups, more men than women were involved, and the overall ratio was 5.4: 1 (Table 2). a j p s . c o m Page 767 TABLE 1: DISTRIBUTION OF MAXILLOFACIAL FRACTURES ACCORDING TO ETIOLOGY Number of cases Percent RTA 207 64.7 Fall 60 18.8 Assault 26 8.1 Sports 17 5.3 FAI 3 0.9 Industrial 2 0.6 Other Causes 5 1.6 Total 320 100.0 The remaining fractures resulted from various causes (n = 5: 1.6%), such as bomb explosion, animal injuries, etc. 236 The patient's age at the time of injury ranged from 2 years. -76 years, with an average age of 25 + 1-13 years. In virtually all age groups, more men than women were involved, and the overall ratio was 5.4: 1 (Table 2). TABLE 1: DISTRIBUTION OF MAXILLOFACIAL FRACTURES ACCORDING TO ETI The remaining fractures resulted from various causes (n = 5: 1.6%), such as bomb explosion, animal injuries, etc. 236 The patient's age at the time of injury ranged from 2 years. -76 years, with an average age of 25 + 1-13 years. In virtually all age groups, more men than women were involved, and the overall ratio was 5.4: 1 (Table 2). w w w . i a j p s . RESULTS: c o m Sabina Alim et al ISSN 2349-7750 IAJPS 2020, 07 (08), 766-769 TABLE 2: GENDER DISTRIBUTION OF MAXILLOFACIAL FRACTURES Number of cases Percent Male 270 84.4 Female 50 15.6 Total 320 100.0 TABLE 2: GENDER DISTRIBUTION OF MAXILLOFACIAL FRACTURES In most cases, the patient was 21-30 years of age (n = 105; 32.8%). Only 12.8% of patients were below 11 years of age, and 1.3% were above 60 years of age (Table 3). In most cases, the patient was 21-30 years of age (n = 105; 32.8%). Only 12.8% of patients were below 11 years of age, and 1.3% were above 60 years of age (Table 3). TABLE 3: DISTRIBUTION OF MAXILLOFACIAL FRACTURES ACCORDING TO AGE Age (years) Number of cases Percent 1-10 41 12.8 11-20 92 28.8 21-30 105 32.8 3140 50 15.6 41-50 20 6.3 51-60 8 2.5 Over 60 4 1.3 Total 320 100.0 TABLE 3: DISTRIBUTION OF MAXILLOFACIAL FRACTURES ACCORDING TO AGE REFERENCES: 1. Khan, Ahmad, Sertaj Khan, Mohammad Tariq Khan, Khadija Syed, Syed Asad Shah, Ruqayyah Younis, Muhammad Sohail, and Umar Khitab. "Pattern of maxillary fractures in patients presented to tertiary care hospital-a study." Pakistan Oral & Dental Journal 39, no. 3 (2019): 247-250. 11. Partiali, Benjamin, Sandra Oska, Antonio Barbat, Joseph Sneij, and Adam Folbe. "Injuries to the Head and Face from Skateboarding: a 10 Year Analysis from NEISS Hospitals." Journal of Oral and Maxillofacial Surgery (2020). 2. Alam, Beenish Fatima, and MFDS RCSED. "Patterns of Facial Fractures Associated with Socio-demographic and Causative Factors: A Multi-Center Analysis from Karachi." JPDA 28, no. 03 (2019): 104. 12. 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Tetteh, Sophia, Richard J. Bibb, and Simon J. Martin. "Maxillofacial prostheses challenges in resource constrained regions." Disability and rehabilitation 41, no. 3 (2019): 348-356. 5. Mohamed, Abdulrahman, Jeffrey Mulcaire, and Anthony James P. Clover. "Head and neck injury in major trauma in Ireland: a multicentre retrospective analysis of patterns and surgical workload." Irish Journal of Medical Science (1971-) (2020): 1-7. 15. Chung, Shin Hye, Kyung A. Chun, Hae-Young Kim, Young-Sun Kim, and Juhea Chang. "Periapical Healing in Single-visit Endodontics under General Anesthesia in Special Needs Patients." Journal of endodontics 45, no. 2 (2019): 116-122. 6. Li, Lifeng, Hongrui Zang, Demin Han, Bentao Yang, Shaun C. Desai, and Nyall R. London. "Nasal Bone Fractures: Analysis of 1193 Cases with an Emphasis on Coincident Adjacent Fractures." Facial Plastic Surgery & Aesthetic Medicine (2020). 7. Cohn, Jason E., Jordan J. Licata, Sammy Othman, Tom Shokri, and Seth Zwillenberg. CONCLUSION: The conducted studies showed that the highest frequency of mandible fractures occurred in the age group of 21-30 years, and the most common cause was R.T.As (64.7%). The male to female ratio was 5.4: 1. The most common site was fracture of the mandibular body (30.3%), followed by the condylar region (24.2%). The results show that there is concern about the high percentage of RTA jaw injuries as few people use seat belts, an awareness campaign to educate the public, especially drivers, about the importance of restraints and protective measures in motor Page 768 w w w . i a j p s . c o m Sabina Alim et al IAJPS 2020, 07 (08), 766-769 ISSN 2349-7750 pidemic of e-scooters." Trauma surgery & acute care open 4, no. 1 (2019): e000337. vehicles should be implemented. These arrangements should alert authorities to the need to enforce existing traffic laws to control speeding on motorways and careless driving, smooth roads and the use of seat belts must be mandatory. 10. Park, Han-Kyul, Jin-Young Park, Na-Rae Choi, Uk-Kyu Kim, and Dae-Seok Hwang. "Sports-related oral and maxillofacial injuries: a 5-year retrospective study, Pusan National University Dental Hospital." Journal of Oral and Maxillofacial Surgery (2020). REFERENCES: "Comparison of Maxillofacial Trauma Patterns in the Urban Versus Suburban Environment: A Pilot Study." Craniomaxillofacial Trauma & Reconstruction (2020): 1943387520910338. 8. Söz, Gülbahar, and Zeynep Karakaya. "The evaluation of geriatric patients who presented with trauma to the emergency department." Archives of Medical Science: AMS 15, no. 5 (2019): 1261. 9. Kobayashi, Leslie M., Elliot Williams, Carlos V. Brown, Brent J. Emigh, Vishal Bansal, Jayraan Badiee, Kyle D. Checchi, Edward M. Castillo, and Jay Doucet. "The e-merging e- Page 769 w w w . i a j p s . c o m
https://openalex.org/W2215921584	https://www.nature.com/articles/srep16808.pdf	English	null	Mesoporous Silica Gel–Based Mixed Matrix Membranes for Improving Mass Transfer in Forward Osmosis: Effect of Pore Size of Filler	Scientific reports	2,015	cc-by	8,031	www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Mesoporous Silica Gel–Based Mixed Matrix Membranes for Improving Mass Transfer in Forward Osmosis: Effect of Pore Size of Filler received: 10 December 2014 accepted: 10 March 2015 Published: 23 November 2015 received: 10 December 2014 accepted: 10 March 2015 Published: 23 November 2015 Jian-Yuan Lee1,2, Yining Wang2, Chuyang Y. Tang1,2,3 & Fengwei Huo4 The efficiency of forward osmosis (FO) process is generally limited by the internal concentration polarization (ICP) of solutes inside its porous substrate. In this study, mesoporous silica gel (SG) with nominal pore size ranging from 4–30 nm was used as fillers to prepare SG-based mixed matrix substrates. The resulting mixed matrix membranes had significantly reduced structural parameter and enhanced membrane water permeability as a result of the improved surface porosity of the substrates. An optimal filler pore size of ~9 nm was observed. This is in direct contrast to the case of thin film nanocomposite membranes, where microporous nanoparticle fillers are loaded to the membrane rejection layer and are designed in such a way that these fillers are able to retain solutes while allowing water to permeate through them. In the current study, the mesoporous fillers are designed as channels to both water and solute molecules. FO performance was enhanced at increasing filler pore size up to 9 nm due to the lower hydraulic resistance of the fillers. Nevertheless, further increasing filler pore size to 30 nm was accompanied with reduced FO efficiency, which can be attributed to the intrusion of polymer dope into the filler pores. Forward osmosis (FO) is one of the promising membrane filtration technologies for potable water pro- duction1,2, wastewater treatment and membrane bioreactor (MBR)3–11, seawater/brackish water desal- ination12–14, drug delivery15, food/juice processing16,17, and valuable materials enrichment17–20. In FO processes, an osmotic pressure difference is created between a low concentration solution and a high concentration solution to draw water through a semi-permeable FO membrane. Osmotically-driven FO membrane processes can potentially reduce the energy consumption of current technologies1,2 com- pared to high pressure-driven reverse osmosis (RO) and nanofiltration (NF) membrane processes. Based on previous studies, several additional advantages of FO processes has been reported, including more reversible fouling due to the no requirement of pressure, high rejection of various solutes and contami- nants, and the lack of high temperature or pressure that can potential destroy some sensitive feed stock in certain applications. 1Nanyang Environment & Water Research Institute, Interdisciplinary Graduate School, Nanyang Technological University, Singapore, 639798. 2Singapore Membrane Technology Centre, Nanyang Environment & Water Research Institute, Nanyang Technological University, Singapore, 637141. Mesoporous Silica Gel–Based Mixed Matrix Membranes for Improving Mass Transfer in Forward Osmosis: Effect of Pore Size of Filler Membrane Membrane Thickness (μm) Contact Angle (ο) Membrane Bulk Porosity (%) Substrate Water Permeabilityb (L/m2 h bar) Membrane Water Permeabilityb (L/m2 h bar) Salt Rejectionc (%) Structural Parameter (mm) Control 42 ± 2 54 ± 4 80 ± 2 171 ± 18 5.4 ± 0.6 83 ± 3 0.36 ± 0.03 F40 43 ± 1 47 ± 3 78 ± 2 218 ± 6 9.2 ± 1.2 80 ± 3 0.22 ± 0.01 F60 46 ± 1 39 ± 2 77 ± 4 244 ± 23 9.4 ± 0.7 74 ± 6 0.19 ± 0.02 F90 43 ± 1 33 ± 3 78 ± 3 307 ± 17 9.9 ± 0.5 68 ± 2 0.17 ± 0.01 F300 49 ± 1 42 ± 4 79 ± 2 273 ± 12 8.9 ± 0.6 76 ± 4 0.24 ± 0.01 Table 2. Membrane thickness, contact angle, membrane bulk porosity, substrate water permeability, membrane water permeability salt rejection, and structural parameter of SG-based MMMs with different pore size of silica gel particlesa. aThe experiment errors are reported as the standard deviation of at least 3 repeated measurements. bMeasured at an applied pressure of 5 bar with ultrapure water as feed water in the pressurized cross flow filtration testing mode. cMeasured at an applied pressure of 5 bar with 5 mM MgCl2 as feed water in the pressurized cross flow filtration testing mode. Table 2. Membrane thickness, contact angle, membrane bulk porosity, substrate water permeability, membrane water permeability salt rejection, and structural parameter of SG-based MMMs with different pore size of silica gel particlesa. aThe experiment errors are reported as the standard deviation of at least 3 repeated measurements. bMeasured at an applied pressure of 5 bar with ultrapure water as feed water in the pressurized cross flow filtration testing mode. cMeasured at an applied pressure of 5 bar with 5 mM MgCl2 as feed water in the pressurized cross flow filtration testing mode. Several reviews on FO pointed to the critical need for fabricating high performance FO mem- branes1,2,21. One commonly adopted approach to enhance FO performance is to create rejection lay- ers with higher water permeability and/or lower salt permeability. Existing attempts include interfacial polymerization (IP) optimization22–25, membrane post-modification by chlorination26, and non-IP based approaches, such as layer by layer (LbL)27–29 and integral asymmetric FO membranes30–32. Another approach is based on nanoparticle incorporation33–36. Mesoporous Silica Gel–Based Mixed Matrix Membranes for Improving Mass Transfer in Forward Osmosis: Effect of Pore Size of Filler 3Department of Civil Engineering, the University of Hong Kong, Pokfulam, Hong Kong. 4Key Laboratory of Flexible Electronics (KLOFE) & Institute of Advanced Materials (IAM), Jiangsu National Synergistic Innovation Center for Advanced Materials (SICAM), Institute of Advanced Materials, Nanjing Tech University (NanjingTech), Nanjing, 211816, P. R. China. Correspondence and requests for materials should be addressed to C.Y.T. (email: tangc@hku.hk) or F.H. (email: iamfwhuo@njtech.edu.cn) Scientific Reports \| 5:16808 \| DOI: 10.1038/srep16808 1 www.nature.com/scientificreports/ SGa Nominal Pore Size (Å) Measured Pore Size (Å) BET Surface Area (m2/g) SG F40 40 58 433 SG F60 60 59 424 SG F90 90 106 318 SG F300 300 173 68 Table 1. The properties of different pore size and BET surface area of silica gel filler. aAll the SG particles have comparable particle sizes, with each ranging from 40–63 μ m. SGa Nominal Pore Size (Å) Measured Pore Size (Å) BET Surface Area (m2/g) SG F40 40 58 433 SG F60 60 59 424 SG F90 90 106 318 SG F300 300 173 68 Table 1. The properties of different pore size and BET surface area of silica gel filler. aAll the SG particles have comparable particle sizes, with each ranging from 40–63 μ m. SGa Nominal Pore Size (Å) Measured Pore Size (Å) BET Surface Area (m2/g) SG F40 40 58 433 SG F60 60 59 424 SG F90 90 106 318 SG F300 300 173 68 Table 1. The properties of different pore size and BET surface area of silica gel filler. aAll the SG particles have comparable particle sizes, with each ranging from 40–63 μ m. Table 1. The properties of different pore size and BET surface area of silica gel filler. aAll the SG particles have comparable particle sizes, with each ranging from 40–63 μ m. Mesoporous Silica Gel–Based Mixed Matrix Membranes for Improving Mass Transfer in Forward Osmosis: Effect of Pore Size of Filler Inspired by the great success of nanocomposite membranes for reverse osmosis applications37,38, Ma et al. developed microporous zeolite loaded thin film nanocomposite (TFN) FO membranes and demonstrated their significantly higher water permea- bility and thus FO water flux as a result of different zeolite loading39. In a typical design, the pore size of the nanoparticles is chosen to be smaller than the hydrated radius of target solutes to maintain or even enhance solute rejection37,38. j In parallel to the development of TFN FO membranes, recent studies have shown that the loading of porous fillers to FO substrates are highly effective in enhancing FO performance40–42. These porous par- ticles in these mixed matrix substrate (MMS) are shown to provide preferential water paths, which effec- tively enhances the mass transfer in the membrane support layer and mitigates internal concentration polarization (ICP)40. The pore size of the fillers plays a critical role on their effectiveness in enhancing FO performance. Unlike nanoparticle fillers for TFN rejection layers where smaller particle pore size is favored due to solute rejection considerations37,38, fillers for MMS with large pores are presumably more effective “water channels” due to their much reduced hydraulic resistance. Unfortunately, there is lack of guiding principle for the selection of pore size of filler in the FO substrate.hf g g p p pi Therefore, the objective of current study is to systematically study the effects of pore size of substrate fillers on the physiochemical properties, intrinsic separation properties, and FO performance of mixed matrix FO membranes. More specifically, silica gel (SG) particles with a variety of pore size were incor- porated into polyacrylonitrile (PAN) to prepare the SG-based mixed matrix membranes (MMMs) due to their hydrophilic nature and a variety of pore size are available. To the best knowledge of the authors, this is the first study to systematically investigate the effects of filler pore size on SG-based mixed matrix FO membranes. Results Th The nitrogen adsorption and desorption isotherms of different pore size of silica gel is shown in Fig. S1 of the Supporting Information. The measured pore size and BET area are presented in Table 1. For the different pore size of silica gel, the nominal pore size ranged from SG F40 (40 Å) to SG F300 (300 Å). The measured pore size agreed reasonably well with the nominal pore size. Larger pore size of silica gel was Scientific Reports \| 5:16808 \| DOI: 10.1038/srep16808 2 www.nature.com/scientificreports/ Figure 1. The effect of different pore size of SG-based mixed matrix FO membranes on (a) substrate water permeability and A value; (b) substrate water permeability and S value; and (c) the plot of A value against the 1/S value. Testing conditions: DI water as the feed solution; applied pressure: 10 bars, error bar was based on the standard deviation of 3 replicate measurements. Figure 1. The effect of different pore size of SG-based mixed matrix FO membranes on (a) substrate water permeability and A value; (b) substrate water permeability and S value; and (c) the plot of A value against the 1/S value. Testing conditions: DI water as the feed solution; applied pressure: 10 bars, error bar was based on the standard deviation of 3 replicate measurements. generally associated with smaller BET surface area. For example, SG F300 had the largest nominal pore size (300 Å) but also the smallest BET surface area (68 m2/g).hf The properties of SG-based mixed matrix FO membranes with different pore size are shown in Table 2. The membrane thickness was not significantly influenced by the pore size of SG particles. The average thickness of SG-based mixed matrix membrane (42–49 μ m) was similar to the thickness of com- mercial available HTI cellulose acetate FO membranes (50 μ m) but was much thinner than HTI TFC FO membranes (100 μ m) and traditional TFC RO membranes (150 μ m)25,43. In all cases, the incorporation of SG particles had positive effect in improving the hydrophilicity of the substrate. The contact angle of SG-loaded substrates ranged from 33–47°, as compared to 54° for the control without SG addition. The SG-based mixed matrix FO membranes were also more hydrophilic compared to the commercial HTI CA FO membranes (contact angle ~62°). Results Th Based on the observation of the current study, an optimal filler pore size of ~9 nm is recommended. It is worthwhile to note that this optimal value may depend on the properties of the polymer dope and the filler particles. For example, classical theory predicts that the liquid (or in this case the polymer dope) entry pressure into a pore is given by 4γcosθ/dfiller, where γ is surface tension of the polymer dope and θ is the contact angle. Thus, a more viscous polymer dope with higher surface tension may lead to a higher optimal dfiller value. Further systematic studies are required on understanding the effect of polymer dope properties on the optimal filler pore size.i i Both substrate PWP and S values are presented in Fig. 1b as a function of filler pore size. Clearly, PWP and S value were strongly correlated. The PWP curve and S value curve were nearly mirror images to each other. As discussed earlier in the section, the substrate PWP is proportional to the ratio of εs/τ. Meanwhile, based on classical ICP theory54,55, S value is proportional to tortuosity, and it is inversely pro- portional to the substrate porosity. Recent research further reveals that the S value is generally dominated by the skin layer contribution (as a result of much lower surface porosity compared to the average bulk porosity)32,40,56. Thus, it is reasonable to expect a strong dependence of S value on τ/εs, which explains the inverse relationship between substrate PWP and S value. Figure 1c plots the water permeability (A value) of SG-based mixed matrix FO membranes as a function of 1/S value. A value and S value are two key parameters dictating the FO water flux. A linear relationship between the A value and 1/S was observed. The current study clearly demonstrated that the feasibility of using mixed matrix substrates to simultaneously increase A value and reduce S value by enhancing the substrate mass transfer.ffl y y g Figure 2 demonstrates the effect of different pore size of SG on FO water flux and the Js/Jv ratio of SG-based mixed matrix FO membranes is shown in Fig. S3 of the Supporting Information. The FO water flux was tested for both AL-DS and AL-FS orientations using 0.5 M MgCl2 as DS and DI water as FS. Results Th p g p y It is also interesting to note the substantially reduced structural parameter (S) for the SG-based mixed matrix FO substrates (Table 2). Structural parameter is a critical FO membrane property, with a lower S value preferred for controlling ICP in the FO substrate. In the current study, the S value was reduced from 0.36 mm (control) to 0.17–0.24 mm (MMMs) as a result of SG loading. In all cases, the SG-based mixed matrix FO substrates had much lower S values compared to the commercial HTI FO membranes (0.47 mm for CTA membrane and 0.62 mm for TFC membrane)25,43. While their S value was similar to that of the-state-of-the-art FO membranes supported by highly porous nanofiber substrates48,49, MMMs generally offer much better mechanical stability45,50 that can potentially allow these membranes to be used for more demanding applications such as pressure retarded osmosis51,52 and pressure assisted osmosis53.hfi Å g pp p p The effect of filler pore size (dfiller) on S value is shown in Fig. 1b. Increasing dfiller up to 90 Å (F90) helped to reduce the S value, which is consistent with the hypothesis that the mesoporous silica gels with larger dfiller are more effective in enhancing mass transfer in the substrate since they are more per- meably to water and solutes. Nevertheless, the S value increased from 0.17 ± 0.01 mm to 0.24 ± 0.01 mm when dfiller was further increased from 90 Å (F90) to 300 Å (F300). One plausible explanation is that, at very large filler pore size, the polymer molecules can enter into the filler pores to result in partial pore blockage and therefore reduced effectiveness of the fillers. Figure S2 shows the nitrogen adsorption and desorption isotherms of clean F300 silica gels and that of F300 fillers separated from a polymer dope solution by centrifugation. The results clearly indicate a significant reduction of both the filler pore size from 17 nm to 15 nm and BET surface area from 68 m2/g to 54 m2/g, which providing a direct support to the filler pore blockage hypothesis. The current study seems to suggest that the optimal dfiller value is dependent on the competing effects of the reduced resistance to water/solute transport for larger pore size and the simultaneously increased risk of blockage of filler pores by the polymer chains. Results Th While their S value was similar to that of the-state-of-the-art FO membranes supported by highly porous nanofiber substrates48,49, MMMs generally offer much better mechanical stability45,50 that can potentially allow these membranes to be used for more demanding applications such as pressure retarded osmosis51,52 and pressure assisted osmosis53. The effect of filler pore size (dfiller) on S value is shown in Fig. 1b. Increasing dfiller up to 90 Å (F90) helped to reduce the S value, which is consistent with the hypothesis that the mesoporous silica gels with larger dfiller are more effective in enhancing mass transfer in the substrate since they are more per- meably to water and solutes. Nevertheless, the S value increased from 0.17 ± 0.01 mm to 0.24 ± 0.01 mm when dfiller was further increased from 90 Å (F90) to 300 Å (F300). One plausible explanation is that, at very large filler pore size, the polymer molecules can enter into the filler pores to result in partial pore blockage and therefore reduced effectiveness of the fillers. Figure S2 shows the nitrogen adsorption and desorption isotherms of clean F300 silica gels and that of F300 fillers separated from a polymer dope solution by centrifugation. The results clearly indicate a significant reduction of both the filler pore size from 17 nm to 15 nm and BET surface area from 68 m2/g to 54 m2/g, which providing a direct support to the filler pore blockage hypothesis. The current study seems to suggest that the optimal dfiller value is dependent on the competing effects of the reduced resistance to water/solute transport for larger pore size and the simultaneously increased risk of blockage of filler pores by the polymer chains. Based on the observation of the current study, an optimal filler pore size of ~9 nm is recommended. It is worthwhile to note that this optimal value may depend on the properties of the polymer dope and the filler particles. For example, classical theory predicts that the liquid (or in this case the polymer dope) entry pressure into a pore is given by 4γcosθ/dfiller, where γ is surface tension of the polymer dope and θ is the contact angle. Thus, a more viscous polymer dope with higher surface tension may lead to a higher optimal dfiller value. Further systematic studies are required on understanding the effect of polymer dope properties on the optimal filler pore size. Results Th The beneficial effect of nanoparticle loading on hydrophilicity enhancement is consistent with prior studies39,44, and it is expected the increased substrate hydrophilicity could enhance FO water flux.f l Adding SG particles to the substrate had no obvious effect on the membrane bulk porosity (Table 2). Nevertheless, the substrate pure water permeability (PWP) was substantially increased for the SG-loaded substrates. For the substrate F90, its PWP was nearly double of that of the control. Since both substrates had similar thickness and bulk porosity, the enhanced PWP was most likely attributed to increased surface porosity (εs) and/or reduced pore tortuosity (τ). According to the classical Hagen-Poiseuille equation, the substrate PWP is proportional to the ratio of εs/τ. The water permeability of the resulting LbL membranes (A value) followed a similar trend to that of the substrate PWP (Fig. 1a), suggesting Scientific Reports \| 5:16808 \| DOI: 10.1038/srep16808 3 www.nature.com/scientificreports/ an important dependence of the rejection layer properties on the substrate pore structure. This obser- vation is consistent with previous studies40,45, which can be partially attributed to the reduced effective water transport distance through the rejection layer for a substrate with higher PWP. The solute (MgCl2) rejection decreased as the membrane permeability increased, showing a typical permeability-selectivity tradeoff46,47. This observation suggests that salt rejection of a mixed matrix membrane is sensitive to the pore size of the SG particles even though these particles are only present in the membrane substrate and not in the rejection layer. Therefore, additional research studies are needed to further investigate the mechanisms involved and further study is needed to optimize the salt rejection of SG-based of mixed matrix FO membrane without compromising the water permeability. matrix FO membrane without compromising the water permeability. It is also interesting to note the substantially reduced structural parameter (S) for the SG-based mixed matrix FO substrates (Table 2). Structural parameter is a critical FO membrane property, with a lower S value preferred for controlling ICP in the FO substrate. In the current study, the S value was reduced from 0.36 mm (control) to 0.17–0.24 mm (MMMs) as a result of SG loading. In all cases, the SG-based mixed matrix FO substrates had much lower S values compared to the commercial HTI FO membranes (0.47 mm for CTA membrane and 0.62 mm for TFC membrane)25,43. Results Th Without the presence of SG particle in the substrate, the control membrane had an FO water flux of 78.1 L/m2 h in AL-DS. A lower flux of 28.7 L/m2 h was obtained in AL-FS due to the more severe dilu- tive ICP in this orientation1. For both AL-DS and AL-FS orientations, the FO water flux of the control membrane was significantly lower than those values of the SG-based MMM membranes, suggesting that the incorporation of SG particles is an effective approach for enhanced FO water flux performance. The optimal FO water flux was obtained for the filler pore size of 90 Å, and further increasing dfiller to 300 Å caused an FO water flux reduction. As explained in the previous section, the pores of SG particle could be clogged with polymer due to the large pore of SG particle, which lead to less effective water transport inside the substrate.hl The FO water flux of SG-based mixed matrix FO membranes (F90) is presented in Fig. S4 for a vari- ety of DS concentrations (0.1, 0.3, 0.5, 1.0, 3.0 M MgCl2) and FS concentrations (0, 10, 100 mM NaCl). From the viewpoint of driving force, higher the concentration of the DS should result in higher the FO water flux for the SG-based mixed matrix FO membranes. This is because of the larger osmotic pressure Scientific Reports \| 5:16808 \| DOI: 10.1038/srep16808 4 ntificreports/ Figure 2. The effect of different pore size and of silica gel loading on FO water flux of SG-based mixed matrix FO membranes. Testing conditions: 0.5 M MgCl2 as the draw solution and DI water as the feed solution, error bar was based on the standard deviation of 3 replicate measurements. www.nature.com/scientificreports/ Figure 2. The effect of different pore size and of silica gel loading on FO water flux of SG-based mixed matrix FO membranes. Testing conditions: 0.5 M MgCl2 as the draw solution and DI water as the feed solution, error bar was based on the standard deviation of 3 replicate measurements. Figure 3. Proposed mechanism for the enhanced FO water flux in SG-based mixed matrix FO membranes. Figure 3. Proposed mechanism for the enhanced FO water flux in SG-based mixed matrix FO membranes. was created across the membrane regardless of the membrane orientation. Results Th In AL-DS orientation, the FO water flux of SG-based mixed matrix FO membranes (F90) was 42.3 L/m2 h for a 0.1 M MgCl2 and it increased to 117.2 L/m2 h at 0.5 M MgCl2 as a result of increased the osmotic driving force. The highest FO water flux of 126 L/m2 h was achieved by using the 1.0 M MgCl2 as the DS and DI water as the FS, this clearly demonstrates the potential of SG-based mixed matrix FO membranes for high flux FO applica- tion. However, further increase the concentration of DS to 3.0 M MgCl2 did not result in additional gain in FO water flux. This is consistent with prior studies14,57 that ICP increases and FO efficiency decreases at higher DS concentrations. The membrane also worked well at higher FS concentration (82.5 L/m2 h and 67.4 L/m2 h in AL-DS orientation and 46.2 L/m2 h to 41.2 L/m2 h in AL-FS orientation using 10 mM or 100 mM NaCl solution as FS and 1.0 M MgCl2 as DS). Scientific Reports \| 5:16808 \| DOI: 10.1038/srep16808 Methods i l Materials. Unless otherwise stated, all chemicals and reagents were used as received. Polyacrylonitrile (PAN, Sigma–Aldrich, Mw ~ 150,000, Lot# MKBD6325V) was used for polymer solution preparation due to its ease of processing, good chemical resistance, mechanical strength, and thermal stability PAN in the current study27,60. N,N-dimethylformamide (DMF, purity ≥ 99.8%, Merck) was used as solvent to dissolve PAN and lithium chloride (anhydrous LiCl, Merck) was added to promote the formation of finger-like pores42. In order to increase the surface hydrophilicity and surface charge, sodium hydroxide (anhydrous NaOH, purity ≥ 98%, Merck) was used to prepare alkali solution for base treatment of PAN substrate. Silica gel (SiliCycle Inc., Canada) were used for MMMs preparation due to its high thermal and chemical stability, high surface area, neutral pH, non-toxic, tunable particle and pore size, readily available in kilogram scale, commercial viable and well characterized properties61,62. According to the product characteristics provided by the company, the pore size of silica gel ranged from 40 to 300 Å respectively (Table 1). p y For the layer-by-layer (LbL) self-assembly, poly(allylamine hydrochloride) (PAH, Mw ∼ 112,000– 200,000, Polyscience, Lot# 639458) was used as positively charged polyelectrolyte and poly(sodium 4-styrene-sulfonate) (PSS, Mw ~ 70,000, 30 wt.% in H2O, Sigma–Aldrich, Batch# 09622HH) was used as negatively charged polyelectrolyte for the formation of rejection layer. The ionic strength of the pol- yelectrolyte solution was controlled by adding sodium chloride (NaCl, 99%, Merck). Glutaraldehyde (GA, 25% in water, Sigma–Aldrich) is used as a crosslinking reagent to make a salt rejection layers with crosslinked layer-by-layer (xLbL) film, which have relatively high FO water flux and good rejection to divalent ions and may have potential applications in biomass concentration, food processing, etc27–29,63. Fabrication of SG-based Mixed Matrix FO Substrates. The detailed procedure for the fabrica- tion of SG-based mixed matrix FO substrates can be found elsewhere42. First of all, silica gel particles (1.0 wt.% based on the total weight of dope solution) were added into DMF followed by ultrasonicating the solution for 1 h to prevent the aggregation of SG particles in the DMF. Next, adding LiCl (2 wt.%) into the mixture prior to the addition of PAN (18 wt.%). The mixture was stirred at 60 °C for 24 h to obtain the homogenous solution, which was left at room temperature for 12 h to release gas bubbles trapped inside the solution. Discussion SG-based mixed matrix FO membranes with different pore size of SG particles were systematically fab- ricated and characterized in this current study. The pore size of SG particles played an important role in the current study. For SG particles with larger pore size (up to ~9 nm), the corresponding membrane had higher water permeability as well as lower structural parameter than the SG particles with smaller pore size. This clearly indicates the importance of water transport through the internal pores of the SG particles, which provide additional pathways (i.e., shortcuts) for water molecules to pass through the sup- port layer (Fig. 3). While earlier works have focused on the use of microporous fillers (dfiller < 2 nm)40, the current study clearly demonstrates the beneficial effect of larger dfiller. Unlike the case of TFN membranes where the fillers are designed in such a way that they retain solutes while allowing water to permeate through them, larger dfiller is preferred for fillers to the membrane substrate as the latter is designed to be conduits to both water and solute molecules. Nevertheless, caution should be exercised to avoid overly large filler pores to prevent their intrusion by polymer dope. The current study shows that a simultaneous Scientific Reports \| 5:16808 \| DOI: 10.1038/srep16808 5 www.nature.com/scientificreports/ improvement of membrane water permeability and reduction in structural parameter can be realized by the porous filler based MMM approach, thereby providing an additional degree of freedom to FO performance enhancement. A similar strategy might be used for the fabrication of SG-based mixed matrix pressure-retarded osmosis (PRO) membranes to control the ICP effect in the PRO substrate. Compared to FO, PRO has great requirement of membrane mechanical stability51,52. In this regard, the MMM approach is more promising approach (say compared to nanofiber based approach) for preparing membranes to greater mechanical strength. Existing works showed that mixed matrix strategy could significantly enhance not only the tensile strength but also the toughness of the membrane for a variety of applications, such as pervaporation and direct methanol fuel cells58,59. Methods i l A thin polymer film was spread out uniformly onto a clean glass plate followed by phase inversion in the tap water bath at room temperature. The resulting substrates were named as Control, F40, F60, F90, and F300 based on the nominal pore size of silica gel (refer to Table 1). In order to partially hydrolyze the surface of SG-based mixed matrix FO membrane substrate, the membrane substrates were immersed into the 1.5 M NaOH solution at room temperature for 1.5 h to before the processes of LbL self-assembly and crosslinking. Preparation of Rejection Layer of SG-based Mixed Matrix FO Membranes. The preparation of LbL rejection layer can be found in details elsewhere27,28,63,64. In brief, one side of the SG-based mixed matrix FO membrane substrates were alternatively immersed into the PAH and PSS solution for 15 min followed by soaking the substrate into the DI water after each polyelectrolyte soaking step to remove the excess charged polyelectrolyte. In order to achieve membranes rejection layer with reasonable salt rejection and high water permeability for FO applications, the PAH/PSS treatment was repeated three times based on the optimal condition of previous studies27,28,42. SG-based mixed matrix FO membranes were then cross-linked by immersing the membranes in a 0.1 wt.% GA solution for 30 min followed by soaking with DI water for 5 min to remove excess GA. Characterization of SG particle and SG-based Mixed Matrix FO Membranes. The detailed procedure for the characterization of SG particle and SG-based mixed matrix FO membranes can be found in details elsewhere42. Briefly, the surface roughness of the SG-based mixed matrix FO mem- branes was characterized by atomic force microscope (AFM, Park Systems XE-100, Korea)42,65. The Brunauer-Emmett-Teller (BET) surface area and the pore size of SG particles were characterized by Micromeritics ASAP 2020 instrument (USA). The membrane porosity (ε) were measured using gravi- metric measurementand the surface hydrophilicity were tested by OCA contact angle goniometer system (DataPhysics Instruments GmbH, Germany)25,66. Performance Evaluation of SG-based Mixed Matrix FO Membranes. The pressurized crossflow system used for determining membrane water permeability and salt rejection has been reported in the Scientific Reports \| 5:16808 \| DOI: 10.1038/srep16808 6 www.nature.com/scientificreports/ previous studies25,39,55. Briefly, the water permeability of SG-based mixed matrix membranes was deter- mined by weighing the amount of permeates at the fixed interval. References L. & Elimelech, M. Energy requirements of ammonia-carbon dioxide forward osmosis desalination. Desalination 207, 370–382 (2007).h , ( ) 5. Theeuwes, F. & Yum, S. I. Principles of the design and operation of generic osmotic pumps for the delivery of semisolid or liquid 15. Theeuwes, F. & Yum, S. I. Principles of the design and operation of generic osmotic pum drug formulations. Ann. Biomed. Eng. 4, 343–353 (1976). 15. Theeuwes, F. & Yum, S. I. Principles of the design and operation of generic osmotic pumps for the delivery of semisolid or liquid drug formulations. Ann. Biomed. Eng. 4, 343–353 (1976). 15. Theeuwes, F. & Yum, S. I. Principles of the design and opera drug formulations. Ann. Biomed. Eng. 4, 343–353 (1976). g g 6. Petrotos, K. B., Quantick, P. & Petropakis, H. A study of the direct osmotic concentration of tomato juice in tubular membrane— module configuration I The effect of certain basic process parameters on the process performance J Membr Sci 150 99 110 g g 16. Petrotos, K. B., Quantick, P. & Petropakis, H. A study of the direct osmotic concentration of tomato juice in tubular membrane— module configuration. I. The effect of certain basic process parameters on the process performance. J. Membr. Sci. 150, 99–110 (1998). Q p y j module configuration. I. 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For the evaluation of FO performance, a small piece of membrane with the membrane area of 60 cm2 was tested for both the active-layer-facing-DS (AL-DS) and active-layer-facing-FS (AL-FS) orientations. Concentrated MgCl2 solutions (0.1, 0.3, 0.5, 1.0, or 3.0 M) were used as draw solution and the DI water, 10 mM or 100 mM NaCl solution was used as feed solution. To determine the FO water flux, the weight change of the FS tank was measured with a weighing machine at fixed time intervals. On the other hand, the FO solute flux was determined by measuring the conductivity of the feed solution. 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In addition, we also thank the Singapore Ministry of Education (Grant #MOE2011-T2-2-035, ARC3/12) for the financial support of the work. The authors also thank Mr. Huang Jijiang and Mr. Zhang Suoying for their help in BET measurement. Author Contributions J.-Y.L. conceived the idea, designed and carried out the experiments, analyzed the data and co-drafted the manuscript. Y.W. analyzed the data and co-drafted the manuscript. F.H. and C.Y.T. supervised the project, helped design the experiments and co-drafted the manuscript. All the authors contributed to the interpretation of the results and writing the manuscript. Scientific Reports \| 5:16808 \| DOI: 10.1038/srep16808 www.nature.com/scientificreports/ Effect of feed spacer induced membrane deformation on the performance of pressure retarded osmosis (PRO) Implications for PRO process operation. J. Membr. Sci. 445, 170–182 (2013). p p p 3. Blandin, G. et al. 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Double-skinned forward osmosis membranes based on layer-by-layer assembly-FO performance and fouling behavior. J. Membr. Sci. 405, 20–29 (2012). Scientific Reports \| 5:16808 \| DOI: 10.1038/srep16808 8 www.nature.com/scientificreports/ 4. Qi, S. R. et al. Influence of the properties of layer-by-layer active layers on forward osmosis performance. J. Membr. Sci. 423 536–542 (2012). 64. Qi, S. R. et al. Influence of the properties of layer-by-layer active layers on forward osmosis performance. J. Membr. Additional Information Supplementary information accompanies this paper at http://www.nature.com/srepihi Competing financial interests: The authors declare no competing financial interests. How to cite this article: Lee, J.-Y. et al. Mesoporous Silica Gel–Based Mixed Matrix Membranes for Improving Mass Transfer in Forward Osmosis: Effect of Pore Size of Filler. Sci. Rep. 5, 16808; doi: 10.1038/srep16808 (2015). 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https://openalex.org/W3147379882	https://www.nature.com/articles/s42003-021-01938-0.pdf	English	null	mmsig: a fitting approach to accurately identify somatic mutational signatures in hematological malignancies	Communications biology	2,021	cc-by	12,603	1 Myeloma Service, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA. 2 Institute for Cancer Research, Oslo University Hospital Radiumhospitalet, Oslo, Norway. 3 Patologia Molecular de Neoplàsies Limfoides, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain. 4 Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), Madrid, Spain. 5 School of Medicine, Systems Immunity Research Institute, Cardiff University, Cardiff, UK. 6 Department of Molecular Biotechnologies and Health Sciences, University of Turin, Turin, Italy. 7 Myeloma Program, Sylvester Comprehensive Cancer Center, University of Miami, Miami, FL, USA. 8 Department of Oncology and Hemato-Oncology, University of Milan, Milan, Italy. 9 Unità Operativa Complessa di Ematologia, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy. 10 Departamento de Bioquimica y Biologia Molecular, Instituto Universitario de Oncologia (IUOPA), Universidad de Oviedo, Oviedo, Spain. 11 Unitat Hematopatologia, Hospital Clínic of Barcelona, Universitat de Barcelona, Barcelona, Spain. ✉email: col15@miami.edu; fxm557@med.miami.edu COMMUNICATIONS BIOLOGY \| (2021) 4:424 \| https://doi.org/10.1038/s42003-021-01938-0 \| www.nature.com/commsbio mmsig: a ﬁtting approach to accurately identify somatic mutational signatures in hematological malignancies T from a practical perspective, it is most important to identify those signatures that have a clear biological correlate and may serve as clinical biomarkers. When algorithms are developed with optimal reconstruction accuracy as the principal goal, this often comes at the cost of overﬁtting, resulting in false positive signature calls10. We developed the novel algorithm mmsig with this in mind to optimize speciﬁcity at a minimal loss of reconstruction accuracy (“Methods”). To illustrate the challenges of mutational signature ﬁtting and the unique features of mmsig, we analyzed a thoroughly characterized WGS dataset with 82 MM samples11,17–19. We included the 8 mutational signatures previously identiﬁed in MM: SBS1 and SBS5, mutations related to the cell aging (i.e., clock- like); SBS2 and SBS13, resulting from APOBEC cytidine deaminase activity; SBS9, attributed to the non-canonical genome-wide action of AID (nc-AID); SBS8 of unknown etiology; SBS18, which may be related to DNA damage from reactive oxygen species; and SBS-MM1, the mutational footprint of melphalan therapy (Fig. 1B)11. A subset of patients with MM show mutational proﬁles dominated by APOBEC mutagenesis, with high proportion of mutations in TCT and TCA context that are well-explained by SBS2 (C>T) and SBS13 (C>G) (e.g., patient PD26419a in Fig. 1A). Patients with lower APOBEC were dominated by SBS5, with distinctive peaks in T>G attributable to SBS9 (e.g., patient V0D58T in Fig. 1A). As a ﬁrst step toward standardization of mutational signature analysis in hematological malignancies, we recently proposed a three-step workﬂow of (1) de novo signature extraction (e.g., by non-negative matrix factorization, NMF); (2) assignment to a set of reference signatures (e.g., COSMIC v3.1; https://cancer.sanger. ac.uk/cosmic/signatures/); and (3) applying a ﬁtting algorithm to determine which of the signatures deﬁned in steps 1–2 are present in each tumor10. We previously focused on the challenges and pitfalls of the ﬁrst two steps, which may result in falsely identi- fying signatures that are not active in a given disease, such as HRD-SBS3 in multiple myeloma (MM)10,14. Successfully apply- ing the most recent de novo extraction tools and assignment of mutational signatures to a large set of samples with a speciﬁc cancer will reveal the main mutational processes active in that disease2. However, as previously reported, this approach may be affected by several issues leading to incorrect quantiﬁcation of signature contribution10. mmsig: a ﬁtting approach to accurately identify somatic mutational signatures in hematological malignancies One such problem is inter-bleeding of signatures, where a mutational signature present in only a subset of cases are incorrectly called in the others as well. Furthermore, de novo extraction requires large cohorts of patients, limiting their applicability in the clinic. In cancers with well-characterized mutational signature landscapes, ﬁtting algorithms are highly suited for clinical application, as they can be applied to individual samples with short run-times5,12,15,16. Although technically straightforward to perform, mutational signature ﬁtting requires rigorous interpretation to ensure the accuracy and validity of results. ( g , p g ) Using the latest COSMIC reference (i.e., COSMIC v3.1; https:// cancer.sanger.ac.uk/cosmic/signatures/SBS/index.tt) and applying three different ﬁtting algorithms (deconstructSigs, mutationalPat- terns, and mmsig) to each of the 82 MM samples without the use of error-correction showed similar results (Fig. 2A). Accordingly, pairwise comparisons of the reconstructed mutational proﬁle for each sample by the different algorithms showed median cosine similarity of >0.998. Moreover, the median cosine similarity between the reconstructed and original mutational proﬁles was 0.97 for all three algorithms. However, when the ﬁtted mutational signatures were compared with actual exposure of patients to melphalan, it became clear that SBS-MM1 was falsely identiﬁed in >90% of samples from untreated patients. This problem of overﬁtting is well known, and a common approach to avoid false positives is to remove all signatures with estimated contribution below a given percentage15,16. A threshold of 6% is the standard setting for deconstructSigs, and we applied the same threshold to mutationalPatterns as well. Although removing false positive SBS-MM1 in most cases, this approach introduced problems of its own, because the COSMIC v3.1 versions of APOBEC and SBS1 often fell below the 6% threshold despite being clearly present (Fig. 2B). SBS1 has been identiﬁed in all cancers and normal individuals, and APOBEC is known to be active in the vast majority of patients with MM7,11,20–23. Moreover, the APOBEC signatures have highly distinct proﬁles, making it possible to identify them at low contributions even by visual inspection of the mutational proﬁle (see Fig. S1). The error- correction approach built into mmsig takes advantage of the distinctiveness of each mutational signature, by keeping in the ﬁnal output only those signatures that lead to considerably lower reconstruction accuracy when removed (>1% reduction in cosine similarity is the standard setting). mmsig: a ﬁtting approach to accurately identify somatic mutational signatures in hematological malignancies Even H. Rustad1,2, Ferran Nadeu 3,4, Nicos Angelopoulos5, Bachisio Ziccheddu6,7, Niccolò Bolli8,9, Xose S. Puente 10, Elias Campo 3,4,11, Ola Landgren 1,7✉& Francesco Maura 1,7✉ Even H. Rustad1,2, Ferran Nadeu 3,4, Nicos Angelopoulos5, Bachisio Ziccheddu6,7, Niccolò Bolli8,9, Xose S. Puente 10, Elias Campo 3,4,11, Ola Landgren 1,7✉& Francesco Maura 1,7✉ Mutational signatures have emerged as powerful biomarkers in cancer patients, with prog- nostic and therapeutic implications. Wider clinical utility requires access to reproducible algorithms, which allow characterization of mutational signatures in a given tumor sample. Here, we show how mutational signature ﬁtting can be applied to hematological cancer genomes to identify biologically and clinically important mutational processes, highlighting the importance of careful interpretation in light of biological knowledge. Our newly released R package mmsig comes with a dynamic error-suppression procedure that improves speciﬁcity in important clinical and biological settings. In particular, mmsig allows accurate detection of mutational signatures with low abundance, such as those introduced by APOBEC cytidine deaminases. This is particularly important in the most recent mutational signature reference (COSMIC v3.1) where each signature is more clearly deﬁned. Our mutational signature ﬁtting algorithm mmsig is a robust tool that can be implemented immediately in the clinic. 1 ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-01938-0 T he mutational proﬁle of a tumor represents an archive of all mutational processes that have been active throughout tumor phylogeny, dating back to the fertilized egg1,2. Analyzing the mutational signatures associated with distinct mutagenic processes has revealed key insights into cancer pathogenesis, evolution, and potential therapeutic strategies1,3–6. To make up a mutational proﬁle, single nucleotide variants (SNVs) are commonly divided in 96 classes based on their tri- nucleotide context, deﬁned by the mutated base and the most proximal bases in the 5′ and 3′ direction1,3. Recent whole-genome sequencing (WGS) studies have revealed more than 40 signatures of single base substitutions (SBS), representing processes active in all tissues (e.g., ageing related clock-like processes), as well as cell- type-speciﬁc intrinsic processes (e.g., APOBEC-family cytidine deaminases, such as activation-induced cytidine deaminase, AID), processes related to exogenous agents (e.g., smoking and che- motherapeutic agents), and deﬁciencies of speciﬁc DNA repair mechanisms (e.g., homologous repair deﬁciency, HRD)1–3,7–12. Several tools for mutational signature analysis have been pro- posed, without a single accepted standard having emerged. Resulting from the lack of methodological consensus, different studies have often reported conﬂicting and biologically implau- sible results10,11,13,14. mmsig: a ﬁtting approach to accurately identify somatic mutational signatures in hematological malignancies Because SBS1 and SBS5 are known to always be present, we kept them in the ﬁnal proﬁle no matter what their contribution was to the overall reconstruction accuracy. The results of all three algorithms after error-correction can be seen in Fig. 2B, along with their reconstruction accuracy before and after error-correction (Fig. 2C, D). In contrast to the other algorithms, mmsig with error-correction was able to correctly identify APOBEC in the majority of samples while avoiding false positive SBS-MM1, at the cost of a 0.005 reduction in median reconstruction accuracy. Here, we show how mutational signature ﬁtting can be used to identify clinically relevant mutational processes in hematological malignancies. We highlight the differences between commonly used algorithms and introduce the novel algorithm mmsig, developed speciﬁcally to solve difﬁcult cases where it is unclear whether a given signature is present or absent. COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-01938-0 In these cases, the mutational proﬁles were well-explained by the ﬁtted signatures (high reconstruction accuracy), indicated by cosine similarity of >0.97 between observed and reconstructed proﬁles. Fig. 1 Mutational ﬁtting in multiple myeloma. A Mutational proﬁles from two example patients with MM. B Mutational signature ﬁtting reveals the main APOBEC signature (SBS2) along with the clock-like signatures SBS1 and SBS5 in both samples. Sample PD26419a (left) also had evidence of SBS13, which is most often detected in patients with high APOBEC mutational burden, as well as SBS8, a common mutational signature in MM that is of unknown etiology. Sample V0D58T (right) showed contributions from non-canonical activation-induced cytidine deaminase (nc-AID; SBS9), melphalan-induced mutagenesis (SBS-MM1), and damage by reactive oxygen species (ROS; SBS18). C To measure how well the ﬁtted signatures account for the actual mutational proﬁle in each sample, the ﬁrst step is to reconstruct a mutational proﬁle by multiplying the weight assigned to each signature with the reference proﬁle of that signature. D Subtracting the reconstructed proﬁle from the original proﬁle illustrates which parts of the mutational spectrum are well-explained and may point to the presence of additional signatures that were not included in the analysis. In these cases, the mutational proﬁles were well-explained by the ﬁtted signatures (high reconstruction accuracy), indicated by cosine similarity of >0.97 between observed and reconstructed proﬁles. were the estimates for SBS2, SBS8, and SBS9. A decrease in SBS5 was seen with the old reference, most likely reciprocal to the presence of a ﬂat “background” in several other muta- tional signatures (Fig. 3C). Conversely, SBS18 was considerably higher with the new reference, as was SBS-MM1. This latter point is important because the SBS-MM1 reference was the same in both analyses, but the assignment of mutations to SBS-MM1 was affected by the changes to other signature references. As expected, updating to a reference set where each mutational signature is more clearly deﬁned resulted in a clear overall increase in reconstruction accuracy for all three ﬁtting algorithms (Fig. 3D). Applying the same ﬁlters as described above resulted in lower reconstruction accuracy, with similar performance for all three algorithms (Fig. 3E). This is in contrast to results from the new reference, where mmsig performed considerably better (Fig. 2D). We note that deconstructSigs and mutationalPatterns were ﬁrst released before the latest mutational signature reference was released and thus optimized for the less speciﬁc COSMIC v2 reference. COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-01938-0 COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-01938-0 Fig. 1 Mutational ﬁtting in multiple myeloma. A Mutational proﬁles from two example patients with MM. B Mutational signature ﬁtting reveals the main APOBEC signature (SBS2) along with the clock-like signatures SBS1 and SBS5 in both samples. Sample PD26419a (left) also had evidence of SBS13, which is most often detected in patients with high APOBEC mutational burden, as well as SBS8, a common mutational signature in MM that is of unknown etiology. Sample V0D58T (right) showed contributions from non-canonical activation-induced cytidine deaminase (nc-AID; SBS9), melphalan-induced mutagenesis (SBS-MM1), and damage by reactive oxygen species (ROS; SBS18). C To measure how well the ﬁtted signatures account for the actual mutational proﬁle in each sample, the ﬁrst step is to reconstruct a mutational proﬁle by multiplying the weight assigned to each signature with the reference proﬁle of that signature. D Subtracting the reconstructed proﬁle from the original proﬁle illustrates which parts of the mutational spectrum are well-explained and may point to the presence of additional signatures that were not included in the analysis. In these cases, the mutational proﬁles were well-explained by the ﬁtted signatures (high reconstruction accuracy), indicated by cosine similarity of >0.97 between observed and reconstructed proﬁles. Fig. 1 Mutational ﬁtting in multiple myeloma. A Mutationa Fig. 1 Mutational ﬁtting in multiple myeloma. A Mutational proﬁles from two example patients with MM. B Mutational signature ﬁtting reveals the main APOBEC signature (SBS2) along with the clock-like signatures SBS1 and SBS5 in both samples. Sample PD26419a (left) also had evidence of SBS13, which is most often detected in patients with high APOBEC mutational burden, as well as SBS8, a common mutational signature in MM that is of unknown etiology. Sample V0D58T (right) showed contributions from non-canonical activation-induced cytidine deaminase (nc-AID; SBS9), melphalan-induced mutagenesis (SBS-MM1), and damage by reactive oxygen species (ROS; SBS18). C To measure how well the ﬁtted signatures account for the actual mutational proﬁle in each sample, the ﬁrst step is to reconstruct a mutational proﬁle by multiplying the weight assigned to each signature with the reference proﬁle of that signature. D Subtracting the reconstructed proﬁle from the original proﬁle illustrates which parts of the mutational spectrum are well-explained and may point to the presence of additional signatures that were not included in the analysis. Results Mutational signature ﬁtting and mmsig. In principle, muta- tional signature ﬁtting is a mathematical procedure aimed to determine the combination of known signatures that best explains the observed mutational proﬁle (Fig. 1A, B)3. This is often measured by the cosine similarity of the original mutational proﬁle with the proﬁle that is reconstructed based on the ﬁtted signatures (reconstruction accuracy; Fig. 1C, D)3,16. However, COMMUNICATIONS BIOLOGY \| (2021) 4:424 \| https://doi.org/10.1038/s42003-021-01938-0 \| www.nature.com/commsbio 2 ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-01938-0 The iterative ﬁltering approach of mmsig, based on cosine similarity rather than a hard cut-off, may be particularly advantageous in identifying mutational signatures that are highly distinctive but account for a low proportion of the mutational proﬁle (e.g. APOBEC and SBS1). Moreover, irrespective of which mutational signature reference was used, only mmsig could avoid Importance of the reference signatures. Since the concept of mutational signatures was ﬁrst introduced in 2012, the most commonly used signature reference has been updated from the initial 30 COSMIC signatures (https://cancer.sanger.ac.uk/ cosmic/signatures_v2.tt) (i.e., COSMIC v2)1,4 to a new catalog of 49 reference signatures (i.e., COSMIC v3.1)2, with additional signatures having been reported as the consequence of exogenous agents9,11,24. Importantly, the updated COSMIC reference led to sharper signature deﬁnitions, removing background noise and contamination from other signatures. For example, the muta- tional signature ﬁrst reported as SBS1 included a ﬂat background similar to SBS5 that has been removed in the new version, leaving only the characteristic and biologically accurate C>T in CpG mutations (see Fig. S2A)2,25. Moreover, the APOBEC mutational signature (i.e., SBS2 and SBS13) proﬁles showed a degree of overlap in the previous reference and have since been clearly separated into one C>T signature (SBS2) and one C>G signature (SBS13) (see Fig. S2B)2,26. To illustrate the effects of these changes on mutational signature ﬁtting, we repeated the above analysis of 82 MM samples using the original COSMIC v2 reference with the addition of SBS-MM1. The estimated con- tribution of each signature was signiﬁcantly different depending on which reference was used (Fig. 3A). As expected, the estimated SBS1 contribution was higher using the old reference (Fig. 3B), as COMMUNICATIONS BIOLOGY \| (2021) 4:424 \| https://doi.org/10.1038/s42003-021-01938-0 \| www.nature.com/commsbio 3 ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-01938-0 COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-01938-0 Fig. 2 Mutational signature ﬁtting with mmsig shows high sensitivity, speciﬁcity, and reconstruction accuracy. A Without any ﬁltering procedures, the results from deconstructSigs, mutationalPatterns, and our novel algorithm mmsig are highly similar. Of note, all of the algorithms falsely identify the mutational signature of melphalan-induced mutagenesis (i.e., SBS-MM1) in >90% of patients who were not exposed to melphalan, representing false positives. B After applying the recommended ﬁltering procedures, mmsig shows 100% speciﬁcity for SBS-MM1 while retaining the contributions from SBS1 and SBS2. Filtering of output from deconstructSigs and mutationalPattern is less ﬂexible, relying on a relative contribution threshold, which results in false negative SBS1 and SBS2 while retaining some cases of false positive SBS-MM1. C The reconstruction accuracy of deconstructSigs, mutationalPatterns, and mmsig are virtually identical when no ﬁltering is applied. D When ﬁltering procedures are applied, mmsig shows superior reconstruction accuracy >0.95 for all samples. Filtered output from deconstructSigs and mutationalPatterns was scaled for the sum of mutational processes to equal 1. ture ﬁtting with mmsig shows high sensitivity, speciﬁcity, and reconstruction accuracy. A Without any ﬁltering proc 2 Mutational signature ﬁtting with mmsig shows high sensitivity, speciﬁcity, and reconstruction accuracy. A Witho Fig. 2 Mutational signature ﬁtting with mmsig shows high sensitivity, speciﬁcity, and reconstruction accuracy. A Without any ﬁltering procedures, the results from deconstructSigs, mutationalPatterns, and our novel algorithm mmsig are highly similar. Of note, all of the algorithms falsely identify the mutational signature of melphalan-induced mutagenesis (i.e., SBS-MM1) in >90% of patients who were not exposed to melphalan, representing false positives. B After applying the recommended ﬁltering procedures, mmsig shows 100% speciﬁcity for SBS-MM1 while retaining the contributions from SBS1 and SBS2. Filtering of output from deconstructSigs and mutationalPattern is less ﬂexible, relying on a relative contribution threshold, which results in false negative SBS1 and SBS2 while retaining some cases of false positive SBS-MM1. C The reconstruction accuracy of deconstructSigs, mutationalPatterns, and mmsig are virtually identical when no ﬁltering is applied. D When ﬁltering procedures are applied, mmsig shows superior reconstruction accuracy >0.95 for all samples. Filtered output from deconstructSigs and mutationalPatterns was scaled for the sum of mutational processes to equal 1. Fig. 2 Mutational signature ﬁtting with mmsig shows high sensitivity, speciﬁcity, and reconstruction accuracy. A Without any ﬁltering procedures, the results from deconstructSigs, mutationalPatterns, and our novel algorithm mmsig are highly similar. COMMUNICATIONS BIOLOGY \| (2021) 4:424 \| https://doi.org/10.1038/s42003-021-01938-0 \| www.nature.com/commsbio COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-01938-0 Of note, all of the algorithms falsely identify the mutational signature of melphalan-induced mutagenesis (i.e., SBS-MM1) in >90% of patients who were not exposed to melphalan, representing false positives. B After applying the recommended ﬁltering procedures, mmsig shows 100% speciﬁcity for SBS-MM1 while retaining the contributions from SBS1 and SBS2. Filtering of output from deconstructSigs and mutationalPattern is less ﬂexible, relying on a relative contribution threshold, which results in false negative SBS1 and SBS2 while retaining some cases of false positive SBS-MM1. C The reconstruction accuracy of deconstructSigs, mutationalPatterns, and mmsig are virtually identical when no ﬁltering is applied. D When ﬁltering procedures are applied, mmsig shows superior reconstruction accuracy >0.95 for all samples. Filtered output from deconstructSigs and mutationalPatterns was scaled for the sum of mutational processes to equal 1. false positive SBS-MM1 in patients without prior melphalan exposure (see Fig. S3). highly likely that the signature is actually present. Below, we illustrate this approach in settings where we have prior knowledge of which signatures are present in each sample: chemotherapy- related signatures in MM and acute myeloid leukemia (AML), and nc-AID activity in chronic lymphocytic leukemia (CLL). Having established above that mutationalPatterns and decon- structSigs provide near-identical results, we moved forward comparing mmsig with deconstructSigs alone. Resolving uncertainty in mutational signature ﬁtting. When it is particularly important to determine if a mutational process is active, two additional steps can be taken to increase speciﬁcity (“Methods”). First, 95% conﬁdence intervals (CI) for each mutational signature estimate can be constructed by resampling with replacement from the original mutational proﬁle of a sam- ple. Second, transcriptional strand bias can be considered for signatures associated with transcription-coupled repair2,24. If the lower bound of the 95% CI for a mutational signature is above zero, and the typical transcriptional strand bias is present, it is First, we conﬁrmed the presence of SBS-MM1 in melphalan- exposed patients with MM (Fig. 4A and Fig. S4). In all cases with >10% estimated signature contribution, the presence of SBS-MM1 was stable in the face of random resampling of mutations (i.e., the CI clearly did not include zero). In all but one of these samples, there was also evidence of transcriptional strand bias in C>T MMUNICATIONS BIOLOGY \| (2021) 4:424 \| https://doi.org/10.1038/s42003-021-01938-0 \| www.nature.com/commsbio 4 COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-01938-0 ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-01938-0 The choice of mutational signature reference version signiﬁcantly impacts the performance of ﬁtting algorithms. Unﬁltered mutational signature utions for each sample were determined as the mean of estimates from deconstructSigs, mutationalPatterns, and mmsig (the three algorithms Fig. 3 The choice of mutational signature reference version signiﬁcantly impacts the performance of ﬁtting algorithms. Unﬁltered mutational signature contributions for each sample were determined as the mean of estimates from deconstructSigs mutationalPatterns and mmsig (the three algorithms Fig. 3 The choice of mutational signature reference version signiﬁcantly impacts the performance of ﬁtting algorithms. Unﬁltered mutational signature Fig. 3 The choice of mutational signature reference version signiﬁcantly impacts the performance of ﬁtting algorithms. Unﬁltered mutational signature contributions for each sample were determined as the mean of estimates from deconstructSigs, mutationalPatterns, and mmsig (the three algorithms produced almost identical results). A The mutational signature contributions were signiﬁcantly different depending on whether the original COSMIC v2 1 2 ce of mutational signature reference version signiﬁcantly impacts the performance of ﬁtting algorithms. Unﬁltered m Fig. 3 The choice of mutational signature reference version signiﬁcantly impacts the performance of ﬁtting algorithms. Unﬁltered mutational signature contributions for each sample were determined as the mean of estimates from deconstructSigs, mutationalPatterns, and mmsig (the three algorithms produced almost identical results). A The mutational signature contributions were signiﬁcantly different depending on whether the original COSMIC v2 reference or the latest updated COSMIC v3.1 from Alexandrov et al. was used for ﬁtting1,2. Boxplots show the relative contribution of each signature in each sample, displaying the median and interquartile range with outlier samples drawn as dots. P-values were estimated by paired Wilcoxon tests. Dashed black lines mark the 6% relative contribution threshold used for ﬁltering of results from deconstructSigs and mutationalPatterns. B, C Showing individual sample data from A for SBS1 and SBS5. B Estimates for SBS1 were generally higher using the old reference, where SBS1 contains “contamination” of a ﬂat background that may be better explained by SBS5. Notably, this resulted in SBS1 ending up above the 6% ﬁltering threshold (dashed black line) in a higher fraction of samples using the old reference. C Estimates of SBS5 were generally higher using the new reference. D The overall cleaner mutational signatures in the new reference resulted in higher reconstruction accuracy compared with when the old reference was used. COMMUNICATIONS BIOLOGY \| (2021) 4:424 \| https://doi.org/10.1038/s42003-021-01938-0 \| www.nature.com/commsbio COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-01938-0 COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-01938-0 deconstructSigs mmsig 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 Relative contribution * * p 0.0 0.2 0.4 0.6 0.8 SBS35 (prop) * * * * * * ** * * * * * * 0.0 0.1 0.2 0.3 SBS MM1 (prop) C>A C>G C>T T>A T>C T>G TCGAAB3001 Platinum PD37515 Platinum PD34280 A[C>A]A A[C>A]C A[C>A]G A[C>A]T C[C>A]A C[C>A]C C[C>A]G C[C>A]T G[C>A]A G[C>A]C G[C>A]G G[C>A]T T[C>A]A T[C>A]C T[C>A]G T[C>A]T A[C>G]A A[C>G]C A[C>G]G A[C>G]T C[C>G]A C[C>G]C C[C>G]G C[C>G]T G[C>G]A G[C>G]C G[C>G]G G[C>G]T T[C>G]A T[C>G]C T[C>G]G T[C>G]T A[C>T]A A[C>T]C A[C>T]G A[C>T]T C[C>T]A C[C>T]C C[C>T]G C[C>T]T G[C>T]A G[C>T]C G[C>T]G G[C>T]T T[C>T]A T[C>T]C T[C>T]G T[C>T]T A[T>A]A A[T>A]C A[T>A]G A[T>A]T C[T>A]A C[T>A]C C[T>A]G C[T>A]T G[T>A]A G[T>A]C G[T>A]G G[T>A]T T[T>A]A T[T>A]C T[T>A]G T[T>A]T A[T>C]A A[T>C]C A[T>C]G A[T>C]T C[T>C]A C[T>C]C C[T>C]G C[T>C]T G[T>C]A G[T>C]C G[T>C]G G[T>C]T T[T>C]A T[T>C]C T[T>C]G T[T>C]T A[T>G]A A[T>G]C A[T>G]G A[T>G]T C[T>G]A C[T>G]C C[T>G]G C[T>G]T G[T>G]A G[T>G]C G[T>G]G G[T>G]T T[T>G]A T[T>G]C T[T>G]G T[T>G]T 0.0 2.5 5.0 7.5 10.0 0 10 20 30 0 10 20 30 40 Mutations (n) A C SBS35 Signature SBS1 SBS5 SBS-HSPC Melphalan No melphalan B * Transcriptional strand bias in typical pattern for SBS-MM1 Platinum No Platinum D * Transcriptional strand bias in C[C>A]C and C[C>T]C Platinum No Platinum Untranscribed strand Transcribed strand Fig. 4 Resolving uncertainty in mutational signature ﬁtting. A Bar chart showing the estimated SBS-MM1 contribution in 82 MM samples with an without prior melphalan exposure by mmsig (similar to Fig. 2B), with 95% CI estimated from 1000 bootstrapping iterations. Asterisks indicate statistic signiﬁcant transcriptional strand bias in characteristic trinucleotide contexts (Poisson test p < 0.05). B Stached bar chart showing mutational signatu proﬁles of 49 patients with acute myeloid leukemia (AML) with (n = 2, left) and without (n = 47, right) prior platinum exposure, estimated using deconstructSigs (top) and mmsig (bottom). SBS-HSPC is a mutational signature characteristic of hematopoietic stem cells. C Similar to A for 49 AM patients with and without prior platinum exposure. D Bar chart showing the number of mutations on the transcribed (blue) and untranscribed (red) str in each trinucleotide context for three patients. On the top we show one patient who did not have prior history of platinum exposure, but where SBS35 w identiﬁed with the whole 95% CI above zero. COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-01938-0 B Stached bar chart showing mutational signature proﬁles of 49 patients with acute myeloid leukemia (AML) with (n = 2, left) and without (n = 47, right) prior platinum exposure, estimated using deconstructSigs (top) and mmsig (bottom). SBS-HSPC is a mutational signature characteristic of hematopoietic stem cells. C Similar to A for 49 AML patients with and without prior platinum exposure. D Bar chart showing the number of mutations on the transcribed (blue) and untranscribed (red) strand in each trinucleotide context for three patients. On the top we show one patient who did not have prior history of platinum exposure, but where SBS35 was identiﬁed with the whole 95% CI above zero. The mutational signature proﬁle does not ﬁt the classic pattern of SBS35, however, and there is no sign of the characteristic transcriptional strand bias, suggesting a false positive call. The two lower panels show the patients with therapy-related AML who were exposed to platinum. Their mutational proﬁles are highly dominated by SBS35 with strong transcriptional strand bias. Fig. 4 Resolving uncertainty in mutational signature ﬁtting. A Bar chart showing the estimated SBS-MM1 contribution in 82 MM samples with and without prior melphalan exposure by mmsig (similar to Fig. 2B), with 95% CI estimated from 1000 bootstrapping iterations. Asterisks indicate statistically signiﬁcant transcriptional strand bias in characteristic trinucleotide contexts (Poisson test p < 0.05). B Stached bar chart showing mutational signature proﬁles of 49 patients with acute myeloid leukemia (AML) with (n = 2, left) and without (n = 47, right) prior platinum exposure, estimated using deconstructSigs (top) and mmsig (bottom). SBS-HSPC is a mutational signature characteristic of hematopoietic stem cells. C Similar to A for 49 AML patients with and without prior platinum exposure. D Bar chart showing the number of mutations on the transcribed (blue) and untranscribed (red) strand in each trinucleotide context for three patients. On the top we show one patient who did not have prior history of platinum exposure, but where SBS35 was identiﬁed with the whole 95% CI above zero. The mutational signature proﬁle does not ﬁt the classic pattern of SBS35, however, and there is no sign of the characteristic transcriptional strand bias, suggesting a false positive call. The two lower panels show the patients with therapy-related AML who were exposed to platinum. Their mutational proﬁles are highly dominated by SBS35 with strong transcriptional strand bias. for deconstructSigs. COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-01938-0 E When using the COSMIC v.2 reference, the reconstruction accuracy was similar irrespective of which ﬁtting algorithm was used: deconstructSigs, mutationalPatterns or mmsig. This is in contrast to the new reference, where mmsig was superior. procedure of mmsig. We prefer to be conservative in these cases and tolerate false negatives to a geater degree than false positives. In patients without prior melphalan exposure, SBS-MM1 with its characteristic transcriptional strand bias was consistently absent (Fig. 4A). mutations across the trinucleotide contexts typically associated with SBS-MM1: CCA, GCA, GCC, GCG, and GCT. Not all patients previously exposed to melphalan had detectable SBS- MM1, because this requires a single melphalan-exposed cell to be positively selected and expand beyond the limit of detection of WGS, which does not always occur11,24,27. An alternative explanation is the engraftment model, where myeloma cells are infused during autologous stem cell transplantation, thus avoiding melphalan exposure27. There will also be a range of exposures (e.g., <10%) where there is insufﬁcient evidence to ascertain whether SBS-MM1 is really present (i.e., transcriptional strand bias and CIs). In such ambiguous cases, SBS-MM1 may not be essential to explain the overall mutational proﬁle, leading to SBS-MM1 being removed from the ﬁnal proﬁle by the error-suppression Platinum-based chemotherapy is the underlying cause of mutational signature SBS352,24. Here, we applied deconstructSigs and mmsig with standard cutoffs to two patients with therapy- related AML previously exposed to platinum-based chemother- apy as well as 47 patients with de novo AML (platinum naive) (Fig. 4B). Point estimates of the SBS35 contribution (i.e., standard mutational signature ﬁtting) identiﬁed SBS35 in both patients with prior platinum exposure. Strikingly, the false positive rate for SBS35 among de novo AML cases was 21% for mmsig and 70% COMMUNICATIONS BIOLOGY \| (2021) 4:424 \| https://doi.org/10.1038/s42003-021-01938-0 \| www.nature.com/commsbio 5 ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-01938-0 The mutational signature proﬁle does not ﬁt the classic pattern of SBS35, however, and there is no sign of characteristic transcriptional strand bias, suggesting a false positive call. The two lower panels show the patients with therapy-related AML who we exposed to platinum. Their mutational proﬁles are highly dominated by SBS35 with strong transcriptional strand bias. * * p 0.0 0.2 0.4 0.6 0.8 SBS35 (prop) * * * * * * ** * * * * * * 0.0 0.1 0.2 0.3 SBS MM1 (prop) A C Melphalan No melphalan * Transcriptional strand bias in typical pattern for SBS-MM1 * Transcriptional strand bias in C[C>A]C and C[C>T]C Platinum No Platinum * * p 0.0 0.2 0.4 0.6 0.8 SBS35 (prop) C * Transcriptional strand bias in C[C>A]C and C[C>T]C Platinum No Platinum * * * * * * ** * * * * * * 0.0 0.1 0.2 0.3 SBS MM1 (prop) A Melphalan No melphalan * Transcriptional strand bias in typical pattern for SBS-MM1 A deconstructSigs mmsig 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 Relative contribution SBS35 Signature SBS1 SBS5 SBS-HSPC B p yp p Platinum No Platinum D B D B D C>A C>G C>T T>A T>C T>G TCGAAB3001 Platinum PD37515 Platinum PD34280 A[C>A]A A[C>A]C A[C>A]G A[C>A]T C[C>A]A C[C>A]C C[C>A]G C[C>A]T G[C>A]A G[C>A]C G[C>A]G G[C>A]T T[C>A]A T[C>A]C T[C>A]G T[C>A]T A[C>G]A A[C>G]C A[C>G]G A[C>G]T C[C>G]A C[C>G]C C[C>G]G C[C>G]T G[C>G]A G[C>G]C G[C>G]G G[C>G]T T[C>G]A T[C>G]C T[C>G]G T[C>G]T A[C>T]A A[C>T]C A[C>T]G A[C>T]T C[C>T]A C[C>T]C C[C>T]G C[C>T]T G[C>T]A G[C>T]C G[C>T]G G[C>T]T T[C>T]A T[C>T]C T[C>T]G T[C>T]T A[T>A]A A[T>A]C A[T>A]G A[T>A]T C[T>A]A C[T>A]C C[T>A]G C[T>A]T G[T>A]A G[T>A]C G[T>A]G G[T>A]T T[T>A]A T[T>A]C T[T>A]G T[T>A]T A[T>C]A A[T>C]C A[T>C]G A[T>C]T C[T>C]A C[T>C]C C[T>C]G C[T>C]T G[T>C]A G[T>C]C G[T>C]G G[T>C]T T[T>C]A T[T>C]C T[T>C]G T[T>C]T A[T>G]A A[T>G]C A[T>G]G A[T>G]T C[T>G]A C[T>G]C C[T>G]G C[T>G]T G[T>G]A G[T>G]C G[T>G]G G[T>G]T T[T>G]A T[T>G]C T[T>G]G T[T>G]T 0.0 2.5 5.0 7.5 10.0 0 10 20 30 0 10 20 30 40 Mutations (n) D Untranscribed strand Transcribed strand Mutations (n) mmsig Fig. 4 Resolving uncertainty in mutational signature ﬁtting. A Bar chart showing the estimated SBS-MM1 contribution in 82 MM samples with and without prior melphalan exposure by mmsig (similar to Fig. 2B), with 95% CI estimated from 1000 bootstrapping iterations. Asterisks indicate statistically signiﬁcant transcriptional strand bias in characteristic trinucleotide contexts (Poisson test p < 0.05). COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-01938-0 This highlights the importance of additional measures to improve speciﬁcity. Presence of SBS35 was conﬁrmed with high conﬁdence in both of the platinum-exposed patients (i.e., non-zero 95% CI and strong transcriptional strand bias in C [C>A]C and C[C>T]C); but in none of the de novo AML cases (Fig. 4C). In one de novo AML sample (TCGAAB3001) the 95% CI was above zero (estimated SBS35 contribution 14.1%; 95% CI 7.8–20.5%), but the characteristic transcriptional strand bias was always absent. In ambiguous cases like this, it can be helpful to visually review the 96-class mutational proﬁle for presence of the key features of the mutational signature in question. As illustrated in Fig. 4D, sample TCGAAB3001 lacked the characteristic peaks of SBS35 in both C>A and C>T (the tall peak in A[C>T]G is most likely attributable to the age-related deamination of 5- methylcytosine to thymine, i.e., SBS1). This is in contrast to the two samples with known platinum exposure, showing the classical proﬁle with dominant peaks in C>T with mutations strongly favoring the transcribed strand. b l ﬁd b d h b f whole-genome sequencing data from 142 patients with CLL with mutated (n = 68) or unmutated (n = 74) IGHV gene. Using mmsig with standard ﬁlters, three patients with unmutated CLL had evidence of SBS9 (Fig. 5A); one of which also had a non-zero CI (CLL1078; Fig. 5B). This sample had an estimated SBS9 contribu- tion of 30% (95% CI 27–34%) and 1.7% of the bases in IGHV were mutated by Sanger sequencing. Unmutated IGHV status was conﬁrmed by the novel algorithm IgCaller applied to WGS data32. This patient had been classiﬁed as memory-CLL based on the epigenetic proﬁle, consistent with having passed through the germinal center30. Visual inspection of the mutational signature proﬁle did indeed show the characteristic proﬁle of SBS9, indicating that considerable genome-wide nc-AID activity can be observed despite relatively few coding mutations in the IGHV region (Fig. 5C and Fig. S5)10. It remains to be seen whether the degree of genome- wide nc-AID activity may further reﬁne the prognostic classiﬁcation of CLL. for deconstructSigs. This highlights the importance of additional measures to improve speciﬁcity. Presence of SBS35 was conﬁrmed with high conﬁdence in both of the platinum-exposed patients (i.e., non-zero 95% CI and strong transcriptional strand bias in C [C>A]C and C[C>T]C); but in none of the de novo AML cases (Fig. 4C). COMMUNICATIONS BIOLOGY \| (2021) 4:424 \| https://doi.org/10.1038/s42003-021-01938-0 \| www.nature.com/commsbio COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-01938-0 Next, we added increasing levels of noise to the simulated MM genomes and estimated the effects on mutational signature estimates (Fig. 6C). The results show that SBS-MM1, SBS5, SBS8, and SBS9 are accurately estimated by all algorithms in the absence of noise, with increasing over-estimation of particularly SBS5 by all algorithms with increasing background noise. As expected, decreasing the number of mutations also increases variability; this was also observed in real data. When the number of mutations fell below 250, there was a tendency for mmsig to over- estimate SBS5, most likely because this signature was always kept in the ﬁnal proﬁle irrespective of random variations in the data, which may lead to dropout of other mutational signatures. For observations from real data, all three algorithms showed virtually identical results when no ﬁlters were applied. After applying standard ﬁltering, the sensitivity of mutationalPatterns and deconstructSigs centered around 6% contribution, with >95% sensitivity achieved at 7–8% contribution. For mmsig, given the unique features of its error-correction approach, the sensitivity varied between mutational signatures. The sensitivity was higher for distinctive mutational signatures such as SBS2, with 100% sensitivity achieved at 4% contribution (Fig. 6A), and lower for less well-deﬁned signatures such as SBS-MM1, with >95% sensitivity achieved at 12% contribution (Fig. 6B). Next, we added increasing levels of noise to the simulated MM genomes and estimated the effects on mutational signature estimates (Fig. 6C). The results show that SBS-MM1, SBS5, SBS8, and SBS9 are accurately estimated by all algorithms in the absence of noise, with increasing over-estimation of particularly SBS5 by all algorithms with increasing background noise. As expected, decreasing the number of mutations also increases variability; this was also observed in real data. When the number of mutations fell below 250, there was a tendency for mmsig to over- estimate SBS5, most likely because this signature was always kept in the ﬁnal proﬁle irrespective of random variations in the data, which may lead to dropout of other mutational signatures. For Mutational signature ﬁtting with low mutational burden. Until this point, we have addressed mutational signature ﬁtting of complete mutational catalogs derived from WGS, consisting of thousands of mutations. However, there are many situations where it is desirable to perform mutational signature ﬁtting with much smaller numbers of mutations; whether for whole-exome or targeted sequencing data, branches in a phylogenetic tree or other applications11,23,27,33,34. COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-01938-0 A Signature SBS1 SBS5 SBS8 SBS9 deconstructSigs mmsig 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 Relative contribution IGHV mutated Unmutated C B IGHV mutated Unmutated 0.0 0.2 0.4 0.6 0.8 SBS9 (prop) B A mmsig C C>A C>G C>T T>A T>C T>G UNMUT (100 %) CLL30 UNMUT (98.3 %) CLL1078 MUT (87.8 %) CLL373 A[C>A]A A[C>A]C A[C>A]G A[C>A]T C[C>A]A C[C>A]C C[C>A]G C[C>A]T G[C>A]A G[C>A]C G[C>A]G G[C>A]T T[C>A]A T[C>A]C T[C>A]G T[C>A]T A[C>G]A A[C>G]C A[C>G]G A[C>G]T C[C>G]A C[C>G]C C[C>G]G C[C>G]T G[C>G]A G[C>G]C G[C>G]G G[C>G]T T[C>G]A T[C>G]C T[C>G]G T[C>G]T A[C>T]A A[C>T]C A[C>T]G A[C>T]T C[C>T]A C[C>T]C C[C>T]G C[C>T]T G[C>T]A G[C>T]C G[C>T]G G[C>T]T T[C>T]A T[C>T]C T[C>T]G T[C>T]T A[T>A]A A[T>A]C A[T>A]G A[T>A]T C[T>A]A C[T>A]C C[T>A]G C[T>A]T G[T>A]A G[T>A]C G[T>A]G G[T>A]T T[T>A]A T[T>A]C T[T>A]G T[T>A]T A[T>C]A A[T>C]C A[T>C]G A[T>C]T C[T>C]A C[T>C]C C[T>C]G C[T>C]T G[T>C]A G[T>C]C G[T>C]G G[T>C]T T[T>C]A T[T>C]C T[T>C]G T[T>C]T A[T>G]A A[T>G]C A[T>G]G A[T>G]T C[T>G]A C[T>G]C C[T>G]G C[T>G]T G[T>G]A G[T>G]C G[T>G]G G[T>G]T T[T>G]A T[T>G]C T[T>G]G T[T>G]T 0 50 100 0 20 40 60 0 50 100 Fig. 5 Identifying non-canonical AID-induced mutagenesis in chronic lymphocytic leukemia. A Mutational signature proﬁles of 142 patients with chronic lymphocytic leukemia (CLL) with mutated (n = 68, left) or unmutated (n = 74, right) IGHV gene, estimated using deconstructSigs (top) and mmsig (bottom). B Bar chart showing the estimated proportion of mutations caused by non-canonical AID (nc-AID; SBS9) with 95% CI generated by 1000 bootstrapping iterations. C 96-class mutational proﬁles of three patients from top to bottom: unmutated CLL without evidence of nc-AID activity; unmutated CLL based on IGHV identity (<2% mutations), but evidence of genome-wide nc-AID activity; CLL with highly mutated IGHV as well as strong nc-AID signature genome-wide. COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-01938-0 C C>A C>G C>T T>A T>C T>G UNMUT (100 %) CLL30 UNMUT (98.3 %) CLL1078 MUT (87.8 %) CLL373 A[C>A]A A[C>A]C A[C>A]G A[C>A]T C[C>A]A C[C>A]C C[C>A]G C[C>A]T G[C>A]A G[C>A]C G[C>A]G G[C>A]T T[C>A]A T[C>A]C T[C>A]G T[C>A]T A[C>G]A A[C>G]C A[C>G]G A[C>G]T C[C>G]A C[C>G]C C[C>G]G C[C>G]T G[C>G]A G[C>G]C G[C>G]G G[C>G]T T[C>G]A T[C>G]C T[C>G]G T[C>G]T A[C>T]A A[C>T]C A[C>T]G A[C>T]T C[C>T]A C[C>T]C C[C>T]G C[C>T]T G[C>T]A G[C>T]C G[C>T]G G[C>T]T T[C>T]A T[C>T]C T[C>T]G T[C>T]T A[T>A]A A[T>A]C A[T>A]G A[T>A]T C[T>A]A C[T>A]C C[T>A]G C[T>A]T G[T>A]A G[T>A]C G[T>A]G G[T>A]T T[T>A]A T[T>A]C T[T>A]G T[T>A]T A[T>C]A A[T>C]C A[T>C]G A[T>C]T C[T>C]A C[T>C]C C[T>C]G C[T>C]T G[T>C]A G[T>C]C G[T>C]G G[T>C]T T[T>C]A T[T>C]C T[T>C]G T[T>C]T A[T>G]A A[T>G]C A[T>G]G A[T>G]T C[T>G]A C[T>G]C C[T>G]G C[T>G]T G[T>G]A G[T>G]C G[T>G]G G[T>G]T T[T>G]A T[T>G]C T[T>G]G T[T>G]T 0 50 100 0 20 40 60 0 50 100 Fig. 5 Identifying non-canonical AID-induced mutagenesis in chronic lymphocytic leukemia. A Mutational signature proﬁles of 142 patients with chronic lymphocytic leukemia (CLL) with mutated (n = 68, left) or unmutated (n = 74, right) IGHV gene, estimated using deconstructSigs (top) and mmsig (bottom). B Bar chart showing the estimated proportion of mutations caused by non-canonical AID (nc-AID; SBS9) with 95% CI generated by 1000 bootstrapping iterations. C 96-class mutational proﬁles of three patients from top to bottom: unmutated CLL without evidence of nc-AID activity; unmutated CLL based on IGHV identity (<2% mutations), but evidence of genome-wide nc-AID activity; CLL with highly mutated IGHV as well as strong nc-AID signature genome-wide. SBS1, given its highly distinctive proﬁle, the estimates by mmsig were highly accurate irrespective of added noise and the number of mutations; this was also the case for SBS2. Finally, using simulated genomes, we conﬁrmed that the 95% CI estimated by mmsig contained the pre-determined contribution of that mutational signature ~94% of the time. observations from real data, all three algorithms showed virtually identical results when no ﬁlters were applied. After applying standard ﬁltering, the sensitivity of mutationalPatterns and deconstructSigs centered around 6% contribution, with >95% sensitivity achieved at 7–8% contribution. For mmsig, given the unique features of its error-correction approach, the sensitivity varied between mutational signatures. The sensitivity was higher for distinctive mutational signatures such as SBS2, with 100% sensitivity achieved at 4% contribution (Fig. 6A), and lower for less well-deﬁned signatures such as SBS-MM1, with >95% sensitivity achieved at 12% contribution (Fig. 6B). COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-01938-0 In one de novo AML sample (TCGAAB3001) the 95% CI was above zero (estimated SBS35 contribution 14.1%; 95% CI 7.8–20.5%), but the characteristic transcriptional strand bias was always absent. In ambiguous cases like this, it can be helpful to visually review the 96-class mutational proﬁle for presence of the key features of the mutational signature in question. As illustrated in Fig. 4D, sample TCGAAB3001 lacked the characteristic peaks of SBS35 in both C>A and C>T (the tall peak in A[C>T]G is most likely attributable to the age-related deamination of 5- methylcytosine to thymine, i.e., SBS1). This is in contrast to the two samples with known platinum exposure, showing the classical proﬁle with dominant peaks in C>T with mutations strongly favoring the transcribed strand. Our observations thus far have suggested that mmsig is able to conﬁdently identify highly characteristic mutational signatures such as SBS2 at low abundance, whereas the sensitivity is slightly lower for less distinctive signatures. To quantify this effect, we simulated a series of MM genomes based on the median contribution of each mutational signature observed in WGS data. Starting with such an average genome as background, we estimated the sensitivity of each algorithm to detect signatures at a given admixture (Fig. 6A, B). In concordance with our CLL can be classiﬁed based on the presence or absence of somatic hypermutation of the immunoglobulin heavy chain variable region (IGHV) (threshold at >2% mutated bases), where unmutated IGHV is strongly associated with poor outcomes2,10,28–30. We have previously shown, using mutational signature extraction algorithms, that mutated IGHV status is associated with the genome-wide footprint of nc-AID activity (i.e., SBS9), whereas the unmutated subgroup lacks this mutational process10,31. Here, we analyzed UNICATIONS BIOLOGY \| (2021) 4:424 \| https://doi.org/10.1038/s42003-021-01938-0 \| www.nature.com/commsbio 6 COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-01938-0 ARTICLE COMMUNICATIONS BIOLOGY \| (2021) 4:424 \| https://doi.org/10.1038/s42003-021-01938-0 \| www.nature.com/commsbio COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-01938-0 A B Signature SBS1 SBS5 SBS8 SBS9 IGHV mutated Unmutated deconstructSigs mmsig 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 Relative contribution 0.0 0.2 0.4 0.6 0.8 SBS9 (prop) IGHV mutated Unmutated C C>A C>G C>T T>A T>C T>G UNMUT (100 %) CLL30 UNMUT (98.3 %) CLL1078 MUT (87.8 %) CLL373 A[C>A]A A[C>A]C A[C>A]G A[C>A]T C[C>A]A C[C>A]C C[C>A]G C[C>A]T G[C>A]A G[C>A]C G[C>A]G G[C>A]T T[C>A]A T[C>A]C T[C>A]G T[C>A]T A[C>G]A A[C>G]C A[C>G]G A[C>G]T C[C>G]A C[C>G]C C[C>G]G C[C>G]T G[C>G]A G[C>G]C G[C>G]G G[C>G]T T[C>G]A T[C>G]C T[C>G]G T[C>G]T A[C>T]A A[C>T]C A[C>T]G A[C>T]T C[C>T]A C[C>T]C C[C>T]G C[C>T]T G[C>T]A G[C>T]C G[C>T]G G[C>T]T T[C>T]A T[C>T]C T[C>T]G T[C>T]T A[T>A]A A[T>A]C A[T>A]G A[T>A]T C[T>A]A C[T>A]C C[T>A]G C[T>A]T G[T>A]A G[T>A]C G[T>A]G G[T>A]T T[T>A]A T[T>A]C T[T>A]G T[T>A]T A[T>C]A A[T>C]C A[T>C]G A[T>C]T C[T>C]A C[T>C]C C[T>C]G C[T>C]T G[T>C]A G[T>C]C G[T>C]G G[T>C]T T[T>C]A T[T>C]C T[T>C]G T[T>C]T A[T>G]A A[T>G]C A[T>G]G A[T>G]T C[T>G]A C[T>G]C C[T>G]G C[T>G]T G[T>G]A G[T>G]C G[T>G]G G[T>G]T T[T>G]A T[T>G]C T[T>G]G T[T>G]T 0 50 100 0 20 40 60 0 50 100 Fig. 5 Identifying non-canonical AID-induced mutagenesis in chronic lymphocytic leukemia. A Mutational signature proﬁles of 142 patients with chronic lymphocytic leukemia (CLL) with mutated (n = 68, left) or unmutated (n = 74, right) IGHV gene, estimated using deconstructSigs (top) and mmsig (bottom). B Bar chart showing the estimated proportion of mutations caused by non-canonical AID (nc-AID; SBS9) with 95% CI generated by 1000 bootstrapping iterations. C 96-class mutational proﬁles of three patients from top to bottom: unmutated CLL without evidence of nc-AID activity; unmutated CLL based on IGHV identity (<2% mutations), but evidence of genome-wide nc-AID activity; CLL with highly mutated IGHV as well as strong nc-AID signature genome-wide. COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-01938-0 To systematically evaluate the accuracy of mutational signature ﬁtting in real data as the number of mutations decreases, we generated sets of progressively fewer mutations by random downsampling of the WGS data presented previously and estimated signature contributions using mmsig. Reducing the number of mutations had the main effect of increasing variability in the mutational signature estimates, as illustrated here in two 7 COMMUNICATIONS BIOLOGY \| (2021) 4:424 \| https://doi.org/10.1038/s42003-021-01938-0 \| www.nature.com/commsbio 7 ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-01938-0 ed multiple myeloma genomes. Mutational signature contributions were estimated for simulated mutational catalogs corresponding to le myeloma (MM) genomes. A, B Dilution series of 1–10% SBS2 (A) and 1–20% SBS-MM1 (B) contribution in a background similar to an enome. 100 simulations of 5000 mutations were performed at each dilution. Mutational signature ﬁtting was performed by mmsig, rns, and deconstructSigs without ﬁlters and using standard ﬁlters. The lines corresponding to the sensitivity without ﬁlters are superimposed orithms consistent with virtually identical results. Regarding the results after ﬁltering, mutationalPatterns and deconstructSigs showed virtually Fig. 6 Simulated multiple myeloma genomes. Mutational signature contributions were estimated for simulated mutational catalogs corresponding to average multiple myeloma (MM) genomes. A, B Dilution series of 1–10% SBS2 (A) and 1–20% SBS-MM1 (B) contribution in a background similar to an average MM genome. 100 simulations of 5000 mutations were performed at each dilution. Mutational signature ﬁtting was performed by mmsig, mutationalPatterns, and deconstructSigs without ﬁlters and using standard ﬁlters. The lines corresponding to the sensitivity without ﬁlters are superimposed for all three algorithms consistent with virtually identical results. Regarding the results after ﬁltering, mutationalPatterns and deconstructSigs showed virtually identical performance, leading to the sensitivity curves being superimposed, whereas mmsig showed either higher (A) or lower (B) sentisitivity than the other algorithms. C 100 simulations were generated of 5000, 2000, 1000, 500, 250, and 100 mutations (rows) and mutational signature ﬁtting performed by mmsig, mutationalPatterns and deconstructSigs with standard ﬁlters (columns). Boxplots show median and interquartile range with outliers drawn as dots. Each data point represents the estimated signature contribution in one simulated mutation catalog. illustrative examples: SBS2 and SBS-MM1 in MM and SBS9 in CLL, each with a distinct mutational proﬁle as well as different abundance when present (mean contribution of 6.1%, 18.7%, and 35.6%, respectively) (“Methods”). COMMUNICATIONS BIOLOGY \| (2021) 4:424 \| https://doi.org/10.1038/s42003-021-01938-0 \| www.nature.com/commsbio COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-01938-0 Strikingly, all of the three mutational signatures could be identiﬁed with high sensitivity and speciﬁcity all the way down to sets of 100 mutations using mmsig with standard settings, requiring a non-zero estimated contribu- tion for a positive signature call (Fig. 7B). With sets of 500 or more mutations, the results were similar to those obtained from the full WGS data. Applying a more stringent criterion of non- zero 95% CI resulted in ~100% speciﬁcity across the board, but at a considerable loss of sensitivity, particularly with lower mutation counts. example patients (Fig. 7A). Of note, variability markedly increased below 500 mutations, disproportionately affecting ﬂat muta- tional signatures such as SBS5, while the APOBEC-signatures and SBS1 were relatively spared. The mean contribution of each signature also changed slightly, which may be explained by the fact that SBS5 and SBS1 are always included in the ﬁnal signature proﬁle from mmsig, while other mutational signatures may disappear in some mutation sets due to random variation. Consequently, the observed means of SBS5 and SBS1 were slightly higher and the means for other signatures slightly lower than what was estimated from the full mutational catalog. We went on to estimate how the sensitivity and speciﬁcity of the mutational signature ﬁtting is affected by the variability of mutational proﬁles due to random sampling as the number of mutations decreases. Three mutational signatures were selected as Finally, we compared mutational signature ﬁtting in the full WGS catalogs with subsets of mutations corresponding to whole- 8 ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-01938-0 COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-01938-0 g. 7 Mutational signature ﬁtting with low numbers of mutations. Mutational signature contributions were estimated for each patient in 100 randomly awn catalogs of 2000, 1000, 500, 250, and 100 mutations. Boxplots show median and interquartile range with outliers drawn as dots. A Mutational gnature proﬁles of two example patients (rows), showing the estimated mutational signature contributions (columns). Each data point represents the ntribution of a given signature in one randomly drawn mutation set. B Sensitivity and speciﬁcity of mmsig to identify SBS-MM1 in MM (left), SBS2 in MM middle) and SBS9 in CLL (right) when different criteria were applied (color legend). Each data point represents the mean estimated sensitivity or eciﬁcity across 100 replicates for a given patient and mutation count. Fig. 7 Mutational signature ﬁtting with low numbers of mutations. Mutational signature contributions were estimated for each patient in 100 randomly drawn catalogs of 2000, 1000, 500, 250, and 100 mutations. Boxplots show median and interquartile range with outliers drawn as dots. A Mutational signature proﬁles of two example patients (rows), showing the estimated mutational signature contributions (columns). Each data point represents the contribution of a given signature in one randomly drawn mutation set. B Sensitivity and speciﬁcity of mmsig to identify SBS-MM1 in MM (left), SBS2 in MM (middle) and SBS9 in CLL (right) when different criteria were applied (color legend). Each data point represents the mean estimated sensitivity or speciﬁcity across 100 replicates for a given patient and mutation count. exome and targeted sequencing capture kits (“Methods”; Fig. 8). Going from WGS to WES reduced the mean number of mutations from 5437 to 245 per sample for MM and 2391 to 108 for CLL. Targeted capture panels did not yield sufﬁcient mutation counts to proceed with mutational signature analysis. The mean numbers of mutations per sample within the capture regions were 20 for MM and 1 for CLL, with a subset of patients lacking mutations altogether (2.4% for MM and 56% for CLL). Exome-based analysis of SBS-MM1 showed excellent perfor- mance compared with WGS, consistent with results from random downsampling of WGS data (Fig. 8C). However, exome-based analysis of SBS9 in CLL showed considerably lower concordance with WGS than expected from our random exome and targeted sequencing capture kits (“Methods”; Fig. 8). Discussion low cosine similarity reduction. Thus, mmsig inherently requires a larger contribution from indistinctive mutational signatures before they are called. In practice, this dynamic threshold means that mmsig is able to call low contribution of APOBEC with high accuracy, while avoiding extensive false positive calls of other (less distinctive) mutational signatures. This could be particularly relevant considering the emerging critical role of APOBEC in predicting myeloma precursor condition progression and MM clinical outcome23,38–40. We have shown how mutational signature ﬁtting can be applied to hematological cancers, highlighting the importance of careful interpretation in light of biological knowledge10. Our newly released R package mmsig is highly speciﬁc and provides a range of tools to resolve the presence or absence of a mutational sig- nature in difﬁcult cases. When done properly, signature ﬁtting is a powerful tool that can be implemented immediately in the clinic. The application of mutational signature analysis has the potential to improve prognostic models and deﬁne individualized treat- ment strategies5,6,23,35–38. Because different mutational processes occupy largely the same feature space, mutational signature analysis is subject to a degree of uncertainty. We have proposed two objective measures to control uncertainty: estimating 95% CI for the contribution of each mutational signature and checking for transcriptional strand bias typical of signatures associated with transcription-coupled repair. As we have shown empirically, requiring non-zero 95% CI to call a mutational signature as present was highly effective at improving speciﬁcity. Relying only on the error-suppression built into mmsig resulted in excellent sensitivity and speciﬁcity, parti- cularly when applied to catalogs of more than 500 mutations, which is virtually always the case with WGS data. Mutational signature analysis can also be performed on whole-exome sequencing data, with the caveat that the expected signature proﬁle in coding and non-coding regions can be quite different. Targeted capture sequencing panels covering a few megabases often yield too low mutational burdens to allow meaningful mutational signature ﬁtting for individual patients. In its essence, a mutational signature is a set of mutation types that show a characteristic pattern of co-occurrence across tumors3. Mutational signatures can be deﬁned using the 96-class system of base substitutions in their trinucleotide context but may be applied to any set of features that is biologically meaningful. Although some mutational signatures have important biological implications, they are indirect representations of underlying processes. COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-01938-0 Performance 0.0 0.2 0.4 0.6 0.8 1.0 Relative contribution B IGHV Mutated Unmutated Sample C 0.0 0.2 0.4 0.6 0.8 1.0 Relative contribution A B Melphalan No Melphalan Sample Sensitivity Specificity MM SBS MM1 MM SBS2 CLL SBS9 0.00 0.25 0.50 0.75 1.00 0.00 0.25 0.50 0.75 1.00 Performance C C B A Fig. 8 Mutational signature ﬁtting applied to whole exomes. Mutational signature contributions in mutations overlapping the SureSelect V6 + UTR exome capture kit in A MM and B CLL samples. Mutational signatures from the full whole-genome sequencing data from the same samples are shown in Figs. 2 and 5, respectively. C Performance of mutational signature ﬁtting in exome data (shown in A, B) as compared with the full whole-genome sequencing data as gold standard. COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-01938-0 Going from WGS to WES reduced the mean number of mutations from 5437 to 245 per sample for MM and 2391 to 108 for CLL. Targeted capture panels did not yield sufﬁcient mutation counts to proceed with mutational signature analysis. The mean numbers of mutations per sample within the capture regions were 20 for MM and 1 for CLL, with a subset of patients lacking mutations altogether (2.4% for MM and 56% for CLL). Exome-based analysis of SBS-MM1 showed excellent perfor- mance compared with WGS, consistent with results from random downsampling of WGS data (Fig. 8C). However, exome-based analysis of SBS9 in CLL showed considerably lower concordance with WGS than expected from our random downsampling analysis. This observation can be explained by the known behavior of the SBS9 (nc-AID) mutational process, which is predominantly active in the non-coding regions of the genome11. Indeed, exome-based estimates in our data were consistently lower than paired WGS-based estimates (mean 12%; paired T-test, p < 0.001). Enrichment of non-synonymous mutations has been described for the APOBEC mutational signatures (i.e., SBS2 and SBS13)11, but the overall APOBEC contribution in this analysis was similar in the whole-genome and exome. This propensity of different mutational signatures to affect coding vs. non-coding regions may result in differences between genome- and exome-based analysis that reﬂect under- lying biology. COMMUNICATIONS BIOLOGY \| (2021) 4:424 \| https://doi.org/10.1038/s42003-021-01938-0 \| www.nature.com/commsbio 9 9 COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-01938-0 ARTICLE Sensitivity Specificity MM SBS MM1 MM SBS2 CLL SBS9 0.00 0.25 0.50 0.75 1.00 0.00 0.25 0.50 0.75 1.00 Performance 0.0 0.2 0.4 0.6 0.8 1.0 Relative contribution 0.0 0.2 0.4 0.6 0.8 1.0 Relative contribution A B IGHV Mutated Unmutated Melphalan No Melphalan Sample Sample C SBS-MM1 Mutational signature SBS1 SBS2 SBS13 SBS5 SBS8 SBS9 SBS18 Criterion Prop > 0 95 % CI > 0 Fig. 8 Mutational signature ﬁtting applied to whole exomes. Mutational signature contributions in mutations overlapping the SureSelect V6 + UTR exome capture kit in A MM and B CLL samples. Mutational signatures from the full whole-genome sequencing data from the same samples are shown in Figs. 2 and 5, respectively. C Performance of mutational signature ﬁtting in exome data (shown in A, B) as compared with the full whole-genome sequencing data as gold standard. COMMUNICATIONS BIOLOGY \| (2021) 4:424 \| https://doi.org/10.1038/s42003-021-01938-0 \| www.nature.com/commsbio Methods Downsampling of mutational catalogs. To evaluate the effect of mutation counts on signature ﬁtting performance, we performed downsampling of the full mutation catalogs. Patient samples from MM and CLL were included in this analysis since they both had mutational signatures present in a subset of the cohort where we could deﬁne a ground truth regarding the presence or absence of the underlying mutational process (SBS-MM1 in MM and SBS9 (nc-AID) in IGHV mutated CLL). We also included SBS2 in the analysis, representing a highly distinctive signature with relatively low abundance in most cases. For each sample, we performed 100 independent draws, without replacement, of 2000 (MM only), 1000, 500, 250, 100. We also generated mutation sets similar to those obtained by whole-exome and targeted sequencing, using BED ﬁles of a commonly used exome capture kit (Agilent SureSelect V6 + UTR) and custom targeted capture panels speciﬁcally developed for MM47 and CLL48,49. Mutational signature ﬁtting with estimation of 95% CI was performed independently for each mutation set as described above. To estimate sensitivity and speciﬁcity for signature detection, we used signature calls from WGS as a gold standard (requiring non-zero 95% CI, and for SBS-MM1, the presence of transcriptional strand bias). Samples with ambiguous signature calls were removed (e.g., if the 95% CI included zero). Patients and data. Publicly available SNV data from WGS of patient samples were included in the study: 142 CLL (EGAS00000000092)28,30, 82 MM from 45 patients (EGAD00001003309 and phs000348.v2.p1)11,17,18 (7 samples out of the original 89 were removed due to incomplete data on treatment history), 47 AML (phs000178. v1.p1)43, and two therapy-related AML (EGAD00001005028)10. Mutational signature ﬁtting with mmsig. We developed mmsig as a tool for ﬂexible and easily interpretable mutational signature analysis11. At its core, mmsig takes a set of reference mutational signatures and estimates their contribution in each sample employing an expectation maximization algorithm. For each sample, mmsig attempts to reduce the number of features (i.e., reference signatures) used to explain the observed mutational proﬁle. In an iterative process, we reconstructed the 96-class mutational proﬁle for each sample after excluding each reference signature in turn. The least contributing mutational signature was censored for that sample if removal resulted in a cosine similarity reduction of <0.01. This process was subsequently repeated until no reference signatures could be removed without incurring a cosine similarity reduction of more than 0.01. Data availability p g p gy CI were generated by drawing 1000 mutational proﬁles from the multinomial distribution, where the probability that a mutation belongs to a given class (e.g., C [C>T]G) was equal to the proportion of mutations belonging to that class in the original mutational proﬁle. The number of mutations in each random set of mutations was the same as in the original proﬁle. For each random set of mutations, we repeated the entire mutational signature ﬁtting procedure as described above, ﬁnally taking the 2.5th and 97.5th percentile of the estimates for each signature. Transcriptional strand bias was assessed using a Poisson test (p < 0.05). We applied the test both to individual mutation classes independently as well as to the combinations of mutational classes most characteristic of speciﬁc mutational signatures, in order to increase power. For SBS-MM1 (melphalan signature) we combined C[C>T]A, G[C>T]A, G[C>T]C, G[C>T]G and G[C>T]T; and for SBS35 (platinum signature) we combined C[C>A]C and C[C>T]C. CI were generated by drawing 1000 mutational proﬁles from the multinomial distribution, where the probability that a mutation belongs to a given class (e.g., C [C>T]G) was equal to the proportion of mutations belonging to that class in the original mutational proﬁle. The number of mutations in each random set of mutations was the same as in the original proﬁle. For each random set of mutations, we repeated the entire mutational signature ﬁtting procedure as described above, ﬁnally taking the 2.5th and 97.5th percentile of the estimates for each signature. All the raw data used in the study are already publicly available: EGAS00000000092: 142 CLL. EGAD00001003309 and phs000348.v2.p1: 82 MM from 45 patients, 7 samples out of the original 89 were removed due to incomplete data on treatment history. phs000178. v1.p1: 47 de novo AML. EGAD00001005028: two therapy-related AML. References 1. Alexandrov, L. B. et al. Signatures of mutational processes in human cancer. Nature 500, 415–421 (2013). 2. Alexandrov, L. B. et al. The repertoire of mutational signatures in human cancer. Nature 578, 94–101 (2020). 3. Alexandrov, L. B., Nik-Zainal, S., Wedge, D. C., Campbell, P. J. & Stratton, M. 3. Alexandrov, L. B., Nik-Zainal, S., Wedge, D. C., Campbell, P. J. & Stratton, M. R. Deciphering signatures of mutational processes operative in human cancer. Cell Rep. 3, 246–259 (2013). R. Deciphering signatures of mutational processes operative in human cancer. Cell Rep. 3, 246–259 (2013). Comparison of mmsig and established mutational signature ﬁtting algorithms. To benchmark mmsig against established tools, we selected the commonly used mutational signature ﬁtting packages deconstructSigs15 and mutationalPatterns16 in R. The same reference signature catalogs were applied for all three algorithms. Comparison of mmsig and established mutational signature ﬁtting algorithms. To benchmark mmsig against established tools, we selected the commonly used mutational signature ﬁtting packages deconstructSigs15 and mutationalPatterns16 in R. The same reference signature catalogs were applied for all three algorithms. Both mmsig and deconstructSigs have built-in ﬁltering options, which can be altered or turned off entirely. mmsig supports dynamic ﬁltering based on cosine similarity as described above. deconstructSigs applies a hard threshold of signature contributions in each sample, below which all signatures are removed. mutationalPatterns does not have a built-in ﬁltering option, leading us to implement the same ﬁlter as applied by deconstructSigs as a post-processing step. 4. Nik-Zainal, S. et al. Mutational Processes Molding the Genomes of 21 Breast Cancers. Cell 149, 979–993 (2012). 5. Davies, H. et al. HRDetect is a predictor of BRCA1 and BRCA2 deﬁciency based on mutational signatures. Nat. Med. 23, 517–525 (2017). g g pp g Both mmsig and deconstructSigs have built-in ﬁltering options, which can be altered or turned off entirely. mmsig supports dynamic ﬁltering based on cosine similarity as described above. deconstructSigs applies a hard threshold of signature contributions in each sample, below which all signatures are removed. mutationalPatterns does not have a built-in ﬁltering option, leading us to implement the same ﬁlter as applied by deconstructSigs as a post-processing step. 6. Davies, H. et al. Whole-genome sequencing reveals breast cancers with mismatch repair deﬁciency. Cancer Res. 77, 4755–4762 (2017). mismatch repair deﬁciency. Cancer Res. 77, 4755–4762 (2017 7. Alexandrov, L. B. et al. Clock-like mutational processes in human somatic cells. Nat. Genet 47, 1402–1407 (2015). 8. Code availability Transcriptional strand bias was assessed using a Poisson test (p < 0.05). We applied the test both to individual mutation classes independently as well as to the combinations of mutational classes most characteristic of speciﬁc mutational signatures, in order to increase power. For SBS-MM1 (melphalan signature) we combined C[C>T]A, G[C>T]A, G[C>T]C, G[C>T]G and G[C>T]T; and for SBS35 (platinum signature) we combined C[C>A]C and C[C>T]C. mmsig is an R package and is available on GitHub: https://github.com/evenrus/mmsig. Received: 17 October 2020; Accepted: 3 March 2021; mmsig is an R package and is available on GitHub: https://github.com/evenrus/ mmsig. mmsig is an R package and is available on GitHub: https://github.com/evenrus/ mmsig. Mutational signature references. We used the two versions of the COSMIC mutational signature reference (i.e., COSMIC v2 and COSMIC v3.1), with the addition of SBS-MM1 and SBS-HSPC as previously described by our group and others1,2,10,11,44–46. The appropriate catalog of reference signatures to include in the analysis for each hematological cancer type was based on de novo signature extraction as previously reported10,11. Methods Since SBS1 and SBS5 are known to always be present in all human tumors and normal cells alike, we forced their inclusion in all samples. Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article. p The same set of reference mutational signatures can be used for each sample, or a different subset of reference signatures be speciﬁed for each sample according to prior knowledge of sample biology or de novo extraction results. ARTICLE ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-01938-0 pinpoint the mutational machinery and therapeutic suscept- ibilities in a given tumor5,10,35–37. a poisson distribution3 with lambda = 2. The weight of the noise signature relative to other signatures was pre-determined (e.g., 5%) and the relative contributions of each mutational signature was scaled accordingly. Discussion Distinguishing one signature from another is most straightforward when they involve entirely different parts of the feature-space (i.e., mutational classes), such as the two APOBEC- associated signatures SBS2 and SBS13 in the most recent COS- MIC v3.1 reference. Conversely, SBS5 and SBS8 have highly overlapping “ﬂat” proﬁles; and SBS-MM1 shows some degree of overlap with both SBS5 and SBS9. Highly distinct signatures behave differently from those who are partially overlapping and/ or “ﬂat”, depending on which mutational signature ﬁtting algo- rithm and post-processing ﬁlters are applied. g g p In conclusion, we have shown how the novel mutational sig- nature ﬁtting algorithm mmsig can be applied to identify biolo- gically and clinically important mutational processes acting in hematological cancers, including chemotherapy-related muta- tional signatures, APOBEC activity in MM and nc-AID in CLL. The tools and principles outlined here may be applicable in other cancers with a well-characterized mutational signature landscape, taking into account the speciﬁc biology and exogenous exposures of each disease. The accuracy of mutational signature ﬁtting, in general, is optimal when applied to catalogs of more than 500 mutations, which can be consistently obtained by WGS. With WGS there is also the opportunity to integrate multiple data types, such as patterns structural variation31,41,42, to better Highly characteristic mutational signatures in the latest refer- ence (i.e., COSMIC v3.1) such as SBS2 and SBS13 will stand out over the background even if their relative contribution to the mutational proﬁle is low (<6%). Error-suppression using hard cut-offs, such as the standard 6% threshold imposed by decon- structSigs, will be inappropriately strict in these cases. Conversely, mmsig will tend to keep these signatures in the proﬁle because removing them will considerably penalize the cosine similarity between the reconstructed mutational proﬁle and the original. The opposite can be said for ﬂat mutational signatures, which may be falsely removed by mmsig because all the mutations can be re-classiﬁed to another mutational signature with a relatively COMMUNICATIONS BIOLOGY \| (2021) 4:424 \| https://doi.org/10.1038/s42003-021-01938-0 \| www.nature.com/commsbio 10 COMMUNICATIONS BIOLOGY \| (2021) 4:424 \| https://doi.org/10.1038/s42003-021-01938-0 \| www.nature.com/commsbio Additional information 33. Maura, F. et al. Role of AID in the temporal pattern of acquisition of driver mutations in multiple myeloma. Leukemia https://doi.org/10.1038/s41375- 019-0689-0 (2019). Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s42003-021-01938-0. Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s42003-021-01938-0. 34. Ziccheddu, B. et al. 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As a basis for simulated MM genomes, we generated an average MM mutational signature proﬁle where the contribution of each signature was set to the median contribution of that signature in MM samples with SBS- MM1. The resulting signature proﬁle consisted of 4.8% SBS1, 5.1% SBS2, 34.1% SBS5, 18.2% SBS8, 18.9% SBS9 and 18.8% SBS-MM1. We then calculated the expected contribution of each of the 96 mutational classes from the weighted sum of each mutational signature reference. 9. Kucab, J. E. et al. A compendium of mutational signatures of environmental agents. Cell 177, 821–836 (2019). g 10. Maura, F. et al. A practical guide for mutational signature analysis in hematological malignancies. Nat. Commun. 10, 2969 (2019). 11. Rustad, E. H. et al. Timing the initiation of multiple myeloma. Nat. Commun. 11, 1917 (2020). 12. Degasperi, A. et al. A practical framework and online tool for mutational signature analyses show intertissue variation and driver dependencies. Nat. Cancer 1, 249–263 (2020). To generate simulated genomes, each simulated mutation was drawn from a multinomial distribution of 96 mutational classes. The probability to draw a mutation of a given class was equal to the relative contribution of that mutational class in the average MM proﬁle. 13. Hoang, P. H., Cornish, A. J., Dobbins, S. E., Kaiser, M. & Houlston, R. S. Mutational processes contributing to the development of multiple myeloma. Blood Cancer J. 9, 60 (2019). The sensitivity for each mutational signature was estimated by adding a progressively larger contribution of that signature (1–20%) to a background mutational proﬁle consisting of an MM genome without the signature in question. The background proﬁle was scaled to maintain a constant proportion of each signature relative to the others. 14. Hoang, P. H. et al. Whole-genome sequencing of multiple myeloma reveals oncogenic pathways are targeted somatically through multiple mechanisms. Leukemia https://doi.org/10.1038/s41375-018-0103-3 (2018). Leukemia https://doi.org/10.1038/s41375-018-0103-3 (2018). 15. Rosenthal, R., McGranahan, N., Herrero, J., Taylor, B. S. & Swanton, C. deconstructSigs: delineating mutational processes in single tumors To generate random noise, we generated a “noise signature” independently for each simulation, drawing the contribution of each of the 96 mutational classes from 11 COMMUNICATIONS BIOLOGY \| (2021) 4:424 \| https://doi.org/10.1038/s42003-021-01938-0 \| www.nature.com/commsbio Additional information Whole genome sequencing provides evidence of two biologically and clinically distinct entities of asymptomatic monoclonal gammopathies: progressive versus stable myeloma precursor condition. Nat. Commun. https://doi.org/10.1038/s41467-021-22140-0 (2021). 41. Maura, F. et al. Genomic landscape and chronological reconstruction of driver events in multiple myeloma. Nat. Commun. 10, 3835 (2019). 12 COMMUNICATIONS BIOLOGY \| (2021) 4:424 \| https://doi.org/10.1038/s42003-021-01938-0 \| www.nature.com/commsbio
https://openalex.org/W4288474759	https://zenodo.org/records/7541730/files/eBook_Consternacao-suicida.pdf	Portuguese	null	Consternação suicida: manual de apoio para pessoas com depressão e condutas suicidas	null	2,022	cc-by	30,473	Copyright © Pimenta Cultural, alguns direitos reservados. Copyright do texto © 2022 o autor. Copyright da edição © 2022 Pimenta Cultural. Esta obra é licenciada por uma Licença Creative Commons: Atribuição-NãoComercial-SemDerivações 4.0 Internacional - (CC BY-NC-ND 4.0). Os termos desta licença estão disponíveis em: <https://creativecommons. org/licenses/>. Direitos para esta edição cedidos à Pimenta Cultural. O conteúdo publicado não representa a posição oficial da Pimenta Cultural. Doutores e Doutoras Adilson Cristiano Habowski Universidade La Salle, Brasil Adriana Flávia Neu Universidade Federal de Santa Maria, Brasil Adriana Regina Vettorazzi Schmitt Instituto Federal de Santa Catarina, Brasil Aguimario Pimentel Silva Instituto Federal de Alagoas, Brasil Alaim Passos Bispo Universidade Federal do Rio Grande do Norte, Brasil Alaim Souza Neto Universidade Federal de Santa Catarina, Brasil Alessandra Knoll Universidade Federal de Santa Catarina, Brasil Alessandra Regina Müller Germani Universidade Federal de Santa Maria, Brasil Aline Corso Universidade do Vale do Rio dos Sinos, Brasil Aline Wendpap Nunes de Siqueira Universidade Federal de Mato Grosso, Brasil Ana Rosangela Colares Lavand Universidade Federal do Pará, Brasil André Gobbo Universidade Federal da Paraíba, Brasil Andressa Wiebusch Universidade Federal de Santa Maria, Brasil Andreza Regina Lopes da Silva Universidade Federal de Santa Catarina, Brasil Angela Maria Farah Universidade de São Paulo, Brasil Adilson Cristiano Habowski Universidade La Salle, Brasil Adriana Flávia Neu Universidade Federal de Santa Maria, Brasil Adriana Regina Vettorazzi Schmitt Instituto Federal de Santa Catarina, Brasil Aguimario Pimentel Silva Instituto Federal de Alagoas, Brasil Alaim Passos Bispo Universidade Federal do Rio Grande do Norte, Brasil Alaim Souza Neto Universidade Federal de Santa Catarina, Brasil Alessandra Knoll Universidade Federal de Santa Catarina, Brasil Alessandra Regina Müller Germani Universidade Federal de Santa Maria, Brasil Aline Corso Universidade do Vale do Rio dos Sinos, Brasil Aline Wendpap Nunes de Siqueira Universidade Federal de Mato Grosso, Brasil Ana Rosangela Colares Lavand Universidade Federal do Pará, Brasil André Gobbo Universidade Federal da Paraíba, Brasil Andressa Wiebusch Universidade Federal de Santa Maria, Brasil Andreza Regina Lopes da Silva Universidade Federal de Santa Catarina, Brasil Angela Maria Farah Universidade de São Paulo, Brasil Anísio Batista Pereira Universidade Federal de Uberlândia, Brasil Antonio Edson Alves da Silva Universidade Estadual do Ceará, Brasil Antonio Henrique Coutelo de Moraes Universidade Federal de Rondonópolis, Brasil Arthur Vianna Ferreira Universidade do Estado do Rio de Janeiro, Brasil Ary Albuquerque Cavalcanti Junior Universidade do Estado da Bahia, Brasil Asterlindo Bandeira de Oliveira Júnior Universidade Federal da Bahia, Brasil Bárbara Amaral da Silva Universidade Federal de Minas Gerais, Brasil Bernadétte Beber Universidade Federal de Santa Catarina, Brasil Bruna Carolina de Lima Siqueira dos Santos Universidade do Vale do Itajaí, Brasil Bruno Rafael Silva Nogueira Barbosa Universidade Federal da Paraíba, Brasil Caio Cesar Portella Santos Instituto Municipal de Ensino Superior de São Manuel, Brasil Carla Wanessa do Amaral Caffagni Universidade de São Paulo, Brasil Carlos Adriano Martins Universidade Cruzeiro do Sul, Brasil Carlos Jordan Lapa Alves Universidade Estadual do Norte Fluminense Darcy Ribeiro, Brasil Caroline Chioquetta Lorenset Universidade Federal de Santa Catarina, Brasil Anísio Batista Pereira Universidade Federal de Uberlândia, Brasil Antonio Edson Alves da Silva Universidade Estadual do Ceará, Brasil Antonio Henrique Coutelo de Moraes Universidade Federal de Rondonópolis, Brasil Arthur Vianna Ferreira Universidade do Estado do Rio de Janeiro, Brasil Ary Albuquerque Cavalcanti Junior Universidade do Estado da Bahia, Brasil Asterlindo Bandeira de Oliveira Júnior Universidade Federal da Bahia, Brasil Bárbara Amaral da Silva Universidade Federal de Minas Gerais, Brasil Bernadétte Beber Universidade Federal de Santa Catarina, Brasil Bruna Carolina de Lima Siqueira dos Santos Universidade do Vale do Itajaí, Brasil Bruno Rafael Silva Nogueira Barbosa Universidade Federal da Paraíba, Brasil Caio Cesar Portella Santos Instituto Municipal de Ensino Superior de São Manuel, Brasil Carla Wanessa do Amaral Caffagni Universidade de São Paulo, Brasil Carlos Adriano Martins Universidade Cruzeiro do Sul, Brasil Carlos Jordan Lapa Alves Universidade Estadual do Norte Fluminense Darcy Ribeiro, Brasil Caroline Chioquetta Lorenset Universidade Federal de Santa Catarina, Brasil Cássio Michel dos Santos Camargo Universidade Federal do Rio Grande do Sul-Faced, Brasil Christiano Martino Otero Avila Universidade Federal de Pelotas, Brasil Cláudia Samuel Kessler Universidade Federal do Rio Grande do Sul, Brasil Cristiane Silva Fontes Universidade Federal de Minas Gerais, Brasil Daniela Susana Segre Guertzenstein Universidade de São Paulo, Brasil Daniele Cristine Rodrigues Universidade de São Paulo, Brasil Dayse Centurion da Silva Universidade Anhanguera, Brasil Dayse Sampaio Lopes Borges Universidade Estadual do Norte Fluminense Darcy Ribeiro, Brasi Diego Pizarro Instituto Federal de Brasília, Brasil Dorama de Miranda Carvalho Escola Superior de Propaganda e Marketing, Brasil Edson da Silva Universidade Federal dos Vales do Jequitinhonha e Mucuri, Brasi Elena Maria Mallmann Universidade Federal de Santa Maria, Brasil Eleonora das Neves Simões Universidade Federal do Rio Grande do Sul, Brasil Eliane Silva Souza Universidade do Estado da Bahia, Brasil Elvira Rodrigues de Santana Universidade Federal da Bahia, Brasil Éverly Pegoraro Universidade Federal do Rio de Janeiro, Brasil Fábio Santos de Andrade Universidade Federal de Mato Grosso, Brasil Fabrícia Lopes Pinheiro Universidade Federal do Estado do Rio de Janeiro, Brasil Felipe Henrique Monteiro Oliveira Universidade Federal da Bahia, Brasil Fernando Vieira da Cruz Universidade Estadual de Campinas, Brasil Gabriella Eldereti Machado Universidade Federal de Santa Maria, Brasil Germano Ehlert Pollnow Universidade Federal de Pelotas, Brasil Geymeesson Brito da Silva Universidade Federal de Pernambuco, Brasil Giovanna Ofretorio de Oliveira Martin Franchi Universidade Federal de Santa Catarina, Brasil Cássio Michel dos Santos Camargo Universidade Federal do Rio Grande do Sul-Faced, Brasil Christiano Martino Otero Avila Universidade Federal de Pelotas, Brasil Cláudia Samuel Kessler Universidade Federal do Rio Grande do Sul, Brasil Cristiane Silva Fontes Universidade Federal de Minas Gerais, Brasil Daniela Susana Segre Guertzenstein Universidade de São Paulo, Brasil Daniele Cristine Rodrigues Universidade de São Paulo, Brasil Dayse Centurion da Silva Universidade Anhanguera, Brasil Dayse Sampaio Lopes Borges Universidade Estadual do Norte Fluminense Darcy Ribeiro, Brasil Diego Pizarro Instituto Federal de Brasília, Brasil Dorama de Miranda Carvalho Escola Superior de Propaganda e Marketing, Brasil Edson da Silva Universidade Federal dos Vales do Jequitinhonha e Mucuri, Brasil Elena Maria Mallmann Universidade Federal de Santa Maria, Brasil Eleonora das Neves Simões Universidade Federal do Rio Grande do Sul, Brasil Eliane Silva Souza Universidade do Estado da Bahia, Brasil Elvira Rodrigues de Santana Universidade Federal da Bahia, Brasil Éverly Pegoraro Universidade Federal do Rio de Janeiro, Brasil Fábio Santos de Andrade Universidade Federal de Mato Grosso, Brasil Fabrícia Lopes Pinheiro Universidade Federal do Estado do Rio de Janeiro, Brasil Felipe Henrique Monteiro Oliveira Universidade Federal da Bahia, Brasil Fernando Vieira da Cruz Universidade Estadual de Campinas, Brasil Gabriella Eldereti Machado Universidade Federal de Santa Maria, Brasil Germano Ehlert Pollnow Universidade Federal de Pelotas, Brasil Geymeesson Brito da Silva Universidade Federal de Pernambuco, Brasil Giovanna Ofretorio de Oliveira Martin Franchi Universidade Federal de Santa Catarina, Brasil Handherson Leyltton Costa Damasceno Universidade Federal da Bahia, Brasil Hebert Elias Lobo Sosa Universidad de Los Andes, Venezuela Helciclever Barros da Silva Sales Instituto Nacional de Estudos e Pesquisas Educacionais Anísio Teixeira, Brasil Helena Azevedo Paulo de Almeida Universidade Federal de Ouro Preto, Brasil Hendy Barbosa Santos Faculdade de Artes do Paraná, Brasil Humberto Costa Universidade Federal do Paraná, Brasil Igor Alexandre Barcelos Graciano Borges Universidade de Brasília, Brasil Inara Antunes Vieira Willerding Universidade Federal de Santa Catarina, Brasil Ivan Farias Barreto Universidade Federal do Rio Grande do Norte, Brasil Jaziel Vasconcelos Dorneles Universidade de Coimbra, Portugal Jean Carlos Gonçalves Universidade Federal do Paraná, Brasil Jocimara Rodrigues de Sousa Universidade de São Paulo, Brasil Joelson Alves Onofre Universidade Estadual de Santa Cruz, Brasil Jónata Ferreira de Moura Universidade São Francisco, Brasil Jorge Eschriqui Vieira Pinto Universidade Estadual Paulista Júlio de Mesquita Filho, Brasil Jorge Luís de Oliveira Pinto Filho Universidade Federal do Rio Grande do Norte, Brasil Juliana de Oliveira Vicentini Universidade de São Paulo, Brasil Julierme Sebastião Morais Souza Universidade Federal de Uberlândia, Brasil Junior César Ferreira de Castro Universidade Federal de Goiás, Brasil Katia Bruginski Mulik Universidade de São Paulo, Brasil Laionel Vieira da Silva Universidade Federal da Paraíba, Brasil Leonardo Pinheiro Mozdzenski Universidade Federal de Pernambuco, Brasil Lucila Romano Tragtenberg Pontifícia Universidade Católica de São Paulo, Brasil Lucimara Rett Universidade Metodista de São Paulo, Brasil Avaliadores e avaliadoras Ad-Hoc Alessandra Figueiró Thornton Universidade Luterana do Brasil, Brasil Alexandre João Appio Universidade do Vale do Rio dos Sinos, Brasil Bianka de Abreu Severo Universidade Federal de Santa Maria, Brasil Carlos Eduardo Damian Leite Universidade de São Paulo, Brasil Catarina Prestes de Carvalho Instituto Federal Sul-Rio-Grandense, Brasil Elisiene Borges Leal Universidade Federal do Piauí, Brasil Elizabete de Paula Pacheco Universidade Federal de Uberlândia, Brasil Elton Simomukay Universidade Estadual de Ponta Grossa, Brasil Francisco Geová Goveia Silva Júnior Universidade Potiguar, Brasil Indiamaris Pereira Universidade do Vale do Itajaí, Brasil Alessandra Figueiró Thornton Universidade Luterana do Brasil, Brasil Alexandre João Appio Universidade do Vale do Rio dos Sinos, Brasil Bianka de Abreu Severo Universidade Federal de Santa Maria, Brasil Carlos Eduardo Damian Leite Universidade de São Paulo, Brasil Catarina Prestes de Carvalho Instituto Federal Sul-Rio-Grandense, Brasil Elisiene Borges Leal Universidade Federal do Piauí, Brasil Elizabete de Paula Pacheco Universidade Federal de Uberlândia, Brasil Elton Simomukay Universidade Estadual de Ponta Grossa, Brasil Francisco Geová Goveia Silva Júnior Universidade Potiguar, Brasil Indiamaris Pereira Universidade do Vale do Itajaí, Brasil Jacqueline de Castro Rimá Universidade Federal da Paraíba, Brasil Lucimar Romeu Fernandes Instituto Politécnico de Bragança, Brasil Marcos de Souza Machado Universidade Federal da Bahia, Brasil Michele de Oliveira Sampaio Universidade Federal do Espírito Santo, Brasil Samara Castro da Silva Universidade de Caxias do Sul, Brasil Thais Karina Souza do Nascimento Instituto de Ciências das Artes, Brasil Viviane Gil da Silva Oliveira Universidade Federal do Amazonas, Brasil Weyber Rodrigues de Souza Pontifícia Universidade Católica de Goiás, Brasil William Roslindo Paranhos Universidade Federal de Santa Catarina, Brasil Alessandra Figueiró Thornton Universidade Luterana do Brasil, Brasil Alexandre João Appio Universidade do Vale do Rio dos Sinos, Brasil Bianka de Abreu Severo Universidade Federal de Santa Maria, Brasil Carlos Eduardo Damian Leite Universidade de São Paulo, Brasil Catarina Prestes de Carvalho Instituto Federal Sul-Rio-Grandense, Brasil Elisiene Borges Leal Universidade Federal do Piauí, Brasil Elizabete de Paula Pacheco Universidade Federal de Uberlândia, Brasil Elton Simomukay Universidade Estadual de Ponta Grossa, Brasil Francisco Geová Goveia Silva Júnior Universidade Potiguar, Brasil Indiamaris Pereira Universidade do Vale do Itajaí, Brasil Jacqueline de Castro Rimá Universidade Federal da Paraíba, Brasil Lucimar Romeu Fernandes Instituto Politécnico de Bragança, Brasil Marcos de Souza Machado Universidade Federal da Bahia, Brasil Michele de Oliveira Sampaio Universidade Federal do Espírito Santo, Brasil Samara Castro da Silva Universidade de Caxias do Sul, Brasil Thais Karina Souza do Nascimento Instituto de Ciências das Artes, Brasil Viviane Gil da Silva Oliveira Universidade Federal do Amazonas, Brasil Weyber Rodrigues de Souza Pontifícia Universidade Católica de Goiás, Brasil William Roslindo Paranhos Universidade Federal de Santa Catarina, Brasil Jacqueline de Castro Rimá Universidade Federal da Paraíba, Brasil Lucimar Romeu Fernandes Instituto Politécnico de Bragança, Brasil Marcos de Souza Machado Universidade Federal da Bahia, Brasil Michele de Oliveira Sampaio Universidade Federal do Espírito Santo, Brasil Samara Castro da Silva Universidade de Caxias do Sul, Brasil Thais Karina Souza do Nascimento Instituto de Ciências das Artes, Brasil Viviane Gil da Silva Oliveira Universidade Federal do Amazonas, Brasil Weyber Rodrigues de Souza Pontifícia Universidade Católica de Goiás, Brasil William Roslindo Paranhos Universidade Federal de Santa Catarina, Brasil Cássio Michel dos Santos Camargo Universidade Federal do Rio Grande do Sul-Faced, Brasil Christiano Martino Otero Avila Universidade Federal de Pelotas, Brasil Cláudia Samuel Kessler Universidade Federal do Rio Grande do Sul, Brasil Cristiane Silva Fontes Universidade Federal de Minas Gerais, Brasil Daniela Susana Segre Guertzenstein Universidade de São Paulo, Brasil Daniele Cristine Rodrigues Universidade de São Paulo, Brasil Dayse Centurion da Silva Universidade Anhanguera, Brasil Dayse Sampaio Lopes Borges Universidade Estadual do Norte Fluminense Darcy Ribeiro, Brasil Diego Pizarro Instituto Federal de Brasília, Brasil Dorama de Miranda Carvalho Escola Superior de Propaganda e Marketing, Brasil Edson da Silva Universidade Federal dos Vales do Jequitinhonha e Mucuri, Brasil Elena Maria Mallmann Universidade Federal de Santa Maria, Brasil Eleonora das Neves Simões Universidade Federal do Rio Grande do Sul, Brasil Eliane Silva Souza Universidade do Estado da Bahia, Brasil Elvira Rodrigues de Santana Universidade Federal da Bahia, Brasil Éverly Pegoraro Universidade Federal do Rio de Janeiro, Brasil Fábio Santos de Andrade Universidade Federal de Mato Grosso, Brasil Fabrícia Lopes Pinheiro Universidade Federal do Estado do Rio de Janeiro, Brasil Felipe Henrique Monteiro Oliveira Universidade Federal da Bahia, Brasil Fernando Vieira da Cruz Universidade Estadual de Campinas, Brasil Gabriella Eldereti Machado Universidade Federal de Santa Maria, Brasil Germano Ehlert Pollnow Universidade Federal de Pelotas, Brasil Geymeesson Brito da Silva Universidade Federal de Pernambuco, Brasil Giovanna Ofretorio de Oliveira Martin Franchi Universidade Federal de Santa Catarina, Brasil Cássio Michel dos Santos Camargo Universidade Federal do Rio Grande do Sul-Faced, Brasil Christiano Martino Otero Avila Universidade Federal de Pelotas, Brasil Cláudia Samuel Kessler Universidade Federal do Rio Grande do Sul, Brasil Cristiane Silva Fontes Universidade Federal de Minas Gerais, Brasil Daniela Susana Segre Guertzenstein Universidade de São Paulo, Brasil Daniele Cristine Rodrigues Universidade de São Paulo, Brasil Dayse Centurion da Silva Universidade Anhanguera, Brasil Dayse Sampaio Lopes Borges Universidade Estadual do Norte Fluminense Darcy Ribeiro, Brasil Diego Pizarro Instituto Federal de Brasília, Brasil Dorama de Miranda Carvalho Escola Superior de Propaganda e Marketing, Brasil Edson da Silva Universidade Federal dos Vales do Jequitinhonha e Mucuri, Brasil Elena Maria Mallmann Universidade Federal de Santa Maria, Brasil Eleonora das Neves Simões Universidade Federal do Rio Grande do Sul, Brasil Eliane Silva Souza Universidade do Estado da Bahia, Brasil Elvira Rodrigues de Santana Universidade Federal da Bahia, Brasil Éverly Pegoraro Universidade Federal do Rio de Janeiro, Brasil Fábio Santos de Andrade Universidade Federal de Mato Grosso, Brasil Fabrícia Lopes Pinheiro Universidade Federal do Estado do Rio de Janeiro, Brasil Felipe Henrique Monteiro Oliveira Universidade Federal da Bahia, Brasil Fernando Vieira da Cruz Universidade Estadual de Campinas, Brasil Gabriella Eldereti Machado Universidade Federal de Santa Maria, Brasil Germano Ehlert Pollnow Universidade Federal de Pelotas, Brasil Geymeesson Brito da Silva Universidade Federal de Pernambuco, Brasil Giovanna Ofretorio de Oliveira Martin Franchi Universidade Federal de Santa Catarina, Brasil Rosane de Fatima Antunes Obregon Universidade Federal do Maranhão, Brasil Samuel André Pompeo Universidade Estadual Paulista Júlio de Mesquita Filho, Brasil Sebastião Silva Soares Universidade Federal do Tocantins, Brasil Silmar José Spinardi Franchi Universidade Estadual Paulista Júlio de Mesquita Filho, Brasil Simone Alves de Carvalho Universidade de São Paulo, Brasil Simoni Urnau Bonfiglio Universidade Federal da Paraíba, Brasil Stela Maris Vaucher Farias Universidade Federal do Rio Grande do Sul, Brasil Tadeu João Ribeiro Baptista Universidade Federal do Rio Grande do Norte Taiane Aparecida Ribeiro Nepomoceno Universidade Estadual do Oeste do Paraná, Brasil Taíza da Silva Gama Universidade de São Paulo, Brasil Tania Micheline Miorando Universidade Federal de Santa Maria, Brasil Tarcísio Vanzin Universidade Federal de Santa Catarina, Brasil Tascieli Feltrin Universidade Federal de Santa Maria, Brasil Tayson Ribeiro Teles Universidade Federal do Acre, Brasil Thiago Barbosa Soares Universidade Federal de São Carlos, Brasil Thiago Camargo Iwamoto Pontifícia Universidade Católica de Goiás, Brasil Thiago Medeiros Barros Universidade Federal do Rio Grande do Norte, Brasil Tiago Mendes de Oliveira Centro Federal de Educação Tecnológica de Minas Gerais, Brasil Valdir Lamim Guedes Junior Universidade de São Paulo, Brasil Vanessa Elisabete Raue Rodrigues Universidade Estadual de Ponta Grossa, Brasil Vania Ribas Ulbricht Universidade Federal de Santa Catarina, Brasil Wellington Furtado Ramos Universidade Federal de Mato Grosso do Sul, Brasil Wellton da Silva de Fatima Instituto Federal de Alagoas, Brasil Yan Masetto Nicolai Universidade Federal de São Carlos, Brasil Rosane de Fatima Antunes Obregon Universidade Federal do Maranhão, Brasil Samuel André Pompeo Universidade Estadual Paulista Júlio de Mesquita Filho, Brasil Sebastião Silva Soares Universidade Federal do Tocantins, Brasil Silmar José Spinardi Franchi Universidade Estadual Paulista Júlio de Mesquita Filho, Brasil Simone Alves de Carvalho Universidade de São Paulo, Brasil Simoni Urnau Bonfiglio Universidade Federal da Paraíba, Brasil Stela Maris Vaucher Farias Universidade Federal do Rio Grande do Sul, Brasil Tadeu João Ribeiro Baptista Universidade Federal do Rio Grande do Norte Taiane Aparecida Ribeiro Nepomoceno Universidade Estadual do Oeste do Paraná, Brasil Taíza da Silva Gama Universidade de São Paulo, Brasil Tania Micheline Miorando Universidade Federal de Santa Maria, Brasil Tarcísio Vanzin Universidade Federal de Santa Catarina, Brasil Tascieli Feltrin Universidade Federal de Santa Maria, Brasil Tayson Ribeiro Teles Universidade Federal do Acre, Brasil Thiago Barbosa Soares Universidade Federal de São Carlos, Brasil Thiago Camargo Iwamoto Pontifícia Universidade Católica de Goiás, Brasil Thiago Medeiros Barros Universidade Federal do Rio Grande do Norte, Brasil Tiago Mendes de Oliveira Centro Federal de Educação Tecnológica de Minas Gerais, Brasil Valdir Lamim Guedes Junior Universidade de São Paulo, Brasil Vanessa Elisabete Raue Rodrigues Universidade Estadual de Ponta Grossa, Brasil Vania Ribas Ulbricht Universidade Federal de Santa Catarina, Brasil Wellington Furtado Ramos Universidade Federal de Mato Grosso do Sul, Brasil Wellton da Silva de Fatima Instituto Federal de Alagoas, Brasil Yan Masetto Nicolai Universidade Federal de São Carlos, Brasil Manoel Augusto Polastreli Barbosa Universidade Federal do Espírito Santo, Brasil Marcelo Nicomedes dos Reis Silva Filho Universidade Estadual do Oeste do Paraná, Brasil Marcio Bernardino Sirino Universidade Federal do Estado do Rio de Janeiro, Brasil Marcos Pereira dos Santos Universidad Internacional Iberoamericana del Mexico, México Marcos Uzel Pereira da Silva Universidade Federal da Bahia, Brasil Maria Aparecida da Silva Santandel Universidade Federal de Mato Grosso do Sul, Brasil Maria Cristina Giorgi Centro Federal de Educação Tecnológica Celso Suckow da Fonseca, Brasil Maria Edith Maroca de Avelar Universidade Federal de Ouro Preto, Brasil Marina Bezerra da Silva Instituto Federal do Piauí, Brasil Michele Marcelo Silva Bortolai Universidade de São Paulo, Brasil Mônica Tavares Orsini Universidade Federal do Rio de Janeiro, Brasil Nara Oliveira Salles Universidade do Estado do Rio de Janeiro, Brasil Neli Maria Mengalli Pontifícia Universidade Católica de São Paulo, Brasil Patricia Bieging Universidade de São Paulo, Brasil Patricia Flavia Mota Universidade do Estado do Rio de Janeiro, Brasil Raul Inácio Busarello Universidade Federal de Santa Catarina, Brasil Raymundo Carlos Machado Ferreira Filho Universidade Federal do Rio Grande do Sul, Brasil Roberta Rodrigues Ponciano Universidade Federal de Uberlândia, Brasil Robson Teles Gomes Universidade Federal da Paraíba, Brasil Rodiney Marcelo Braga dos Santos Universidade Federal de Roraima, Brasil Rodrigo Amancio de Assis Universidade Federal de Mato Grosso, Brasil Rodrigo Sarruge Molina Universidade Federal do Espírito Santo, Brasil Rogério Rauber Universidade Estadual Paulista Júlio de Mesquita Filho, Brasi Rosane de Fatima Antunes Obregon Universidade Federal do Maranhão, Brasil Samuel André Pompeo Universidade Estadual Paulista Júlio de Mesquita Filho, Brasil Sebastião Silva Soares Universidade Federal do Tocantins, Brasil Silmar José Spinardi Franchi Universidade Estadual Paulista Júlio de Mesquita Filho, Brasil Simone Alves de Carvalho Universidade de São Paulo, Brasil Simoni Urnau Bonfiglio Universidade Federal da Paraíba, Brasil Stela Maris Vaucher Farias Universidade Federal do Rio Grande do Sul, Brasil Tadeu João Ribeiro Baptista Universidade Federal do Rio Grande do Norte Taiane Aparecida Ribeiro Nepomoceno Universidade Estadual do Oeste do Paraná, Brasil Taíza da Silva Gama Universidade de São Paulo, Brasil Tania Micheline Miorando Universidade Federal de Santa Maria, Brasil Tarcísio Vanzin Universidade Federal de Santa Catarina, Brasil Tascieli Feltrin Universidade Federal de Santa Maria, Brasil Tayson Ribeiro Teles Universidade Federal do Acre, Brasil Thiago Barbosa Soares Universidade Federal de São Carlos, Brasil Thiago Camargo Iwamoto Pontifícia Universidade Católica de Goiás, Brasil Thiago Medeiros Barros Universidade Federal do Rio Grande do Norte, Brasil Tiago Mendes de Oliveira Centro Federal de Educação Tecnológica de Minas Gerais, Brasil Valdir Lamim Guedes Junior Universidade de São Paulo, Brasil Vanessa Elisabete Raue Rodrigues Universidade Estadual de Ponta Grossa, Brasil Vania Ribas Ulbricht Universidade Federal de Santa Catarina, Brasil Wellington Furtado Ramos Universidade Federal de Mato Grosso do Sul, Brasil Wellton da Silva de Fatima Instituto Federal de Alagoas, Brasil Yan Masetto Nicolai Universidade Federal de São Carlos, Brasil PARECER E REVISÃO POR PARES Os textos que compõem esta obra foram submetidos para avaliação do Conselho Editorial da Pimenta Cultural, bem como revisados por pares, sendo indicados para a publicação. Patricia Bieging Raul Inácio Busarello Direção editorial Patricia Bieging Editora executiva Landressa Rita Schiefelbein Coordenadora editorial Caroline dos Reis Soares Assistente editorial Raul Inácio Busarello Diretor de criação Ligia Andrade Machado Assistente de arte Lucas Andrius de Oliveira Peter Valmorbida Editoração eletrônica Myoceanstudio - Freepik.com Imagens da capa Caroline dos Reis Soares Revisão Robson Marins de Abreu Autor SUMÁRIO Introdução....................................................................................... 11 Dedicatória...................................................................................... 12 Capítulo 1 Existe um perfil para pessoas suicidas?....................................... 13 Tipos de suicídios................................................................................17 O perigo de uma tentativa de suicídio frustrada.................................18 Intoxicação por informações falsas.....................................................18 Equívocos sobre o suicídio..................................................................19 O drama de um suicida.......................................................................19 Obstáculos que impedem você de ser feliz........................................20 Refugiar-se em uma falsa tranquilidade..............................................21 A dor do tempo....................................................................................23 O trauma nos impede de sermos quem realmente queremos ser............................................................24 Maltrato infantil.....................................................................................24 Traumas de infância.............................................................................25 É possível curar um trauma.................................................................27 Curar a criança interior.........................................................................27 O medo patológico de não ser amado...............................................28 Vozes destrutivas.................................................................................29 Como fazer as pazes com nossa voz interior crítica..............................................................29 Voz do passado..........................................................................................30 Voz do medo...............................................................................................30 Voz da vitimização.......................................................................................30 Voz da ansiedade........................................................................................30 Quais são os tipos de distúrbios de ansiedade?...............................................................31 A selva escura......................................................................................32 O efeito esponja...................................................................................33 Seletividade empática..........................................................................33 É possível ser emocionalmente forte?.................................................34 O desafio de uma personalidade turbulenta.......................................35 O que é personalidade borderline?............................................................35 Causas........................................................................................................35 Sintomas.....................................................................................................36 Exaustão mental...................................................................................36 Como identificar a exaustão mental...........................................................37 Como superar a exaustão emocional.........................................................37 Dependência emocional......................................................................39 Diferença entre dependência e codependência.................................40 Tipos de pessoas.................................................................................41 Melancolia ..................................................................................................41 Capítulo 2 Fatores suicidas............................................................................. 44 Ideação suicida....................................................................................47 Automutilação.............................................................................................49 Controlar as emoções.................................................................................50 O risco de automutilar-se............................................................................50 Quais são os sinais de automutilação?......................................................51 Por que algumas pessoas se automutilam?..............................................52 Qual é a visão do suicida............................................................................52 Lembranças de um suicida........................................................................54 Robson........................................................................................................56 Consequências do abuso...........................................................................57 Suicídio econômico.....................................................................................58 O suicídio de uma pessoa que amamos....................................................58 Capítulo 3 Medidas de prevenção................................................................... 61 Quais seriam as estratégias para evitar o suicídio?..........................................................................62 Capítulo 4 Depressão....................................................................................... 64 O que causa a depressão?.................................................................65 Não julgue...................................................................................................66 Os riscos.....................................................................................................66 O que é tristeza...........................................................................................67 Depressão temporária................................................................................67 Depressão agressiva..................................................................................67 Sentimentos de culpa.................................................................................68 Dependência terapêutica............................................................................68 É possível superar a depressão?................................................................69 Como ajudar uma pessoa com depressão?..............................................69 Diretrizes de comportamento para o bem-estar emocional................................................................70 Capítulo 5 Ansiedade....................................................................................... 75 Melhorando a autoestima....................................................................78 A caixa de crenças 79 Qual é a visão do suicida............................................................................52 Lembranças de um suicida........................................................................54 Robson........................................................................................................56 Consequências do abuso...........................................................................57 Suicídio econômico.....................................................................................58 O suicídio de uma pessoa que amamos....................................................58 Liberdade de consciência...........................................................................81 Técnica para resolver conflitos...................................................................83 Treinamentos e técnicas que deveria usar.................................................84 Outras recomendações para manejar o estresse......................................85 Técnica básica............................................................................................86 Uma reeducação emocional ......................................................................88 Inoculação do estresse...............................................................................89 O que significa estar em crise?...................................................................90 Existem quatro fases em uma crise.....................................................91 Fase de contato...................................................................................98 Fase de calibração do problema.........................................................98 Fase de busca de solução..................................................................99 Fase de realização...............................................................................99 Fase de monitoramento.....................................................................100 Resolução de conflitos ......................................................................101 Aparecimento de respostas fisiológicas e emocionais inadequadas......109 Em nível técnico........................................................................................109 Promova uma boa comunicação..............................................................110 Tomada de decisão por consenso...........................................................111 Atitudes frente ao conflito.........................................................................113 Considerações finais.................................................................... 118 Sobre o autor................................................................................ SUMÁRIO 119 INTRODUÇÃO S U M Á R I O Este não é um livro de autoajuda com orientações sobre como você deve viver, mas um manual de apoio emocional, pois algumas pessoas acreditam que a maioria dos livros de autoajuda nada mais é do que propaganda de marketing para ganhar dinheiro. Mas penso: se livros de autoajuda não ajudam, por que conti- nuamos lendo livros com conteúdo de autoajuda escondido em pe- quenas palavras? Qualquer livro que lemos pode nos ajudar de forma independente, seja de autoajuda ou não, a chave é saber ter um jul- gamento crítico ao ler. Talvez muitos estejam fartos dos típicos livros de melhoria rápida, quando a realidade é diferente. Seja qual for o livro, seja de autoajuda ou espiritual, filosófico, etc. Nenhum desses livros tem o poder de mudar sua vida se você não estiver realmente determinado a se desintoxicar completamente de tudo o que está des- truindo você. A maior frustração é acreditar que só porque funcionou para outra pessoa, pode funcionar para você também. Você tem que estar preparado para as frustrações. Ler um livro em busca de uma solução rápida e milagrosa é trapacear com a vida, se você tem uma crença em algo que está lhe limitando de encontrar a paz que você tanto precisa, então você deve começar a trocar o chip e se refazer de novo. Aprender a administrar as emoções é ser mais responsável com a vida, porque quando não sabemos como administrá-las somos como um carro desgovernado prestes a bater. S U M Á R I O 11 DEDICATÓRIA S U M Á R I O Dedico ao meu grande amigo Fermín Manteca que sempre es- teve disposto a me ajudar em tudo, ao Urtix que me acompanha há anos, a todos os que necessitam apoio e uma palavra amiga em tempo difícil e ao meu querido companheiro Ignácio Comaleras Briegas por ser parte de minha vida. S U M Á R I O 12 Capítulo 1 EXISTE UM PERFIL PARA PESSOAS SUICIDAS?1 EXISTE UM PERFIL PARA PESSOAS SUICIDAS? Capítulo 1 EXISTE UM PERFIL PARA PESSOAS SUICIDAS?1 EXISTE UM PERFIL PARA PESSOAS SUICIDAS? O suicídio pode estar ligado tanto a fatores psicológicos quanto a pessoas que não sofrem de nenhum tipo de patologia psicológica. Pes- soas com tendências suicidas tendem a apresentar alguns padrões ex- tremos de descontrole emocional. Outras já, por sofrerem a pressão do capitalismo brutal e implacável, onde ter e possuir é mais importante que ser, a maior doença que estamos tendo se chama “consumo excessivo”. S U M Á R I O Mas, como posso identificar uma pessoa que tem tendência suicida? Cuidado! Existem pessoas que podemos conhecer há anos, que nunca apresentaram nenhum tipo de sinal de depressão e no final acabam tirando a própria vida. Sinais a serem observados: Sinais a serem observados: Sinais a serem observados: • Fala que sente vontade de desistir de tudo, porque se sente cansado de lutar e não conseguir nada; cansado de lutar e não conseguir nada; • Pensamentos constante de suicídio (acreditam que se estivesse morto tudo seria mais fácil); • Mudança de rotinas diárias; • Não sente vontade de fazer nada; • Passa o dia todo na cama sem querer se levantar; • Alterações de humor; • Sem esperança ou sentido de viver; • Estar ansioso, agitado; • Usa drogas e bebidas alcoólicas (autodestruição); • Quer isolar-se; • Frustração excessiva; • Pensamentos constante de suicídio (acreditam que se estivesse morto tudo seria mais fácil); S U M Á R I O 14 S U M Á R I O • Descuido pela aparência física; • Perda de apetite; • Mudanças extremas de personalidade; • Descuidado com a higiene. Muitas vezes acreditamos que quando uma pessoa diz que quer morrer é porque está chamando a atenção, devemos ter um pouco de empatia, há pessoas que não apresentam sinais de comportamento sui- cida, estas pessoas carregam dentro de si todo o peso de suas emo- ções. Há casos de pessoas que não pretendiam tirar a própria vida, mas infelizmente em um momento de impulso saiu do controle e acabou acontecendo. Por exemplo, uma pessoa que é ativamente suicida, é fácil perceber o comportamento suicida, porém, os suicidas passivos, são difíceis de identificar, pois tentam enfrentar a vida por eles mesmos sem buscar ajuda psicológica, e, quando não há mais ferramentas suficien- tes para enfrentar os problemas que os torturam psicologicamente, entra em um desespero que os faz acreditar que a solução é tirar a própria vida. O que acontece quando alguém acredita que o suicídio é a única maneira de escapar ou descansar? O nível de estresse nos enlouquece, nos fazendo ver as coisas de um ângulo mais catastrófico, circunstân- cias como desemprego, faltam de renda, solidão e maltrato psicológico. S U M Á R I O As tentativas de suicídio podem causar esgotamento psicoló- gico ao suicida, como também a seus familiares, pois os sentimentos suicidas são transitórios, aprender a administrar a impulsividade em momentos de pressão é a chave para não cometer tolices que poste- riormente podem levar ao arrependimento. Pensamentos suicidas: Pensamentos suicidas: • Sentimento de fracasso; • Sentimento de fracasso; 15 • Sentimento de rejeição; • Comparação com outros; • Culpa; • Dependência emocional; • Baixa autoestima; • Medo; • Autoexigência; • Falta de confiança; • Incapacidade de ver as coisas boas que se tem; • Preocupação excessiva pela morte; • Sentindo ou parecendo sentir-se preso ou sem esperança. • Sentimento de rejeição; • Comparação com outros; • Culpa; • Dependência emocional; • Baixa autoestima; • Medo; • Autoexigência; • Falta de confiança; • Incapacidade de ver as cois • Preocupação excessiva pel • Sentindo ou parecendo sen S U M Á R I O • Incapacidade de ver as coisas boas que se tem; • Incapacidade de ver as coisas boas que se tem; • Preocupação excessiva pela morte; • Sentindo ou parecendo sentir-se preso ou sem esperança. • Descuidado com a higiene. Infelizmente, em alguns países da América do Sul, a psicologia é vista como: algo que só os ricos têm acesso, é que a depressão e uma doença de ricos. Já que tem uma visão que a depressão e um problema espiritual e não mental, alguns líderes religiosos acreditam que não precisamos de profissionais que sejam capazes de nos ajudar a enfrentar as situações difíceis do dia a dia. S U M Á R I O Muitas vezes acreditamos que os amigos são o tipo de psicólogo de que precisamos, porque confiamos a eles nossos segredos mais íntimos, mais penso que muitos de nossos amigos não têm ferramen- tas e conhecimentos suficientes para certos tipos de dificuldades. Nem sempre os amigos poderão nos ajudar, pois até eles enfrentam suas batalhas internas e externas. Tenha em mente que nem todos os que têm condutas suicidas são depressivos e nem todos os que sofrem de- pressões são suicidas, sim, pode parecer estranho, mas a maioria das 16 pessoas que são depressivos têm medo de morrer, são como os típicos mortos-vivos, são aqueles que expressam palavras que querem tirar a própria vida, que querem morrer, que estão cansados de viver, mas na realidade a maioria deles tem medo da morte. Então, você me pergun- ta, por que fazem isso? Porque estão viciados, acostumados a jogar o papel de vítimas, buscando manipular seu ambiente para ser sempre o centro das atenções, pois exigem e necessitam atenção, carinho e apoio e por isso penso que eles não estão dispostos a tirar a própria vida sabendo que não serão mais o centro das atenções, o papel de vítima é uma opção e não uma realidade, o vitimizador usa seus sofrimentos como uma apresentação externa para comover outras pessoas. S U M Á R I O TIPOS DE SUICÍDIOS 1. Suicídio impulsivo; 2. Suicídio coletivo (a maioria sendo por motivos religiosos em que eles fazem lavagem cerebral); 2. Suicídio coletivo (a maioria sendo por motivos religiosos em que eles fazem lavagem cerebral); 2. Suicídio coletivo (a maioria sendo por motivos religiosos em que eles fazem lavagem cerebral); 3. Suicídio induzido; 3. Suicídio induzido; S U M Á R I O 4. Tentativas; 5. Envenenamento por drogas; 6. Por arma de fogo. 6. Por arma de fogo. Quantas vezes já ouvimos que um suicida é egoísta, covarde, que não teve coragem de enfrentar as situações da vida, é impossível evitar o sofrimento, porque, se você projeta um mundo perfeito, seria melhor ir para a lua. A vida não tem culpa pelos erros que cometemos e nem mesmo pelas nossas frustrações. Morrer é algo que vai tocar a todos, alguns tem 17 medo da não existência e outros acreditam que exista uma vida melhor depois que partirmos. Independente da crença que você tem, ou não, o fato de viver já é uma dádiva. Tenha em mente que a vida não tem culpa das coisas estúpidas que você fez, e o conceito de que a vida é muito difícil, é uma falsa crença de culpar a vida pelos erros que se cometem, realmente se a vida fosse injusta não o permitiria levantar-se pela manhã. S U M Á R I O O PERIGO DE UMA TENTATIVA DE SUICÍDIO FRUSTRADA Nem todo suicida quer realmente se matar, o que procura é uma forma de acalmar a dor que sente. O suicida frustrado é aquele que tenta cometer o ato, mas depois se arrepende, e, por causa de sua ação, tem uma reação dolorosa como: ficar paraplégico. EQUÍVOCOS SOBRE O SUICÍDIO S U M Á R I O Não gosto da expressão: “chamar atenção”, porque minimiza, muitas vezes fazendo crer que o suicida está procurando atenção sem realmente ter coragem de cometer o ato. Então, quais são as crenças errôneas típicas que conhecemos: • Quem diz não faz (alguns acreditam que é algum tipo de chan- tagem emocional); • Quem pensa em suicídio é porque tem uma doença mental; • Desafiar alguém a cometer suicídio (se uma pessoa tem comporta- mentos suicidas, não a desafie tentando provar se é capaz ou não). Todos os dias há pessoas que cometem suicídio e algumas de- las antes mesmo de cometer o ato, já disseram o que planejavam fazer, mas não levam a sério. Você tem que entender que as pessoas mais velhas cometem menos suicídio do que os jovens. Não é fácil falar so- bre suicídio, pois infelizmente há muitos assuntos que evitamos como: aborto, homossexualidade, AIDS, racismo, uma sociedade onde a de- mocracia e o socialismo mentem mais do que falam. S U M Á R I O INTOXICAÇÃO POR INFORMAÇÕES FALSAS Estamos vivendo momentos muito transcendentais, pois, com o avanço da tecnologia podemos perceber que estamos tendo uma overdose de informações e muitos não conseguem avaliar o que es- tão lendo ou ouvindo simplesmente comem toda a falácia que estão oferecendo sem ao menos pesquisar. As pessoas suicidas muitas ve- zes têm crenças disfuncionais que não são reais, porque elas só têm um excesso de informações tóxicas. Lembre-se de que não existem apenas pessoas tóxicas, mas também muitas informações veneno- sas que destroem, pois informações tóxicas geram uma distorção do que é viver a vida na realidade. S U M Á R I O 18 O DRAMA DE UM SUICIDA O suicida vive um drama sem fim, pois, ao mesmo tempo em que quer morrer, por outro lado não quer. O ato suicida é um ato de vida e não de morte, como muitos pensam. E para isso quero lembrar o que escrevi antes, nem todo suicida quer morrer, só busca uma forma de viver sem sofrimento, o que eu vejo como muito difícil estar em uma sociedade como a que vivemos 19 sem sofrer. O perigo de viver uma vida confortável é que isso impossi- bilita que você cresça como pessoa, já que o lado bom da vida são os desafios que nos fazem moldar nosso caráter e nossa personalidade. sem sofrer. O perigo de viver uma vida confortável é que isso impossi- bilita que você cresça como pessoa, já que o lado bom da vida são os desafios que nos fazem moldar nosso caráter e nossa personalidade. S U M Á R I O OBSTÁCULOS QUE IMPEDEM VOCÊ DE SER FELIZ 1. Desejar o que você não pode ter; 2. Gastar muita energia dando explicação; 3. Querer agradar a todos; 4. Autoexigência; 5. Amizades tóxicas; 6. Procrastinação; 7. O falso julgamento; 8. Transtorno paranoico; 9. Sintoma extremo de desconfiança; 10. A autopunição; 11. Culpa; 12. Medo; 13. Sempre questionar e não buscar uma mudança real; 14. Acreditar nas respostas erradas; 15. Querer se encaixar em certos tipos de padrões que só limitam você S U M Á R I O 20 16. Querer tudo no curto prazo sem esforço; S U M Á R I O 17. Encontrar atalhos na vida; 18. Cercar-se de pessoas pessimistas que só farão você se sentir como elas; 19. Permitir que críticas destrutivas paralisassem sua vida; 20. Não acreditar em suas virtudes; 20. Não acreditar em suas virtudes; 21. Tentar resolver os problemas dos outros, sem dar prioridade aos seus. 21. Tentar resolver os problemas dos outros, sem dar prioridade aos seus. É você quem decide que vida quer ter. Viver a vida dos outros só vai lhe mostrar com o tempo que você não passou de um fantoche. Você tem que entender que não é possível agradar a todos e se você tentar é como pular de um penhasco. Aprenda a dizer não, sem se sentir culpado, faça o que quiser, mesmo sabendo que sempre haverá pessoas sem vida próprias prontas para o criticar por tudo. Você não precisa ser “amigo” de ninguém, pois amigos verda- deiros serão aqueles que sempre estarão ao seu lado quando você mais precisar. Pare de buscar salvadores, você tem que aprender a defender por você mesmo, cada dia, mas tenho visto que as pessoas têm perdido a empatia, a generosidade. Então, se você esperar por alguém o salvar, realmente, sinto muito, mas perderá toda sua vida aprisionado ao sofrimento. S U M Á R I O REFUGIAR-SE EM UMA FALSA TRANQUILIDADE A conexão emocional é um território desconhecido para muitos, uma vez que identificar e interpretar emoções pode produzir conflitos, tan- to racionais quanto emocionais. Evitá-los é o que a maioria faz por medo. 21 O que sinto neste momento? Não é apenas saber responder, mas encontrar uma forma de encontrar a solução sem se julgar. S U M Á R I O É impossível se livrar das emoções, porque fazem parte da nos- sa natureza humana, basta agir com razão, sem permitir que a emoção nos sabote. As pessoas que são rápidas em responder muitas vezes são porque podem ter certo tipo de conhecimento, mas não inteligên- cia emocional e, segundo elas, seu ponto de vista não importa, o que elas dizem é a verdade absoluta. Transformar erros em aprendizado é para poucos, já que a gran- de maioria prefere viver cheia de rancores e raiva... Refugiar-se em uma falsa tranquilidade é tentar camuflar suas emoções para que outras pessoas o vejam como uma boa pessoa e não como você realmente é. Todos nós temos um lado oculto e é nor- mal, o problema é quando criamos certos tipos de personagens para tentar camuflar quem realmente somos. Recentemente percebi coisas que antes me afetavam e agora não me afetam tanto, mas continuo com minha “cruzada”. É como quem faz o Caminho de Aparecida do Norte para se conectar de alguma forma com o lado mais espiritual. Hoje penso que o caminho mais longo que deve- mos traçar é o das nossas emoções. Como posso entrar em contato com o lado espiritual, se meu lado emocional é como um campo minado? S U M Á R I O E se você colocasse um pouco de teatro em seus conflitos emo- cionais? Explico-lhe. Seria como começar a representar um conflito; não dê tanta importância aquele sentimento que antes o destruía e o impossibilitava de avançar, canalizar e vivenciar a experiência emocio- nal sem que ela saia do controle. Quando teremos drama novamente? Você sabia que tem pes- soas que utilizam o teatro como forma de manejar suas emoções, sem- pre olhamos os artistas como pessoas que não tem vergonha de nada, 22 quando na verdade existe alguns que encontraram apoio no teatro para aprender a sentir e transmitir tudo aquilo que por si mesmo não podiam. S U M Á R I O A DOR DO TEMPO O trauma é um fardo emocional quando a criança ou adulto são expostos a algum tipo de situação que produz um evento trau- mático, como: trauma pós-guerra, morte de um membro da família, sequestros, estupro, etc. Temos que entender que nem todo tipo de situação difícil na vida produz trauma, há pessoas que são capazes de enfrentar muitas situações difíceis como: fome, dormir na rua, desemprego, não ter ne- nhum membro da família; e mesmo assim eles levam as coisas com filosofia. Talvez você diga que são pessoas de caráter forte, mas eu prefiro chamá-las de estoicos. O trauma traz consigo culpa, medo, ansiedade, depressão. Quando ocorre na infância, gera um estado de sentimento em que a criança em fase adulta para se defender tem comportamentos ex- plosivos, uma irritabilidade que muitas vezes outras pessoas não en- tendem. Esses estilos de sobrevivência nascem como estratégia de adaptação para lidar com experiências traumáticas. S U M Á R I O Há pessoas que sofrem abuso sexual na infância, e quando crescem têm como resposta ao trauma um comportamento em que lhes custa ter relação sexual, outras não gostam de serem tocadas. Crianças que foram abusadas na infância, na idade adulta têm dificul- dade em se relacionar com pessoas do mesmo sexo. 23 O TRAUMA NOS IMPEDE DE SERMOS QUEM REALMENTE QUEREMOS SER S U M Á R I O Tenha cuidado ao chamar alguém de tóxico sem conhecer seu passado, vejo que é muito elegante chamar de “tóxico” qualquer tipo de relacionamento e amizade, talvez porque as pessoas que não en- tendem de psicologia adorem parecer inteligentes, não confunda uma pessoa com problemas emocionais com tóxicos. MALTRATO INFANTIL O maltrato infantil é um dos grandes problemas da sociedade. Pais frustrados que descarregam tudo em seus filhos, crendo que a disciplina mais adequada seja bater, sem esquecer que isso se chama maltrato. O castigo físico sempre foi a via mais fácil para os pais para educar sem entender o limite que existe, uma coisa é corrigir a conduta de um filho e outra é maltratá-lo. Cada dia conheço jovens, até mesmo pessoas maiores que le- vam uma bagagem pesada de um passado cheio de dor, onde não compreendem porque os seus pais, lhes batiam tanto, talvez você seja daqueles que diz: se batia é porque merecia, algo de ruim fez. Independentemente do que a criança tenha feito existe um limite que devemos deixar claro entre correção e agressão. S U M Á R I O O maltrato marca por toda a vida a pessoa que sofre, limitando sua autoestima, fazendo essa pessoa sentir uma necessidade extrema de amor, a maoria das mortes por maltrato infantil são classificadas como um acidente. Nossa sociedade segue crendo que disciplinar como os seus pais antes faziam e educar, esta falsa crença produz ao 24 menor uma sensação de ódio, medo, frustração, limitação, impedindo até mesmo seu rendimento escolar. S U M Á R I O Qualquer violência física ou mental, lesão ou abuso, negligên- cia ou tratamento negligente, maus-tratos ou exploração, enquanto a criança é considerada maltrato. É POSSÍVEL CURAR UM TRAUMA SÓ é possível curar um trauma se você permitir. Primeiro você tem que tentar sair dos padrões de vitimização. A maioria das pessoas que sofrem qualquer categoria de trauma se isola acreditando que es- tão mais seguros em casa, sem ninguém para machucá-los. Sentem que o exterior os assusta, sem entender que a única maneira de su- perar o trauma é enfrentá-lo. Talvez você possa dizer: “parece fácil”. Confesso que para mim não foi fácil, porque eu tive que passar por muitos traumas, que, com o tempo, eu tive que decidir entre superá-los ou deixá-los me destruir, entre morrer e viver eu escolhi me salvar. S U M Á R I O TRAUMAS DE INFÂNCIA O trauma infantil ocorre quando qualquer criança experimenta um evento negativo real ou ameaçador, uma série de eventos e situa- ções que causam sofrimento emocional e sobrecarrega sua capaci- dade de lidar com estas emoções. A maioria das crianças que sofrem algum tipo de trauma na infância, na idade adulta terá grandes pro- blemas para socializar. O trauma infantil pode causar transtorno de estresse pós-trau- mático, estas lembranças dolorosas fazem com que o menor em sua fase adulta recorde sempre do sucedido, tornando o escravo destes pensamentos negativos, produzindo sintomas como: S U M Á R I O • Pesadelos; • Sudoração noturna; • Ansiedade; • Alterações de humor; • Problemas para dormir; • Dificuldade em relacionar-se; • Comportamentos agressivos; 25 • Dificuldades de concentração. Adolescente com traumas pode: • Estar ansioso ou deprimido; • Envolver-se em comportamentos arriscados ou autodestrutivos (por exemplo, uso indevido de drogas e álcool, direção peri- gosa, promiscuidade sexual, sexo desprotegido, automutilação, atividade ilegal); • Sentir intensa culpa, raiva ou vergonha; • Ter uma visão negativa das pessoas e da sociedade; • Em alguns casos, eles têm pensamentos persistentes sobre sui- cídio ou busca de vingança. Sintomas de trauma infantil na juventude: • Necessidade de querer ajudar aos demais; • Buscar aprovação dos demais; • Ser muito complacente; • Sintomas de codependência; • Sintomas de dependência emocional; • Estar sempre em alerta; • Ter pensamentos que todos estão contra eles; • Tomar tudo como pessoal; • Ter medo do abandono; S U M Á R I O S U M Á R I O 26 • Não dar importância a si mesmo porque acredita que a neces- sidade dos outros é mais importante que a sua; S U M Á R I O • Permitir comportamentos abusivos; • Atrair pessoas com os mesmos sintomas que eles; • Atrair pessoas com os mesmos sintomas que eles; • Ser submisso. • Ser submisso. CURAR A CRIANÇA INTERIOR O nosso “EU” adulto ferido não compreende que aquela criança indefesa agora é uma pessoa capaz de se defender. O “EU” adulto é a consciência, referindo-se a nossa responsabilidade de tomar decisões e assumir nossos atos). Existem três estados do nosso “EU” exemplo: • Estado criança: aquele em que se encontram todas as fanta- sias, emoções; • Estado criança: aquele em que se encontram todas as fanta- sias, emoções; 27 • Estado pai: no qual colocam-se os limites; S U M Á R I O • Estado criança submissa: é aquele que satisfaz a os demais; • Estado criança rebelde: é a que sabe se defender. Aprender a curar sua criança interior é um processo lento, em que você terá de buscar ajuda terapêutica para conseguir, quando eu faço contato com minha criança interior ferida, posso, então, começar a ter uma vida mais tranquila sem tantas paranoias e sentimentos de culpas. VOZES DESTRUTIVAS Quem nunca teve pensamentos negativos, esta pequena voz que sempre o acusa, fazendo-o sentir-se incapaz de fazer nada na vida, estes pensamentos tentam limitá-lo, questioná-lo. Esta voz au- todestrutiva que se manifesta em momentos de tomada de decisão. S U M Á R I O O MEDO PATOLÓGICO DE NÃO SER AMADO Infelizmente às vezes associamos a obediência ao afeto, o afeto é uma das necessidades que temos como seres humanos, a maioria das pessoas que têm esse medo é porque na infância não tiveram a atenção, o afeto e a proteção de seus pais. As crianças que tiveram a devida atenção desenvolverão uma vida saudável e eficaz, enquanto outras terão uma personalidade estruturada de acordo com o que os outros esperam que elas sejam. S U M Á R I O Em alguns casos este medo pode ser devido à baixa autoesti- ma, em outros pode ser uma necessidade de compensar o afeto não recebido dos pais, razão pela qual existem muitos casais conflitantes, pois eles têm que manter o papel de pai/mãe ao mesmo tempo. Quando você desempenha o papel de pai/mãe o relacionamen- to se torna excessivo, porque você começa a dizer o que deveria e não deveria fazer o que deveria ter feito, impedindo assim que a pessoa assuma sua autonomia. As pessoas que sofreram abusos na infância, na fase adulta têm esta necessidade de se sentirem protegidas, procuram alguém para 28 protegê-las, seu inconsciente as faz acreditar que ainda são crianças in- defesas, quando na verdade já são adultos capazes de se defenderem. S U M Á R I O As pessoas com medo de não serem amadas têm tendência a se desvalorizar, são inseguras, sensíveis, têm uma visão irrealista do mundo, sempre assumindo um papel submisso. Elas podem até evitar o contato com os outros e se isolar socialmente por medo de rejeição. Se ao menos soubéssemos amar sem expectativas, sem pretensões, sem esperar ser retribuídos teríamos menos frustração. No entanto, somos humanos, com deficiências e necessida- des. Para trabalhar sobre o medo, a primeira coisa a fazer é entender que você vive em uma sociedade na qual você será infelizmente re- jeitado ou rejeitada um dia. Quando você deixa de criar expectativas e de idealizar as pessoas, tudo flui muito melhor, deixe de desempe- nhar o papel catastrófico. COMO FAZER AS PAZES COM NOSSA VOZ INTERIOR CRÍTICA A primeira coisa é identificar a que tipo de vozes você está preso, reconhecendo que isso o ajudará a ser capaz de lidar com elas e adotar uma mudança de crença mental, isso o ajudará a ver os pensamentos 29 do ponto de vista de um forasteiro e não como declarações verdadeiras. Estes tipos de vozes muitas vezes não pedem sua permissão, mas o dominam, absorvendo sua paz de espírito. S U M Á R I O Que tipo de voz você ouve atualmente? Voz da vitimização S U M Á R I O A voz da vitimização sustenta um papel passivo, com frases como: por que tudo de ruim sempre me acontece? Isso abala a rea- lidade, acreditando que o que acontece com eles não tem controle. Voz do medo A voz do medo é aquela voz irracional que limita você, que o impede de alcançar seus objetivos. Voz do passado Há memórias do passado que são gratificantes, o problema é quando o tipo de voz que você ouve o impede de viver no presente, pois seu passado pode trancá-lo em uma prisão mental. Voz da ansiedade A armadilha mental cheia de perigos e ameaças irrealistas. O sentimento de ansiedade é uma parte normal da vida, o pro- blema surge quando há uma preocupação constante, a ansiedade 30 surge como um sinal de alerta quando a pessoa não consegue lidar com alguma situação na vida, neste momento há um pensamento dis- torcido, um conflito entre a realidade e a fantasia. S U M Á R I O O tipo de fantasia pode ser: medo de morrer, medo de estar so- zinho nesta vida, medo de um membro da família morrer, dando assim uma sensação desagradável ao corpo. QUAIS SÃO OS TIPOS DE DISTÚRBIOS DE ANSIEDADE? A ansiedade pode ser considerada um distúrbio quando o senti- mento é contínuo e não desaparece, interferindo com a vida social, os tipos de distúrbios de ansiedade são: 1. Desordem generalizada de ansiedade; 2. Distúrbios de pânico; 3. Transtorno obsessivo-compulsivo (TOC); 4. Fobias; 5. Transtorno de Estresse Pós-Traumático (PTSD). Note que nem todos os tipos de ansiedade são os mesmos. Os sintomas comuns de ansiedade são os seguintes: • Sentimento de nervosismo, agitação ou tensão; • Sentimento de perigo iminente, pânico ou catástrofe; • Aumento do ritmo cardíaco; 1. Desordem generalizada de ansiedade; S U M Á R I O 31 • Respiração rápida (hiperventilação); • Suando; • Tremendo; • Sentimento de fraqueza ou cansaço. Alguns outros sintomas: • Você se sente culpado depois de comer; • Você come demais e ainda não se sente cheio; • Você come até vomitar. S U M Á R I O • Você come demais e ainda não se sente cheio; • Você come demais e ainda não se sente cheio; • Você come até vomitar. O EFEITO ESPONJA Conhecido como um círculo vicioso de absorver os problemas de outras pessoas. O problema é que essas pessoas são frequente- mente as que mais sofrem, quando assumem toda a responsabilidade pelos outros, não gostam de ver ninguém sofrer, sem entender que a dor é muitas vezes um processo da vida. Se todos nós pudéssemos ter uma vida perfeita, mas a reali- dade é diferente. É bom ajudar os outros, mas quando nos sentimos obrigados a ajudá-los, a obrigação provoca ansiedade, estresse, fadiga mental, etc. S U M Á R I O A SELVA ESCURA Há pessoas que se sentem frustradas porque não conseguem se relacionar com seus colegas de trabalho. Sei que o local de trabalho é muitas vezes como um campo de batalha, não se culpe e não se sin- ta menos do que qualquer outra pessoa só porque você não aguentou tanta pressão e teve de desistir. É melhor para sua saúde mental do que tentar se encaixar em um trabalho que o faz sentir-se péssimo. S U M Á R I O Nosso local de trabalho e como uma escola que nos ensina a conviver e para conviver tem que aprender a respeitar e ser respeita- dos, mas muitas vezes por necessidades somos obrigados a suportar coisas que nos leva a um limite extremo. Escutar fofocas, discussões, ver como seu chefe grita com você, o explora. O bom de tudo é que hoje existe uma lei para o trabalhador você necessita ser forte e, em momentos de injustiça, denunciar qual- quer categoria de maltrato tanto psicológico e físico no trabalho para que não gere uma depressão. 32 O que evitar se você sofre de ansiedade: S U M Á R I O • Café; • Álcool. SELETIVIDADE EMPÁTICA Pouco a pouco estou aprendendo que não posso interromper o processo de responsabilidade dos outros, meu sonho como ado- lescente era ser psicólogo porque queria resolver os problemas dos outros, mas entendi que também não era capaz de resolver os meus. Aprendi, de acordo com as crenças que me haviam ensinado, que deveria pensar sempre nos outros primeiro e que era egoísta pensar em mim mesmo, o problema é que agora percebo qual é a diferença entre “ego” e “eu” (consciência). 33 Independentemente do estado emocional dos outros, devemos aprender que o que temos que fazer é ouvir, dar uma opinião, mas não assumir estes problemas como se fossem nossos. Em qualquer processo terapêutico o psicólogo apenas orienta e é o paciente que decide se quer ou não seguir em frente. Você não pode se sentir res- ponsável pelo que o outro sente, pensa ou faz. S U M Á R I O Há muito tempo eu tinha uma amizade da qual tive que me distanciar. Falar com ele era como pisar em cascas de ovos, tudo o ofendia, ele só queria ouvir coisas legais, eu perdia muito tempo aconselhando-o quando ele tinha seus estados de crise, e ciúmes porque eu não podia ter nenhuma amizade a não ser a dele, e cada vez que eu tentava era inútil. Até que percebi que ele não estava procurando nenhuma mu- dança, apenas queria alguém para passar o seu tempo. Fui perce- bendo que o único que estava perdendo aqui era eu, já que me sentia muito cansado, com muita ansiedade porque muitas vezes eu não sabia como ajudá-lo, até que aprendi a não me responsabilizar pelos problemas dos outros, porque quando era minha vez de resolver os meus, quase ninguém me ouvia. S U M Á R I O É POSSÍVEL SER EMOCIONALMENTE FORTE? O primeiro passo para ser emocionalmente forte é aprender a administrar nossas próprias emoções. O segundo é acompanhar e não assumir a responsabilidade pelos problemas das outras pessoas. O terceiro é aprender a não aceitar qualquer crítica sem analisar se é produtiva para sua vida. Estamos cercados cada dia de assassinos, não com armas e sim com palavras, frases em que você não consegue um monte de 34 afirmação que não tem sentido algum só porque a vida deles não saiu como eles queriam. Ser forte e blindar-se destas palavras que são, mas veloz que uma bala de revólver. S U M Á R I O O que é personalidade borderline? Conhecida como borderline, as pessoas que sofrem de bor- derline têm mudanças emocionais bruscas de felicidade intensa, para irritabilidade, ansiedade, raiva, muitas pessoas confundem esta patologia como se fosse bipolar, pois alguns dos sintomas são muito semelhantes e aqueles que sofrem sintomas constantes. Não há cura para os que sofrem de borderline, mas há tratamen- to, o que muitas vezes leva tempo para alcançar resultados. Causas S U M Á R I O A causa de ter borderline é desconhecida. Pensa-se que esteja relacionado a fatores genéticos, familiares e sociais. Os fatores de risco incluem: Os fatores de risco incluem: • Abandono na infância ou adolescência; • Vida familiar dissociativa; • Má comunicação na família; • Abuso sexual, físico ou emocional. 35 Sintomas S U M Á R I O Comprenda que existen varios tipos de trastornos de personali- dad e este son algunos: • Transtorno limite de personalidade; • Transtorno de personalidades dependente; • Transtorno histriónico de personalidades; • Transtorno narcisista de personalidades; • Trastorno de personalidad obsesivo-compulsiva; • As personas que sofrem de borderline tem comportamentos im- pulsivos, são muito emotivas, quero ressaltar que, apesar desta patologia, o que eles sofrem é verdadeiro, e algo real, não estão montando nenhum teatro. • As personas que sofrem de borderline tem comportamentos im- pulsivos, são muito emotivas, quero ressaltar que, apesar desta patologia, o que eles sofrem é verdadeiro, e algo real, não estão montando nenhum teatro. EXAUSTÃO MENTAL S U M Á R I O As pessoas que sofrem de exaustão mental têm um sentimento de cansaço tão intenso, o que faz até mesmo sentirem dores nas costas, como se estivessem carregando um montão de peso. Isso pode ocorrer no ambiente de trabalho, ou com estudantes que exigem muito de si para tirarem notas boas na universidade, mas também é muito comum em relacionamentos, especialmente em pessoas que sofrem de algum tipo de distúrbio emocional, como depressão, ansiedade crônica, etc. A sobrecarga é causada por fortes demandas emocionais. Há pessoas que parecem ser fortes, mas, com o passar do tempo, elas colapsam porque o estímulo cognitivo é incapaz de suportar a pressão 36 psicológica por muito tempo. Quando você exige demais de si mesmo, acaba tendo um colapso emocional. S U M Á R I O Como identificar a exaustão mental Os sinais parecerão a princípio um pouco confusos, pois podem ser confundidos com cansaço físico. Enquanto o cansaço físico é algo que com uma boa noite de sono, exercício e uma dieta balanceada pode ser recuperado, o esgotamento emocional desmotiva dando a sensação de que tudo o que você faz é porque é obrigado a fazê-lo e não porque você se sente motivado. Alguns dos sintomas são: • Irritabilidade; • Extrema sensação de exaustão sem ter feito nada; • Despersonalização; • Dissociação; S U M Á R I O • Dor de cabeça; • Dor de cabeça; • Sentimento de carregar o peso do mundo em suas costas. • Sentimento de carregar o peso do mundo em suas costas. Como superar a exaustão emocional A primeira coisa é aprender a se desligar de tudo o que o sobre- carrega. Para muitas pessoas isso é difícil, pois estão ocupadas com trabalho, estudos, cuidar de seus filhos... Até mesmo as mídias sociais podem causar exaustão mental. Muitas vezes percebemos que passa- mos todo o tempo que tínhamos disponível para fazer algo produtivo 37 em redes sociais olhando para a vida de pessoas que realmente não se importam conosco. O que recomendo para relaxar. S U M Á R I O • Faça técnicas de relaxamento; • Faça técnicas de relaxamento; • Ouça meditação guiada; • Ouça meditação guiada; • Ouça músicas relaxantes, com sons da natureza, algumas canções budistas (monges budistas); • Ouça músicas celtas como: Enya, Loreena McKennitt, Ima Galguem e Celtic Woman; • Ouça mantras: você sabia que a prática de qualquer tipo de mantra pode ajudá-lo a controlar a ansiedade e as emoções? • Ouça mantras: você sabia que a prática de qualquer tipo de mantra pode ajudá-lo a controlar a ansiedade e as emoções? Na verdade, sou fã de alguns personagens contemporâneos da Nova Era como: • Krishna Das; • Krishna Das; • Deva Premal: talvez ela seja uma das artistas mais conhecidas, já que a maioria das aulas de ioga tocou uma de suas músicas sem saber que ela é a cantora. S U M Á R I O Você é livre para escolher a técnica que melhor lhe convém para se reconectar, alguns preferem a oração, outros saem para o campo, nadam no mar, andam na praia, cantam, dançam..., você vai me dizer que dançar cansa, mas na verdade dançar é um processo de desco- nexão, algumas pessoas quando dançam são capazes de se desco- nectar de tudo o que as preocupa, melhora o humor, a autoestima. Você é livre para escolher o que quiser, não é necessário seguir minhas diretrizes, pois você pode investigar e procurar o que mais lhe convém. 38 DEPENDÊNCIA EMOCIONAL S U M Á R I O Dependência emocional é muito difícil... Porque, se você não tem a ajuda certa, você está como em uma prisão emocional. É uma fase muito destrutiva. Passei por ela e a superei sozinho, porque quan- do procurei ajuda ninguém entendeu o que estava acontecendo comi- go e eu não tinha a resposta de como eles poderiam me ajudar. Às vezes eu pensava que era algo espiritual, que alguém tinha feito algum tipo de ritual de maldição, eu estava completamente de- sesperado; infelizmente, eu não tinha nenhuma informação e nenhuma ajuda de ninguém. Era como uma droga, um vício que, por mais que eu tentasse parar, sempre caía de novo; passei horas chorando, sofrendo, queria tanto essa pessoa que eu não conseguia dormir e nem comer. Infelizmente eu não era consciente das consequências, a de- pendência emocional produz perca de identidade, era como suicidar- -se lentamente. A outra pessoa, esta por quem eu estava apaixonado, não percebia que também necessitava de ajuda, já que ela era posses- siva, ciumenta, a ponto de me causar taquicardia, controlando cada minuto e segundo da minha vida, como se eu não pudesse estar com mais ninguém além dela. Você deve saber que o que estou explicando, a dependência emocional pode ser entre um parceiro ou amigos. S U M Á R I O Quando encontramos alguém pela primeira vez, não temos ideia das cargas emocionais que eles carregam, a menos que as descubra- mos mais tarde. Claro que, no início, tudo é engraçado; depois você percebe que as coisas estão ficando fora de controle e que é hora de corrigir e muitas vezes pensamos que é tarde demais. É preciso entender que a dependência emocional não é ape- nas uma doença, é um processo muito estressante, porque a pessoa que sofre de dependência emocional não está ciente do que está acontecendo com ela. 39 DIFERENÇA ENTRE DEPENDÊNCIA E CODEPENDÊNCIA S U M Á R I O Temos que saber distinguir entre dependência emocional e co- dependência, pois muitas vezes confundimos as duas, a codependên- cia é algo insalubre, pois é um amor patológico, cheio de idealização, acompanhado de sintomas tais como: • Pânico de estar sozinho; • Baixa autoestima; • Insegurança; • Insatisfação pessoal; • Vida sexual inibida ou reprimida; • Comportamento submisso; • Considerar o abuso como normal; • Necessidade excessiva de um parceiro; • Negação da realidade; • Distúrbios alimentares; • Complexos físicos que afetam a autoestima; • Sentimento de culpa. • Pânico de estar sozinho; • Baixa autoestima; • Insegurança; S U M Á R I O 40 40 • Passivas. As pessoas sensíveis são aquelas que se apaixonam rapida- mente, idealizam o homem ou a mulher de sua vida e acreditam que ele ou ela nunca os abandonará. Pessoas insensíveis podem se relacionar, mas... Não podem sentir nada, têm medo de amar, ou não querem, pensam que são su- periores, mostrando falta de empatia. Essas pessoas estão sempre trocando de parceiro como se estivessem trocando de roupa; elas ain- da não descobriram o amor-próprio ou o amor aos outros. O amor passivo é que não se importa com nada; parece não ter personalidade própria, vive cada minuto e cada segundo em uma ilusão, não tem pés no chão. Quando nos relacionamos, compartilha- mos preocupações, inseguranças, algo que pode ter um custo ou um risco, portanto, tenha cuidado ao se relacionar com alguém apenas por causa da atração. S U M Á R I O TIPOS DE PESSOAS S U M Á R I O Posso dizer que existem alguns tipos de pessoas: Posso dizer que existem alguns tipos de pessoas: • Sensíveis; • Insensíveis; • Insensíveis; • Passivas. Melancolia Você sabe qual é muitas vezes o problema de tanta tristeza? Pri- meiro, a corrupção política que faz sofrer os desprivilegiados, depois as decisões erradas que tomamos na vida. Somos seres humanos e não máquinas, portanto, haverá dias e circunstâncias sombrias que às vezes nos tornarão impotentes. 41 Talvez já tenha acontecido com você de encontrar alguém e lhe conta suas tristezas, mas a primeira coisa que ele faz é ir embora, pen- sando que você é um problemático, como se a vida deles fosse um conto de fadas sem problema algum. S U M Á R I O Cada vez que você permite que alguém entre na sua vida, só porque tem medo de estar sozinho perceberá que seus alicerces emo- cionais estarão em perigo. Há uma luta constante enquanto vivemos e essa luta é tanto interna quanto externa. Uma vez ouvi alguém dizer que quando você é muito melancólico, as pessoas ficarão longe de você. Pensei imediatamente que essa pessoa deveria ser a pessoa mais feliz do mundo ou talvez estivesse usando uma máscara para se disfarçar e se sentir aceita pelos outros, pois muitas vezes quando reve- lamos quem realmente somos algumas pessoas preferem se distanciar. Você já viu nas lojas esotéricas todo tipo de besteira, incenso para felicidade, gotas que se você tomar ajuda a serem mais feliz, pedras místicas... Há uma diferença entre ser tóxico e ser uma pessoa que está passando por um mau momento, a maioria destas pessoas que etiquetamos por tóxico muitas vezes o que buscam é alguém que possam escutá-las sem ser julgadas. S U M Á R I O Não tenha vergonha das feridas que o tempo lhe fez, mas nun- ca esconda seus sentimentos por causa do que eles vão pensar de você. Se eles não o aceitam, não se preocupe, eles estão lhe fazendo um favor, porque a verdade é que este tipo de pessoa é egoísta e só pensa em si mesma. É preciso compreender a sensação que você está sentindo como: tristeza, raiva, medo, solidão e ressentimento. Elas são instintos básicos emocionais, então, não tente combater a tristeza, você só deve senti-la, mas nunca deixe que ela o domine. Melancolia Quando comecei um treinamento como terapeuta Gestalt, no iní- cio eu não sabia do que se tratava e tive um momento terrível, porque 42 S U M Á R I O muitas lembranças do passado começaram a vir sobre mim e me des- consolavam, e a única coisa que ouvi de meu professor na frente de meus colegas de classe foi que eu era um manipulador emocional. O problema é que ele não conhecia minha história e ainda assim me julgou só porque eu chorava muito, havia feridas muito profundas e me lembro de que um de meus colegas de classe me defendeu. Com o tempo, comecei a ser mais rígido, não me permitia dar explicação dos problemas, pois percebi que algumas pessoas interpretavam o que elas queriam. Portanto, não tenha vergonha se você é sensível. Se há pessoas que se afastam de você porque pensam que você é problemático, não pense nelas como super-heróis. Eu estava refletindo sobre um dos grandes problemas que vi hoje: temos que viver em uma sociedade na qual não é mais possível mostrar o que sentimos. Tanta tristeza disfarçada de sorrisos! Algumas pessoas, para não se sentirem rejeitadas, preferem se adaptar a regras absurdas (abuso de álcool, drogas, sexo liberal...). Os milhares de livros de au- toajuda que dizem que você pode ser feliz, quando a felicidade é um estado de ânimo e não existe um manual correto para ser feliz. S U M Á R I O 43 Capítulo 2 FATORES SUICIDAS2 FATORES SUICIDAS • Depressão; Capítulo 2 FATORES SUICIDAS2 FATORES SUICIDAS O suicídio é a principal causa dos jovens. Alguns acreditam que os mais velhos estão destinados a morrer mais rápido, já que a maioria dos pais nunca imagina que um de seus filhos seria capaz de tirar a própria vida, independente da classe social que você tenha. Segundo alguns estudos, os homens são mais propensos a morrer por suicídio e as mulheres a tentar o suicídio. S U M Á R I O Temos que deixar claro que existem pessoas que, em momentos de depressão, dizem que querem morrer, mas que não querem, para mim isso seria a fase zero (eu vou chamar assim), é quando qualquer um de nós se encontra em momentos de estresse e sobrecarga e sem- pre salta uma palavra como: • Espero morrer um dia desses; • Quero morrer agora; • Espero morrer amanhã; • Quero dormir e nunca mais acordar; • Tenho a sensação de que não fará falta para ninguém. Frases como essas sem a intenção de realmente querer morrer. Frases como essas sem a intenção de realmente querer morrer. S U M Á R I O Há um erro gravíssimo em dizer que todo suicida tem um trans- torno mental, quando não é verdade. Se você estudar a cultura japo- nesa vai perceber que o suicídio era um ato de honra. Hoje em dia o suicídio é visto como um ato de covardia e, de acordo com alguns reli- giosos, o suicídio faz com que a pessoa que o comete herde o inferno. Você pode me perguntar se é possível justificar o suicídio, mas acredito que todos os seres humanos têm direito à vida. Quais são as causas do suicídio em adolescentes? Capítulo 2 FATORES SUICIDAS2 FATORES SUICIDAS • Depressão; • Depressão; 45 • Sentimento de fracasso; • Medo do futuro; • Autoexigência; • Abuso sexual; • Assédio e vergonha (quando um menino/menina vê sua intimi- dade exposta: fotos íntimas, vídeo sexual); • Pressão psicológica; • Bullying; • Desafios e modas que circulam na internet; • Falta de aceitação e apoio no comportamento sexual (gay, lés- bica, transexual); • Sentimento de fracasso; • Medo do futuro; • Autoexigência; • Abuso sexual; • Assédio e vergonha (quando um menino/menina vê sua intimi- dade exposta: fotos íntimas, vídeo sexual); • Pressão psicológica; • Bullying; • Desafios e modas que circulam na internet; • Falta de aceitação e apoio no comportamento sexual (gay, lés- bica, transexual); S U M Á R I O • Desafios e modas que circulam na internet; • Falta de aceitação e apoio no comportamento sexual (gay, lés- bica, transexual); • Falta de aceitação e apoio no comportamento sexual (gay, lés- bica, transexual); • Falta de desempenho escolar. • Falta de desempenho escolar. • Falta de desempenho escolar. Mas o que seria o ato suicida na adolescência, uma patologia ou um comportamento? Que dilema tem, mas independente de tudo isso devemos aprender a proteger as crianças e adolescentes, ensinando- -os a viver a vida sem autocobrança, aprender a aceitar que às vezes para ganhar é preciso perder, e que é impossível ser perfeito. S U M Á R I O Pais que ensinam seus filhos a serem mais competitivos, e es- quecendo que devem viver uma vida mais holística do que competitiva. Crianças e adolescentes devem aprender a viver a vida sem serem culpados por nossos erros do passado. Assim como a depressão tem seus níveis, classifico o compor- tamento suicida da fase 0 à fase 1. 46 Fase 1 Fase 1 É a fase mais preocupante porque é quando os pensamentos de suicídio são constantes. Se você perguntar a alguém quantas vezes já pensou em suicídio, alguns responderão que nunca, ou que pode ter passado pela cabeça uma vez, mas aqueles que estão na fase 1 vivem constantemente dominada por pensamentos negativos, fazendo-os acreditar que tirar a própria vida estaria fazendo um favor à humanidade. Muitas vezes é difícil discernir entre uma pessoa que está no es- tágio 1 do comportamento suicida porque alguns dos sintomas podem ser confundidos com a depressão já que os dois são muito parecidos. A diferença é que alguns que sofrem de depressão têm mais sentimen- tos de tristeza, angústia, solidão, vazio, ansiedade e raramente querem realmente se machucar. A maioria das pessoas com transtorno de personalidade border- line vive com uma instabilidade emocional muito grande, estes tipos de pessoas se automutilam para conseguir suportar a dor emocional que sentem por dentro. S U M Á R I O Fase 0 S U M Á R I O Como expliquei antes, é o desejo de não sofrer, dizendo com palavras jogadas no ar que você quer morrer, quando não é verdade. IDEAÇÃO SUICIDA A ideação suicida é a intenção constante de querer se matar. O processo começa na mente, são pensamentos recorrentes que ge- ram medo, insegurança, uma voz que fica na sua cabeça o criticando, exigindo. A autodestruição pode se comparar como um suicídio lento, porque o indivíduo com comportamentos indiretos, como se ferir, usar 47 drogas, beber álcool, pode estar provando uma morte lenta, já que provoca em si mesmo uma destruição. S U M Á R I O Quem sofre de ideação suicida tem uma conduta impulsiva devido ao estresse do dia a dia, os pensamentos são constantes e incontroláveis, o processo suicida tem várias fazes que necessitamos compreender: 1. Primeiro é a acumulação de problemas. 2. Vários con- flitos tanto internos quanto externos. 3. Crises de identidade. 4. Pensa- mentos suicidas. 5. Idealização suicida. 6. Suicídio. Tente identificar se antes da ideação suicida você passa por estes sintomas. 1. Falta de sono; 2. Perda de apetite; 3. Ataque de pânico; 4. Sensação de vazio; 5. Cansaço extremo; 6. Perda de interesse; S U M Á R I O 7. Falta de concentração. Compreenda que existem alguns fatores que podem levar uma pessoa a ter ideação suicida, como: 1. Desemprego; 2. Consumo excessivo de álcool; 3. Dor crônica; 4. Morte de algum familiar ou amigo; 48 5. Fofoca que inclua algum tipo de conteúdo sexual da pessoa; 6. Transtornos mentais; 7. Consumo de drogas; 8. Falta de apoio familiar; 9. Qualquer tipo de violência tanto virtual como física; 10. Maltrato psicológico. 5. Fofoca que inclua algum tipo de conteúdo sexual da pessoa; 6. Transtornos mentais; 7. Consumo de drogas; 8. Falta de apoio familiar; 9. Qualquer tipo de violência tanto virtual como física; 10. Maltrato psicológico. S U M Á R I O Automutilação A maioria das pessoas que sofrem de borderline têm a tendência de automutilar-se. Mas porque eles têm esta conduta? Simplesmente porque buscam uma forma de aliviar a dor emocional. A automutilação é uma via de alívio para os sofrimentos psicológicos, o problema e que acaba se tornado um costume que depois irá interferir em todo tipo de processo de socialização. A automutilação pode ser tanto com intenção suicida como sem. O instrumento mais utilizado normalmente é a gilete de barbear, pode parecer uma prática que não leve à morte. Existe também pes- soas que, para tentar controlar a dor emocional, têm o hábito de bater a cabeça na parede, colocar a mão no fogo, dar tapas na própria cara. S U M Á R I O Quando uma pessoa começa a se automutilar é porque se en- contra trasbordada por seus problemas e não sabe como controlá-los. Aprender a lidar com qualquer sentimento negativo e situação negativa o ajuda a evitar de se machucar. As pessoas que se automutilam com o tempo sentirão vergonha das marcas que ficarão, já que será tão vi- sível que as outras pessoas começarão a perguntar por que fez aquilo. 49 O que é a automutilação: • Cortar-se (usando uma navalha, faca ou outro objeto pontiagu- do para cortar sua pele); • Bater em si mesmo ou em coisas (como uma parede); • Queimar-se com cigarros, fósforos ou velas; • Arrancar o cabelo; • Inserir objetos através das aberturas do corpo; • Quebrar ossos ou causar hematomas. S U M Á R I O • Quebrar ossos ou causar hematomas. Controlar as emoções Talvez você me pergunte se é possível controlar uma emoção negativa, e minha resposta e que sim. O problema é que, quando da- mos muita importância a esta sensação que estamos sentindo é então que nos sentimos descontrolados. Se você for uma pessoa impulsi- va entenderá que necessita aprender a controlar sua impulsividade. Quando somos impulsivos não somos capazes de nos relacionar- mos frente a certos tipos de situação, aprender a controlar uma emoção é a chave para poder avançar nesta vida, e como poder se reeducar emocionalmente. S U M Á R I O O risco de automutilar-se Qualquer pessoa corre o risco de automutilar-se, mas segundo os estudos esta conduta começa a partir da adolescência, é frequente em pessoas que/com: 50 • Foram abusados ou traumatizados quando crian • Doenças mentais; • Depressão; • Distúrbios alimentares; • Transtorno de estresse pós-traumático; • Certos transtornos de personalidade; • Abusam de drogas ou álcool; • Amigos em comum que se automutilam; • Baixa autoestima. S U M Á R I O Quais são os sinais de automutilação? Quando uma pessoa se automutila, ela pode apresentar sinais como: • Ter cortes, hematomas ou cicatrizes frequentes; S U M Á R I O • Vestir roupas de manga comprida ou calças, mesmo quando está quente, para ocultar as marcas; • Tentar se desculpar por seus ferimentos; • Ter objetos pontiagudos por perto sem motivo. • Ter objetos pontiagudos por perto sem motivo. Tratamentos de saúde mental que podem ajudar a pessoa: Tratamentos de saúde mental que podem ajudar a pessoa: • Aprender habilidades para resolver problemas; • Aprender novas maneiras de lidar com emoções fortes; 51 • Buscar melhores maneiras de se relacionar, evitando pes- soas tóxicas; • Buscar melhores maneiras de se relacionar, evitando pes- soas tóxicas; S U M Á R I O • Buscar formas de fortalecer a autoestima. Por que algumas pessoas se automutilam? A maioria geralmente tem problemas para lidar ou processar seus sentimentos. E, quando não conseguem resolver algum proble- ma, se prejudicam tentando: • Sentir algo quando se sentem vazios ou indiferentes por dentro; • Desejar bloquear memórias difíceis; • Mostrar que eles precisam de ajuda; • Liberar sentimentos fortes que os sobrecarregam, como raiva, solidão ou desesperança; • Punir-se. • Punir-se. S U M Á R I O Qual é a visão do suicida A visão dos suicidas é de que não há saída para o seu proble- ma e questionam qualquer conselho que recebem, pois sentem que ninguém se importa. Acreditam que fazendo o mal encontrarão uma maneira de superar a dor interior. O cansaço físico e mental é um estado de autoexigência. A maioria dessas pessoas sente que não tem recursos suficientes para enfrentar as difíceis decisões da vida. 52 52 Vou lhe fazer algumas perguntas e ver se você se encaixa em algumas delas como: S U M Á R I O g p g algumas delas como: • Você se sente vazio; • Você se sente sozinho, mesmo estando perto de outras pessoas; • Você se sente fracassado; • Você anseia pelo passado; • Você tem sentimentos constantes de raiva; • Você se sente rejeitado; • Você se afasta de outras pessoas com medo de que elas o ma- chuquem, mas, ao mesmo tempo, você quer ter alguém próximo; • Você sente o desejo de viver ou não se importa se vive ou morre; • Você tem medo do futuro; • Você tem medo da velhice ou da solidão; • Você sempre vê as coisas como se não houvesse saída; • Você se sente totalmente satisfeito profissionalmente e amo- rosamente; • Você sente que não vale a pena lutar pelo que quer porque acha que não vai conseguir; • Você fica desesperado por qualquer coisa; • Você usa substâncias para tentar lidar com o dia a dia; • Você acha que é antissocial por medo de ser magoado ou porque é tímido; • Você se sente vazio; • Você se sente sozinho, mesmo estando perto de outras pessoas; • Você se sente fracassado; • Você anseia pelo passado; S U M Á R I O 53 • Você tem mudanças de comportamento; S U M Á R I O • Você tem pessoas na sua família com problemas mentais. • Você tem pessoas na sua família com problemas mentais. Sabe que a maioria das coisas que nos dão medo existe dentro de nós. Fugir ou lutar, qual é a melhor escolha? Nesta vida nem tudo é um arco-íris completo. Lembranças de um suicida Jonás era um jovem de família humilde e desestabilizada, seus pais eram alcoólatras e sem nenhuma condição financeira, moravam embaixo de uma rodovia, onde construíram uma moradia simples, com um sofá, uma pequena cama de solteiro na qual seus pais dormiam e no chão seus irmãos. Jonás era um menino de apenas 16 anos que eu não achava muito bonito, com olhos verdes e cabelos cacheados com um tom de loiro acinzentado. Ainda me lembro do dia em que o conheci, eu tinha dezessete anos, adorava andar pelas ruas sentindo como se ninguém existisse. Eu estava no meu mundo e, então, vi aquele jovem ali parado assistindo a um show musical que estava rolando, aproximei-me dele e perguntei-lhe. S U M Á R I O - Oi, como vai? Seu olhar era como: “e o que você se importa?” Fiquei ali alguns minutos esperando que ele reagisse, mas se ele ficou calado me olhan- do em silêncio com uma cara que já me contava tudo, então o que você acha que eu fiz? Sim, saí de lá, mas com a curiosidade de saber quem era aquele menino. Alguns dias se passaram, não sei se foi o destino, mas um amigo meu me apresentou a ele, conversamos um pouco, mas ele não se lembrava de mim. Então, pela manhã, veio a triste notícia de que seu pai havia sido morto com vários tiros de pistola, deixando apenas ele, sua mãe e seus irmãos morando ali debaixo de uma ponte. 54 Com o passar dos dias a amizade ficou mais sólida e eu es- tava em sua humilde casa. Jonás seguiu o mesmo padrão de seus pais, ele começou a beber bebidas alcoólicas e começou a ter com- portamentos de raiva muito intensos. Nesta época éramos em cinco adolescentes todos reunidos ali. Lembrei disso, um de nossos amigos dizia que Jonás tinha o comportamento de uma criança que, quando se zangava, queria atenção. Alguns de seus irmãos foram morar com alguns parentes, deixando apenas sua mãe e dois irmãos S U M Á R I O Eliseu era o mais novo de todos eles, não tinha o mesmo com- portamento de Jonás, que era agressivo, mas também gostava de be- ber muito. Caro leitor, vou ter que resumir a história ou será muito longa e não pretendo escrever uma bíblia, mas sim um manual. Lembranças de um suicida Eliseu foi morto e jogado em um buraco, pela manhã saiu a no- tícia de que havia encontrado seu corpo em uma fossa. Jonás levou a vida em meio à bebida e à depressão, talvez você me pergunte se ele procurou ajuda. Sim, ele foi a uma igreja evangélica, porém, por mais que tentasse, as feridas do tempo eram tão grandes e o pobre menino não tinha ferramentas suficientes para lidar com suas frustrações. Sua mãe, bêbada como sempre, teve um infarto em uma tarde e caiu na beira de um rio, afogando-se, mais um duro golpe para o menino que viveu sua vida desejando um milagre. S U M Á R I O Os anos se passaram e, enquanto eu estava viajando, um amigo me ligou à tarde e me disse: - Eu preciso lhe dizer uma coisa, mas por favor, não se desespere. - Eu preciso lhe dizer uma coisa, mas por favor, não se desespere. - Conte-me o que aconteceu - uma parte de mim disse que al- guém da minha família havia morrido. - Conte-me o que aconteceu - uma parte de mim disse que al- guém da minha família havia morrido. Então ele me responde com toda a firmeza. Então ele me responde com toda a firmeza. - Jonás tirou sua vida. 55 55 A única reação que tive foi “como?” Uma raiva entrou dentro de mim, e a única coisa que eu disse foi. S U M Á R I O - Seu idiota, por que ele fez isso? - Seu idiota, por que ele fez isso? Eu não conseguia chorar eu estava simplesmente com raiva do que ele tinha feito, comecei a dizer: “por que ele não me pediu ajuda?”, “por que não conversou comigo?”, “por que ele fez isso?”. Então nosso amigo em comum me respondeu: - O que parece é que ele estava internado e o médico lhe disse algo que o afetou. Quando melhorou voltou para casa, disse à irmã que queria ficar sozinho e se enforcou. - O que parece é que ele estava internado e o médico lhe disse algo que o afetou. Quando melhorou voltou para casa, disse à irmã que queria ficar sozinho e se enforcou. Já sabemos que o suicídio acontece todos os dias, mas nunca imaginamos que alguém em nosso meio seria capaz de fazê-lo. Robson Cansado dos maus tratos do meu pai, fui morar na casa de uma tia materna que morava perto. Era sexta à noite, meus primos saíram para festejar, me deixando apenas com a minha tia. Ela dor- mia sempre em seu quarto com as luzes ligadas. Eu dormia em um sofá velho, não reclamava porque pelo menos ali podia dormir tran- quilo. Nessa mesma noite não sabia que minha vida mudaria comple- tamente. De repente, senti que alguém estava me tocando, enquanto dormia, um cheiro repugnante de álcool impregnava minhas narinas fazendo me acordar e quando viro a cabeça vejo que meu primo estava perto de mim. Talvez o que eu esteja dizendo seja um pouco forte, mas a verdade é que 20% das crianças abusadas sexualmente são por um familiar próximo. Abusos e maus-tratos deixam rastros que nem o tempo é capaz de curar. É mentira dizer que por causa do abuso sexual uma criança se tornou homossexual. S U M Á R I O 56 Voltando à minha história ele tapo a minha boca com a mão para que ninguém ouvisse meus gritos. S U M Á R I O Após o ato, tudo o que senti foi nojo, desprezo e senti que minha inocência havia sido perdida. Senti-me sozinho, perdido, sem poder compartir com ninguém este segredo. A partir deste momento comecei a sofrer fortes crises de depressão e comportamento suicida, causan- do prejuízos acreditando que a culpa era minha. Inúmeras vezes ingeri comprimidos para tentar aliviar minha dor. Eu não queria morrer, eu só queria encontrar alívio para minha dor interna. Eu não entendia por que haviam feito isso comigo, me dava a impressão de que o agres- sor acreditava que era o dono de meu corpo, fazendo com ele o que queria. Os abusos eram sempre noturnos e principalmente nos finais de semana quando o agressor sabia que não havia mais ninguém na casa, facilitando o ato para ele. Passaram-se muitos anos, fui me tor- nando uma pessoa muito depressiva, tinha medo de me relacionar com as pessoas, meus pensamentos estavam constantemente me acusando, me sentia incapaz de ser amado, Suicídio econômico O que significa suicídio econômico? Suicídio econômico tem a ver com adultos que, infelizmente, veem uma direção precária, onde sua estabilidade econômica está em risco, também com pessoas que tiveram tudo e perderam, talvez por um mau investimento, gastos com drogas, álcool e com a frustração de não ter qualquer coisa e acreditar que a única solução é tirar a vida. Vou lhe colocar um exemplo, um em- presário que tinha sua empresa ganhava milhões e de repente vê que perdeu todo, sente que já não poderá sustentar a sua família e busca o suicídio como uma via de escape. S U M Á R I O Consequências do abuso S U M Á R I O O abuso produz mudanças emocionais em suas vítimas, quan- do uma pessoa é exposta a algum tipo de agressão ou maus-tratos, sua saúde emocional é afetada e a probabilidade de ter depressão e comportamento suicida é de 98%. Minimizar o abuso é como dizer que não há nada de errado, que não é tão grave quanto parece, pois muitas pessoas não acreditam que o agressor seria capaz de fazer tal ato. Deixe claro que a maioria das pessoas que foram abusadas se- xualmente são pessoas próximas. Não é a história que você conta ao seu filho sobre não falar com estranhos, não aceitar doces. 57 O abusador é muito próximo, pode ser primo, tio, etc. É difícil dizer, mas infelizmente é a realidade. Muitas vezes acreditamos que nossos filhos têm algum problema mental e levamos a um psiquiatra e o diagnóstico é difícil de encontrar, sabe por quê? Porque muitas vezes essa criança só tem um trauma de infância que lhe causou instabilida- de emocional e não intelectual. Qualquer tipo de violência tanto verbal, quanto física e sexual produz risco de depressão e estresse pós-trau- mático e comportamento suicida. S U M Á R I O O suicídio de uma pessoa que amamos Superar o duelo pela perda de alguém que suicídio não é fácil, é um duelo infinito de perguntas e sentimentos de culpa como: por que você não conseguiu ajudá-lo? A pessoa que sofre de depressão está em uma fase de atenção em que os familiares muitas vezes não enten- dem e acreditam que é apenas uma fase que vai passar e eles nunca esperam o ato suicida. O impacto é tremendo e produz depressão, pois pode desencadear sentimentos tão intensos quanto: 58 • Descrença; • Culpa; • Desespero; • Confusão; • Anseio; • Rejeição do que aconteceu; • Lembranças dolorosas; • Ansiedade; • Fadiga; • Depressão; • Impotência. As sensações são: • Taquicardia; • Falta de ar; • Sentimento de desespero; • Vazio. O que pode acontecer: • Sonhar com o falecido; • Sentir o cheiro da pessoa; • Guardar os pertences do faleci S U M Á R I O • Depressão; • Impotência. S U M Á R I O 59 • Grite em voz alta para a pessoa; • Grite em voz alta para a pessoa; • Alucinar; • Grite em voz alta para a pessoa; S U M Á R I O • Alucinar; • Ter distúrbios do sono; • Ter distúrbios do sono; • Sentir a presença do falecido. • Sentir a presença do falecido. Saiba que esse luto é um processo muitas vezes lento de adap- tação que pode levar alguns anos para que o familiar comece a recons- truir sua vida novamente. As lembranças serão intensas e a ausência da pessoa física transmite uma sensação de desconforto. S U M Á R I O 60 Capítulo 3 MEDIDAS DE PREVENÇÃO3 MEDIDAS DE PREVENÇÃO Capítulo 3 MEDIDAS DE PREVENÇÃO3 MEDIDAS DE PREVENÇÃO prevenir o suicídio é um dever de todos, depois que o Corona Vírus chegou, grande parte das pessoas foi afetada psicologicamen- te. A quarentena fez com que muitos casais entrassem em conflito e outros que não tinham ninguém infelizmente entraram em depressão e acabaram cometendo suicídio. prevenir o suicídio é um dever de todos, depois que o Corona Vírus chegou, grande parte das pessoas foi afetada psicologicamen- te. A quarentena fez com que muitos casais entrassem em conflito e outros que não tinham ninguém infelizmente entraram em depressão e acabaram cometendo suicídio. S U M Á R I O A melhor forma de prevenir o suicídio é fazer uma limpeza men- tal, remover toda informação falsa que o faça acreditar que não vale a pena seguir em frente. Comece a fazer um reset diga a si mesmo que você tem direito à vida e que não importa as circunstâncias da vida, que a vida é um jogo que não só ganha quem sabe ganhar e sem quem sabe vivê-la. QUAIS SERIAM AS ESTRATÉGIAS PARA EVITAR O SUICÍDIO? Concentrando-se em seu principal bem-estar mental, evitan- do completamente a culpa, independentemente de onde você falhou você deve pensar em quão positivo pode ser começar de novo, mes- mo que pareça difícil. S U M Á R I O A necessidade emocional de ter alguém como parceiro. Cuidado com essa parte, você deve primeiro curar suas feridas e depois se rela- cionar com alguém. Procurar uma pessoa para tentar suprir sua neces- sidade de ser amado não ajuda muito já que você está em um conflito emocional, bem uma separação inesperada pode ser como uma explo- são de sentimentos, desencadeando, assim, o desejo de deixar de existir. A maioria das pessoas adora ouvir músicas românticas com le- tras que destroem a alma. Você deve evitar ouvir esse tipo de música por um tempo, em minha opinião, músicas românticas são capazes de despertar sentimentos que muitas vezes achávamos curados. 62 O acompanhamento profissional é mais uma etapa para a pre- venção do suicídio, é preciso aprender que a pessoa que sofre de depressão precisa de acompanhamento total, da família, dos amigos e dos profissionais. S U M Á R I O S U M Á R I O 63 63 Capítulo 4 DEPRESSÃO4 DEPRESSÃO Capítulo 4 DEPRESSÃO4 DEPRESSÃO Estar triste não é sinônimo de depressão, tem gente que não sabe identificar se é tristeza ou depressão, mas a segunda é um dos transtornos mais comuns do nosso tempo. Alguns dos sintomas são: S U M Á R I O • Tristeza; • Perca de interesse para tudo; • Perca de interesse para tudo; • Culpa; • Medo; • Irritabilidade; • Irascibilidade. S U M Á R I O Não julgue S U M Á R I O Não julgue a pessoa que está em conflito interno com suas de- monstrações emocionais, essa pessoa precisa ser fortalecida, você deve ensiná-la a criar uma conexão de segurança e autoconfiança. Todo mundo que sofre de depressão precisa de ferramentas sufi- cientes para superar os conflitos do dia a dia. O religioso deve aprender que a depressão não é espiritual, é um processo emocional, não há problema em meditar, orar, acender incenso, mas se você não tiver fer- ramentas suficientes, não vai adiantar nada. Todo mundo procura sua maneira de superar os desafios da vida, seja na religião, ou seja, lá o que for. Mas cuidado, a religiosidade não lhe garante que um ser espiritual aparecerá em sua vida dizendo que a partir deste momento você não sofrerá mais. Vivemos em sociedade e uma sociedade às vezes mui- to tóxica e cheia de conflitos, de desafios consegue mesmo. Identificar pessoas em risco de depressão já é uma prevenção contra o suicídio. O QUE CAUSA A DEPRESSÃO? A depressão pode ser genética, bem como devido a um senti- mento de desamparo, fracasso, perda de um ente querido, medo do futuro, devido a maus-tratos, abuso sexual, problemas econômicos e autoexigência, que não podem ser alcançados. Situações traumáticas produzem frustração e depois levam à depressão. 65 Não julgue Depressão temporária Para mim, depressão temporária é aquela que não é persistente, não o domina, a pessoa pode controlá-la, enquanto a persistente o faz perder o controle. Podemos chamar de depressão leve e não agres- siva. A temporária é por um período, você a sente, mas logo depois consegue refazer sua vida por completo. S U M Á R I O O que é tristeza S U M Á R I O A tristeza é um sentimento que pode durar minutos ou alguns dias, mas depois a pessoa retoma sua vida, sem nenhum problema, aceitando a situação sem culpa. Já a depressão afeta todos os es- tados emocionais e mentais da pessoa. A única maneira de tratar a depressão é tanto farmacêutica quanto terapêutica, mas acredito que a melhor maneira de estar em paz é aprender a viver com a emoção sem que ela o destrua. Você quer dizer, Robson, que eu tenho que fazer amizade com minhas emoções negativas? Você tem que aprender a conviver com elas, isso implica que não importa o quanto você as sinta e doam, elas não podem fazer você perder o controle, você tem que aprender que é você quem controla seus sentimentos. Os riscos S U M Á R I O Você já sabe que a depressão é uma das doenças, que é muito difícil de superar completamente, é como fazer uma reabilitação de- sintoxicando de todas as drogas só que emocionais. Então o risco de sofrer novamente é imediato. A depressão é uma doença silenciosa, que vem devagar sem bater na porta, por isso devemos estar cientes de que muitas pessoas às vezes podem ter uma recaída, o perigo da psicologia da felicidade é que ela não ensina como entrar em contato com a frustração, aprender como entrar em contato com a frustração ensina como viver e lidar com dias difíceis. 66 Depressão agressiva A depressão agressiva tem sintomas duradouros, impossibilitan- do a pessoa de socializar, afastando-se dos amigos, pois, acredite ou não, há pessoas que podem passar meses em estado catatônico, sem 67 vontade de se limpar, de ver a luz do dia, passam o tempo todo na cama sem desejar nada. Muitas dessas pessoas preferem a noite ao dia. S U M Á R I O As pessoas que sofrem de depressão agressiva acreditam que ninguém se importa com elas, que mal existem e que não há lugar específico para elas nesta vida. Estas pessoas são solitárias, as noites escuras som a companhia perfeita para elas, eles têm um comportamento irascível. Sentimentos de culpa As pessoas que sofrem de depressão agressiva se sentem cul- padas por tudo. Não conseguem administrar a ansiedade e isso produz insatisfação emocional, não conseguem se integrar à sociedade, se culpam por tudo, aceitam os erros dos outros como se fossem sempre a culpa deles. Tentar alcançar a cura é uma obrigação para o doente, não para um psicólogo. É possível superar a depressão? Superar a depressão sem ajuda profissional não é fácil, não acredito naqueles que usam bebidas alucinógenas para superá-la, pois o que esses tipos de substâncias fazem é estimulá-la e potencia- lizá-la ainda mais. A depressão é uma doença que precisa tanto de cuidados men- tais quanto físicos, você deve mudar a rotina sedentária e começar a fazer algo. Tampouco acredito em curas espirituais para a depressão, lembrando que é uma doença mental e não espiritual. Comece a se relacionar com a realidade ou ficará preso na vitimização. S U M Á R I O Dependência terapêutica S U M Á R I O Quando você procura um psicólogo/psicoterapeuta não pode colocar toda a esperança de que ele é quem sempre vai lhe salvar, os terapeutas estão aqui para lhe ajudar a sair desse declínio, mas se você não se esforçar vai passar anos e anos apelando atenção. Mas o que seria a dependência terapêutica? É quando nos tornamos codependentes, há processos que se o paciente se torna codepen- dente de seu terapeuta, ele foca sua necessidade emocional no psi- cólogo. O terapeuta deve compreender sem julgar os pontos de vista do paciente, mas tentando ajudá-lo a sair do círculo vicioso e reagir ensinando-o a tomar decisões racionais e não emocionais. 68 Existem bons terapeutas e também outros que precisam de um pouco mais de aprendizado, entendo que deve haver uma separação entre paciente e terapeuta e que muitos deles não conseguem se envol- ver emocionalmente com os pacientes, porém tive o prazer de conhecer uma pessoa que me ajudou muito e a ele sou grato por me ouvir. S U M Á R I O Se você não se esforçar, não vai adiantar ficarem anos e anos em sessões terapêuticas, o primeiro passo quem tem de dar é você. Como ajudar uma pessoa com depressão? Pela minha própria experiência, por mais que você dê conse- lhos, a pessoa não vai lhe ouvir, talvez ela lhe diga: - Tens razão, farei o que me diz. Tenho claro que em poucos dias essa pessoa esqueceu com- pletamente tudo o que você disse e voltará ao mesmo problema. Não 69 dê conselho e sim opinião, sempre retire a expressão: “se fosse eu, faria isso no seu lugar”. dê conselho e sim opinião, sempre retire a expressão: “se fosse eu, faria isso no seu lugar”. S U M Á R I O Você não está na mesma situação que a pessoa depressiva, en- tão evite conselhos fáceis que são inúteis, apenas não a sobrecarregue e acompanhe-a mesmo que em silêncio, apenas ouvindo e, se possí- vel, sempre lhe abrace, faça-a sentir que não está sozinha. Se você crê que abraçar é algo que pode parecer perturbador para a pessoa que está sofrendo, então segure a mão dela bem firme. Talvez uma das coisas que você possa fazer é abraçá-la, mas não é daqueles abraços rápidos com umas tapinhas nas costas, tente ficar um pouco, abraçando-a ali, fazendo a pessoa sentir que não está sozi- nha. Dialogar com ela, mostre a ela que necessita procurar ajuda profis- sional, e tente convencê-la de que quem procura um psicólogo ou psi- quiatra não significa que está louco. Há pessoas que têm uma visão ruim da psiquiatria e da psicologia, pois acreditam que quem visita é louco. DIRETRIZES DE COMPORTAMENTO PARA O BEM-ESTAR EMOCIONAL S U M Á R I O A primeira coisa é encontrar-se sem a necessidade de mendi- gar o amor de ninguém, a pessoa que sofre de depressão sente-se sozinha, abandonada, muitas vezes desprezada, consegue comprar o amor e a atenção de outras pessoas. A cura emocional é a chave da cura, é se conectar com a solidão e com sua parte mais sombria sem medo e preconceito. Eu lhe digo seriamente que mesmo que você leia o melhor livro sobre autoajuda, se você não tiver paz interior e perseverança será impossível. Já que as pessoas que sofrem de depressão têm dificuldade em ser constantes, e precisam ir devagar até conseguir se conectar consigo mesmas. 70 Ensine-os a enfrentar as adversidades da vida, sem medo de errar, aprenda a superar o medo e a rejeição. Acho que você já ouviu falar, sobre inteligência emocional acredito que a reeducação emocio- nal é a chave para superar a depressão. S U M Á R I O Aprenda a superar a codependência e a dependência emo- cional sem a necessidade de atenção plena, pois quando precisa- mos tanto dessa atenção nos sentimos mentalmente desgastados. Nossos pensamentos não só alimentam nosso ego como também nos sabotam, educam a mente, aprendem a lidar com pensamentos negativos, identificando as causas do processo neurológico que nos impede de seguir em frente. Se você não souber ser um observador, você será apenas a ví- tima que acredita que todos estão contra você quando, muitas vezes, você está alimentando monstros que não existem. Estamos sempre atentos ao que os outros pensam sobre nós, sem entender que minha atenção principal deve estar em mim. Não podemos confundir com narcisismo, estar sempre em plena atenção com nós mesmos, chamei a fase do amor-próprio sem o ego. O ego espera atenção, despreza os outros, se sente o centro de todas as atenções, acredita que é superior aos outros quando não é verdade. Meu bem-estar emocional tem que ser o foco principal da vida, excluindo tudo o que nos impede de seguir em frente, nos impe- dindo de ver o que é a realidade. S U M Á R I O Eu quero fazer uma pergunta, em que momento você está viven- do? Você está ansioso com o futuro ou ancorado no passado? Existem processos emocionais que são muito difíceis de eliminar e para isso precisamos de persistência, dedicação e aceitação. DIRETRIZES DE COMPORTAMENTO PARA O BEM-ESTAR EMOCIONAL Qual é a atenção que você está dando a este sentimento, isto de- mostra que você precisa curar seus pensamentos e sentimentos nega- tivos sobre o que aconteceu, deixando de dar importância às fantasias 71 mentais, porque muitas pessoas sofrem quando um relacionamento ter- mina. A primeira coisa é que passam dias procurando uma resposta do porquê eles terminaram e que se ela não tivesse reagido de tal forma eles ainda estariam juntos, muitas vezes aceitando uma culpa que não é sua. S U M Á R I O Se você vive no momento passado em busca de respostas é mais provável que seja escravizado pela culpa, desperdiçando assim o tempo que deveria estar se concentrando em atividades que não o atrapalhem emocionalmente. Não sei como é a sua visão do mundo, que muitas vezes parece apocalíptica, você tem que saber que a vida é um dom que tem seu cro- nômetro e quando chega a hora tudo acaba. Não é usar a palavra carpe diem, como se fosse um louco aproveitando tudo sem se preocupar com as ações. Se você tem medo do fracasso, você vai falhar você tem que focar sua mente no fato de que você não se importa com o fracasso. Desapegar-se do passado é o primeiro passo para o bem-estar social, desprender-se dos traumas vivenciados. Digo-lhe seriamente que sei que não é fácil, mas tudo é mental, é você quem alimenta o passado com culpa e sentimento tentando consertar algo que já não pode. Assumir que o erro é o primeiro passo. Assuma a respon- sabilidade pelas coisas que fez, talvez tenha vergonha, mas tenha consciência de que é um momento passado e não presente. Talvez você é um dos que desperdiça seu tempo pensando no passado com lembranças angustiantes e deprimentes. Você tem que aprender a fazer as pazes com seu passado, a vida passa tão depressa que você não pode desperdiçar com bobagens. S U M Á R I O Você gasta tempo e energia com coisas que podem ter resulta- dos, vou dar um exemplo em que o passado pode machucar e punir emocionalmente. Há pessoas com câncer lutando para viver, outras que perderam uma parte do corpo, que tiveram um ente querido ferido em um assalto, uma mãe que, por ódio machista, teve que presenciar o castigo de seu ex-marido matando um de seus filhos como castigo. DIRETRIZES DE COMPORTAMENTO PARA O BEM-ESTAR EMOCIONAL 72 Com o passar dos anos, os traumas doerão menos. Isso é ree- ducação emocional. Eu sei que quando você enfrenta uma situação traumática ela o enfraquece e você se sente incapaz de reagir com razão, respire e tome seu tempo, esteja ciente do problema que você está passando e saiba que não é fácil. Você precisa aprender a não ser escravo de suas emoções. S U M Á R I O Aprender a enfrentar o problema de maneira direta fará de você uma pessoa com uma sólida inteligência emocional, aprenda a se co- municar consigo mesmo. Algumas pessoas acreditam que quem fala sozinho é louco, mas percebi que não. Faça a comunicação consciente, não como quem sofre de pa- ranoia, e com uma visão observadora do problema, se for amigo de sua consciência sem culpa, conseguirá acalmar as mensagens de preconceito. Se você for capaz de observar e analisar as emoções, imaginativa você terá uma mente mais tranquila. O passado nos manipula, nos escraviza, nos aprisiona, impe- dindo-nos de obter uma vida plena. Os pensamentos nos sabotam porque muitas vezes nos fazem julgar as pessoas sem conhecê-las, o caminho da consciência é se livrar dos preconceitos. S U M Á R I O As pessoas que sofreram abuso têm a sensação de que pre- cisam de um protetor. Esse sentimento é uma armadilha na vida, de não pode viver sem a de aprovação dos outros. São pensamentos repetitivos carregados de perguntas que nos fazem ancorar a um sentimento angustiante, e muitas vezes queremos consertar o pas- sado e não conseguimos. Evitar um pensamento negativo é impossível, tudo o que temos a fazer é não dar importância a ele, pois esse pensamento só nos afetará se prestarmos atenção a este pensamento ele nos tomara como prisioneiro. 73 Encontrar o silêncio mental é uma tarefa muito difícil, porque leva tempo e prática, estamos sempre cheios de pensamentos, positivos, negativos, de perguntas que podem ir e vir, mas se você ficar sozinho com pensamentos negativos, isso lhe dará uma sensação de descon- forto emocional, é inevitável não sentir preocupado, porém posso me preocupar sem dar atenção emocional a esse pensamento. S U M Á R I O S U M Á R I O 74 Capítulo 5 ANSIEDADE5 ANSIEDADE Capítulo 5 ANSIEDADE5 ANSIEDADE Ansiedade e depressão andam de mãos dadas e sugiro que aprenda a administrá-las sem que ela o aniquile. Pessoas que sofrem de ansiedade podem ter tonturas, vômitos, taquicardia, rigidez no cor- po. A ansiedade é natural em nosso corpo e existe tratamento, uma das melhores técnicas utilizadas é a meditação, as pessoas que pla- nejam muito o futuro são as mais propensas a sofrer de ansiedade, outras têm medo da doença e da morte. S U M Á R I O A única maneira de superar a ansiedade é a mesma coisa que falei sobre a depressão, aprenda a educar os pensamentos. Quando meditamos sempre haverá um momento em que os pensamentos nos questionam, qualquer técnica para silenciar a mente é assertiva, alguns rezam, outros escutam música, áudios sobre motivação emocional. Qual é o ruído mental que o impede de alcançar o silêncio mental? Para superar a ansiedade você precisa mudar completamen- te seu estilo de vida, já que vivemos em meio ao barulho, tanto so- cial quanto emocional. Será impossível alcançar a paz emocional, se você tiver ressentimento, ódio, amargura, isso impossibilitará você de alcançar sua paz interior. Se você passar a vida inteira preocupado com o futuro, isso lhe causará ansiedade, pare de se comparar com outras pessoas. Você é capaz de discernir um evento sem usar julgamento e culpa? Se você não souber determinar o que é verdadeiro ou falso, estará evitando aceitar a realidade. S U M Á R I O Aprender a resistir aos pensamentos negativos o tornará forte, estar ciente de que posso estar em paz entre o sucesso e o fracasso e a ansiedade não me enfraquecerá. Aprenda a ser uma fonte em meio às tensões do dia a dia, observe de fora, possibilitando assim uma postura em que a mente fi- cará calma e você poderá ouvir o som do universo. Aprenda a praticar a meditação ou rezar, a escolha é sua, use um mantra específico, use 76 algo que o ajude a reeducar a sua mente. Diga a si mesmo que você faz parte do universo e que merece viver a vida, sem a necessidade de se sentir pressionado. S U M Á R I O Inspire, respire devagar sem fazer barulho, com calma, esqueça o WhatsApp, a lição de casa, este é o seu tempo, dedique-o a você. Capítulo 5 ANSIEDADE5 ANSIEDADE As pessoas que sofrem de ansiedade devem aprender a controlar a respiração, expirar devagar e contar pensamentos negativos que não podem controlar vocês. Roupas apertadas muitas vezes nos sobrecarregam. Sei que em nosso meio social devemos manter uma boa aparência, mas quan- do estiver sozinho, aproveite para usar algo mais largo, folgado que lhe dê a possibilidade de se sentir livre. Tente evitar estimulantes, isso o ajudará a controlar a ansiedade, a aprender a descansar bem. Feche a janela do seu quarto deixando tudo completamente escuro; as pessoas que dormem com uma luz acesa, na verdade, não descansam completamente, a necessidade de uma luz acesa é por segurança, e outras vezes por algum trauma vivenciado. A luz muitas vezes nos causa desconforto e recomendo dormir com ela apagada, para que seu corpo descanse mais. S U M Á R I O Em momentos de crise, tome um banho quente e depois troque para água muito fria. Você pode também usar a escritura terapêutica, começar a escrever um diário pessoal com tudo que você está sentindo, também pode ajudar cantar músicas que expressem seus sentimentos. Ir para a cama significa descansar e não estar com seu celular contatado no Facebook e Instagram, pois você vai perceber que está gastando seu tempo olhando a vida de pessoas que não tem noção que você exista. A necessidade de dormir sem levar problemas para a cama o ajudará a superar a ansiedade. Nosso corpo precisa relaxar, pois pas- samos o dia inteiro em um estado físico e mental ocupado. 77 MELHORANDO A AUTOESTIMA S U M Á R I O Há uma série de regras que ajudarão a aumentar e melhorar a autoestima: • Detecte pensamentos negativos automáticos e pará-los; • Transforme os pensamentos negativos em positivos; • Não generalize; • Concentre-se nos aspectos positivos; • Esteja ciente de suas conquistas e aproveite-as; • Não se compare com os outros; • Aprenda a diferenciar o comportamento das pessoas; • Aprenda com os erros; • Mude o “eu deveria...” para “eu gostaria...”, “eu prefiro”, “seria melhor se...”; • Aceite limites e capacidades; • Esforce-se para melhorar. • Detecte pensamentos negativos automáticos e pará-los; • Aceite limites e capacidades; S U M Á R I O • Esforce-se para melhorar. • Esforce-se para melhorar. A primeira habilidade que precisamos desenvolver para aumen- tar nossa autoestima é transformar pensamentos negativos limitantes em pensamentos mais motivadores e realistas. São particularmente úteis as folhas de autogestão emocional para poder aplicar a técnica quando surge um pensamento irracional. O sistema consiste basicamente em escrever o pensamento em uma tabela, a fim de analisá-lo e modificá-lo. Podemos levar este registro conosco para que possamos utilizá-lo a qualquer momento e lugar, evitando assim a perda de informações devido ao esquecimento. 78 A CAIXA DE CRENÇAS S U M Á R I O No Brasil 97% da população é muito religiosa. A religião pode ser benéfica como prejudicial, o grande problema da religião são os famosos pastores, sacerdotes que utilizam o poder para manipular. É possível viver sem religião? Claro que sim, o problema é que uma pessoa que vive sem religião é taxada de ateu. A falta de respeito pela decisão dos demais é algo que me preo- cupa. Como temos que crer em tudo naquilo em que os outros creem, somos seres com livre arbítrio, mas infelizmente parece que existe uma escravidão mental que nos obriga a seguir a opinião das outras pessoas. Faz muito tempo que os filhos estavam obrigados a seguir as crenças dos seus pais ou avós, agora devemos repensar antes de dei- xar qualquer pessoa decidir que caminho religioso deseja toma. Mas porque estou falando de religião, o que tem a ver tudo isso com depressão e ansiedade? Eu tive a experiência de conhe- cer pessoas muito religiosas que entraram em colapso mental por causa da sua religiosidade. S U M Á R I O Vou dar um exemplo: há muitos anos eu conheci um filho de pastor que, por se masturbar, teve graves problemas mentais, ele cria que Deus já não ia perdoá-lo. Começo a sentir uma forte culpa, já não conseguia refazer sua vida espiritual porque se sentia sujo. Passou toda a vida aprendendo que era pecado se masturbar, os inúmeros versículos bíblicos em vez de o ajudarem a avançar, o condenavam cada vez mais. Quando eu era religioso, lembro que me impediam de estudar porque diziam que estudar o fazia perder sua espiritualidade com Deus. 79 Hoje compreendo que educação e religião devem andar juntos, sem perder o rumo. O problema é que a maioria dos líderes religiosos se aproveitam da debilidade das outras pessoas para manipulá-los, fazendo acreditar que tudo é espiritual. S U M Á R I O Depressão não é espiritual, condutas suicidas não são demô- nios falando no seu ouvido para você se matar, é um transtorno muito sério e devemos buscar ajuda profissional. Creio que temos medo de questionar as crenças que fomos aprendendo, pensando que qualquer desobediência o levará a um castigo eterno, que Deus o colocará uma doença. Conheci um grande número de pessoas evangélicas que sofreram problemas mentais por acreditarem nas famosas profecias e revelações. A CAIXA DE CRENÇAS Estas pessoas come- çam a ficar com medo extremo e preocupadas. A caixa das crenças é um perigo porque você pode estar apri- sionado a um tipo de pensamento que na verdade não está produzin- do liberdade. Sua mente é como um computador, a mesma informação que você recebeu quando era criança por seus professores, tios, avós não significa que todas sejam realmente válidas. Refazer qualquer crença que recebemos hoje em nossa vida é como aprender a identificar o que realmente são benéficos para mim ou não, os líderes religiosos querem que aceitemos o tipo de crença que eles criaram, sem perguntar se me interessa ou não aceitar. S U M Á R I O Saiba que estas crenças nos limitam, nos impedem de sermos felizes e livres de verdade. O medo de ir ao inferno nos oprime, já que segundo a crença que nos ensinaram temos de ser santos. A maioria das pessoas religiosas sofrem constantemente de depressão e ansie- dade porque estão sempre com autoexigência, resumindo, a palavra se autocastigando buscando uma santidade que não é real. 80 Doença mental não tem nada que ver com espiritual, já que se- gundo a bíblia demônio sai em nome de Jesus. Há alguns anos tenho visto notícias de pastores que tem se suicidado, está notícia, claro, é um tema tabu, porque segundo os religiosos todo suicida vai para o inferno a não ser que existe um privilégio para quem for pastor. S U M Á R I O Necessitamos entender que as crenças são algo que nos im- põem. Isso significa que posso simplesmente averiguar, analisar o tipo de informação e chegar à conclusão se aceito ou não. Mas por que esta crença nos limita? Porque nos faz crer que certo tipo de atos que cometemos é contra a vontade de Deus, sendo assim, você começa a ser escravo de uma ideia incorreta. Um exemplo, quando alguém diz que ir ao psicólogo é coisa de louco e que você deve buscar a Deus, esta é uma crença equivocada, porque eu ao mesmo tempo em que posso buscar a Deus também posso buscar ajuda profissional. O problema dos evangélicos deve ser que por ler a bíblia e ver os apóstolos sofrendo e o único que eles faziam era orar, mas já não vivemos naquela época, estamos no século XXI, hoje temos muitas mais possibilidades que antes. Liberdade de consciência S U M Á R I O Você já pensou para si mesmo, o que são crenças? Esqueça as crenças religiosas; que outros tipos de crenças existem? O que são crenças? Se você fosse procurar a definição de crença, o que você acha que é? Crença é algo em que você está convencido, é algo em que você acredita. É algo de que você tem procurado pro- vas ao longo de sua vida e que se tornou uma ideia profundamente enraizada. Hoje sua vida está cheia de crenças. Crenças, muitas das quais se tornaram hábitos ou costumes, que você herdou que você foi ensinado ou que você desenvolveu. 81 As crenças são muito poderosas porque são suas crenças que o levam a decidir o que é certo e o que é errado, estas crenças devem ser analisadas. Como por exemplo: a mulher foi feita para ter filhos e estar na cozinha, os homossexuais são coisa do diabo, homem que é homem fala grosso. S U M Á R I O Estes tipos de crenças podem ser hereditárias, pessoais, sociais ou por medo. As crenças hereditárias são aquelas crenças em que escu- tamos quando somos crianças, quando nossos pais dizem coisas que ficam marcados em nossa mente como: “você é muito burro”, “nunca conseguirá nada na vida”, normalmente a maioria das pessoas que es- cutam este tipo de comentário levam anos para conseguir esquecer, so- frem constantemente crises de identidade, quando vão praticar qualquer tipo de trabalho sofrem de ansiedade porque têm medo de errar. Aprender a pensar é um ato humano. Você não pode estar apri- sionado a uma crença que o limita, que o aprisiona, que lhe impõe que o faz sofrer, que o faz sentir-se escravizado, nesta vida com certeza terão pessoas que irão lhe criticar só que as críticas podem ser como uma base de aprendizagem. Liberdade de consciência Algumas dicas que eu recomendo: S U M Á R I O • Desconecte-se completamente das redes sociais; • Encontre um lugar privado de intimidade; • Aprenda a agradecer toda vez que acordar; • Ore; • Aprenda a ficar quieto; • Ouça mais e fale menos; • Aprenda o caminho da consciência; • Desconecte-se completamente das redes sociais; • Encontre um lugar privado de intimidade; • Aprenda a agradecer toda vez que acordar; • Ore; • Aprenda a ficar quieto; • Ouça mais e fale menos; • Aprenda o caminho da consciência; • Desconecte-se completamente das redes sociais; • Encontre um lugar privado de intimidade; 82 • Tenha disciplina; • Tenha constância; • Faça caminhadas pela floresta; • Ouça músicas de relaxamento; • Faça exercícios de relaxamento guiado; • Faça algum tipo de exercício físico; • Faça longas caminhadas, pois elas ajudam na ansiedade; • Tenha uma boa dieta; • Peça ajuda quando perceber que não é capaz de se controlar; • Identifique os tipos de pensamentos e desvie a atenção negativa • Tenha disciplina; • Tenha constância; • Faça caminhadas pela floresta; • Ouça músicas de relaxamento; • Faça exercícios de relaxamento guiado; • Faça algum tipo de exercício físico; • Faça longas caminhadas, pois elas ajudam na ansiedade; • Tenha uma boa dieta; • Peça ajuda quando perceber que não é capaz de se controlar; • Identifique os tipos de pensamentos e desvie a atenção negativa S U M Á R I O • Peça ajuda quando perceber que não é capaz de se controlar • Identifique os tipos de pensamentos e desvie a atenção negativa. Saiba que a ansiedade pode ser tanto fisiológica quanto patológi- ca, portanto, independente do que você sinta, não deixe de pedir ajuda. Técnica para resolver conflitos S U M Á R I O A maioria das pessoas que veem tudo como catastrófico não conseguem manejar qualquer conflito e a única técnica que usam é a de evasão e agressão. Quando não sabemos como afrontar certa si- tuação, o sentimento de fracasso trasborda nossa mente fazendo-nos sentirmos oprimidos. O processo para solucionar um problema começa da seguinte maneira: • Analisar que tipo de problema você está sofrendo; • Aprender a diferenciar o tipo de problema; 83 • Ser claro com o objetivo que desejas conseguir; • Fazer uma lista de alternativa; • Buscar um plano B; • Começar a idealizar o plano de ajuda; • Colocar em prática; • Buscar ajuda, se for possível. • Ser claro com o objetivo que desejas conseguir; • Fazer uma lista de alternativa; • Buscar um plano B; • Começar a idealizar o plano de ajuda; • Colocar em prática; • Buscar ajuda, se for possível. S U M Á R I O • Buscar ajuda, se for possível. Treinamentos e técnicas que deveria usar Hoje em dia existe um monte de práticas que podem nos ajudar a manejar a ansiedade como meditação e práticas de mindfulness. Estes tipos de práticas têm a função de relaxarmos em momentos de crise, abaixando a intensidade da ansiedade e nos fazendo estar em nosso momento presente. Cuidado! Nem toda técnica que você encontra pela internet serve para qualquer tipo de transtorno, pessoas com estresse pós- traumático e transtorno compulsivo, transtorno de pânico e ansiedade aguda necessitam de uma atenção mais específica. S U M Á R I O As experiências traumáticas da vida nos fazem relacionar de maneira impulsiva interferindo a nossa vida normal. Um especialista é capaz de identificar o tipo de transtorno que você sofre, não se baseie por testes que existem pela internet. Agora vamos utilizar uma tabela com técnicas para seu bem- -estar emocional, para isso você necessitará ser uma pessoa assertiva evitando todo estilo tóxico que o limita de avançar aprenda a ser, mas tolerante e respeitoso. 84 Técnica básicas • Dieta • Buscar apoio social • Distração, vida social Técnicas cognitivas • Detectar pensamentos negativos • Aprender a relaxar em situações de estresse Técnicas fisiológicas • Relaxação muscular • Usar treinamento autógeno • Aprender a controlar a respiração Técnicas conduta • Aprender a resolver problemas • Aprender autocontrole • Saber manejar conflitos sociais Outras recomendações para manejar o estresse 1. Não trabalhe mais de 8 horas por dia; 2. Tente descansar entre as tarefas; 3. Durma de 7 a 8 horas por dia; 4. Ouça música relaxante; 5. Pratique um hobby criativo; 6. Faça exercícios moderados regularmente; 7. Evite o uso de drogas (incluindo álcool) e outros meios artificiais usados para aliviar o estresse; 8. Planeje férias todo ano, longe do trabalho e do barulho; 9. Visite o médico quando achar necessário; 10. Expresse seus sentimentos, sem antagonismo ou hostilidade; 11. Discuta seus problemas com alguém em quem confia; Técnica básicas • Dieta • Buscar apoio social • Distração, vida social Técnicas cognitivas • Detectar pensamentos negativos • Aprender a relaxar em situações de estresse Técnicas fisiológicas • Relaxação muscular • Usar treinamento autógeno • Aprender a controlar a respiração Técnicas conduta • Aprender a resolver problemas • Aprender autocontrole • Saber manejar conflitos sociais S U M Á R I O Outras recomendações para manejar o estresse 1. Não trabalhe mais de 8 horas por dia; 2. Treinamentos e técnicas que deveria usar Tente descansar entre as tarefas; 3. Durma de 7 a 8 horas por dia; 4. Ouça música relaxante; 5. Pratique um hobby criativo; 6. Faça exercícios moderados regularmente; 7. Evite o uso de drogas (incluindo álcool) e outros meios artificiais usados para aliviar o estresse; 8. Planeje férias todo ano, longe do trabalho e do barulho; 9. Visite o médico quando achar necessário; 10. Expresse seus sentimentos, sem antagonismo ou hostilidade; 11. Discuta seus problemas com alguém em quem confia; S U M Á R I O 85 12. Planeje e peça seu trabalho; 13. Faça apenas uma coisa de cada vez e estabeleça uma ordem de prioridades; 14. Nem sempre esteja ciente do tempo; 15. Faça coisas sem pressa (quando andar, falar, comer etc.); 16. Evite discussões; 17. Esqueça os ressentimentos, queixas e ódios; 18. Evite a competição no trabalho e nos jogos; 19. Concentre-se no presente; 20. Não faça mudanças nos padrões de vida que o afetem muito (pouco a pouco); 21. Nunca perca a fé em si mesmo e em suas faculdades; 22. Aproveite a vida de uma maneira saudável. S U M Á R I O 13. Faça apenas uma coisa de cada vez e estabeleça uma ordem de prioridades; 21. Nunca perca a fé em si mesmo e em suas faculdades; 22. Aproveite a vida de uma maneira saudável. Técnica básica S U M Á R I O Você tem que começar a estar preparado para o estresse. Infe- lizmente é impossível viver uma visa sem ele, mas se sabemos como nos controlar evitará de entrar em um colapso mental. Preparar-se para o estresse parece difícil porque normalmente não temos controle de nossos impulsos. 1. Compreenda que: • É inevitável não se estressar; • Averigue as causas e circunstâncias que me levam a estressar-me; • Averigue as causas e circunstâncias que me levam a estressar-me; 86 • Afronte-o como algo que você necessita resolver; • Busque o lado positivo. 2. Fatores pessoais: • Dieta; • Descanso; • Limites; • Exercícios; • Certos tipos de hábitos que não são saudáveis; • Aprenda a ser mais tolerante; • Aprenda a tomar decisões; • Socialize. 3. Controle no trabalho: • Estabeleça prioridades; • Suprima atividades que o desgasta; • Reduza a exigência pessoal; • Saiba selecionar as metas que deseja; • Aprenda a terminar assuntos pendentes; • Aprenda a expressar as emoções sem atacar; • Não seja muito passivo. • Afronte-o como algo que você necessita resolver; S U M Á R I O • Dieta; • Descanso; • Limites; S U M Á R I O 87 1. Aprenda a lidar com os erros: 1. Aprenda a lidar com os erros: Existem pessoas que são muito exigentes consigo mesmas, es- tão constantemente se culpando por ter fracassado, aprender a corri- gir os nossos erros sem uma culpa extrema nos fará mais saudáveis emocionalmente. S U M Á R I O 2. Autoavaliação: 2. Autoavaliação: 2. Autoavaliação: Aprender a autoavaliar-se o ajudará a ser consciente de cada sensação, emoção, ato. 3. Autoclítico: Uma reeducação emocional S U M Á R I O Para uma melhor reeducação emocional, você terá que com- preender que algumas das crenças são incondicionais, que levam muito tempo decodificando a imagem que temos de nós mesmos. Este erro cognitivo que fomos obtendo, provoca uma conduta ne- gativa com tendência a cometer muitos fracassos. Uma pessoa que sofre de depressão tem a sensação de que é inútil, com uma visão negativa de si mesma, pensando que as outras pessoas não vão te aceitar por ser depressivo. O primeiro passo que você deve tomar como uma pessoa que sofre depressão é aprender a eliminar estes pensamentos que a limi- tam de se aproximar de outras pessoas. Saiba identificar cada emoção e gerenciá-la de maneira mais racional. 1. Aprenda a lidar com os erros: 3. Autoclítico: Ser capaz de elogiar-se por suas próprias conquistas, sem bus- car aprovação das outras pessoas. Quando dependemos da aprovação externa, limitamos nosso potencial. 88 1 ELLIOTT, G.R.; EISDORFER, C. (1982) Stress and Human Health. Springer, New York. Health, v. 8, n. 15, Dec. 9th, 2016. Inoculação do estresse • Ajudar os pacientes a adquirir conhecimentos suficientes para facilitar maneiras de abordar situações estressantes inespera- das (A MENTE É MARAVILHOSA, 2020)2. 2 A MENTE É MARAVILHOSA. Como o treinamento de inoculação de estresse pode nos aju- dar. Online, 29 ago. 2020. Disponível em: https://amenteemaravilhosa.com.br/treinamento- -de-inoculacao-de-estresse/. Acesso em: 18 fev. 2022. 3 WECAREON. O que é a Intervenção na Crise? Online, 23 dez. 2021. Disponível em: https://wecareon.com/blog/intervencao-crise/. Acesso em: 18 fev. 2022. Inoculação do estresse S U M Á R I O Alguma vez você já ouviu falar sobre inoculação do estresse? É uma técnica usada por Eliot e Eisdorfer (1982)1: • Estressores agudos de tempo limitado. São acontecimentos es- tressantes que ocorrem em um determinado momento e são de curta duração (por exemplo, exames médicos pontuais ou prova para tirar carteira de motorista). • Sequências de estresse. Fazem referência a acontecimentos de vida estressantes específicos e significativos (por exemplo, abu- sos sexuais, demissões, perda de um parente querido, etc.). Sequências de estresse. Fazem referência a acontecimentos de vida estressantes específicos e significativos (por exemplo, abu- sos sexuais, demissões, perda de um parente querido, etc.). • Intermitência crônica. Em geral, é provocada pela exposição re- petida a situações estressantes (por exemplo, provas, exames médicos, combates militares, etc.). Estresse crônico continuado. Inclui a luta contra doenças médicas ou psiquiátricas de longa duração ou problemas afetivos prolongados. • Estresse crônico continuado. Inclui a luta contra doenças médicas ou psiquiátricas de longa duração ou problemas afetivos prolongados. S U M Á R I O • Ensinar os pacientes sobre a natureza do estresse e combatê-lo. • Treinar os pacientes para que controlem pensamentos, ima- gens, sentimentos e comportamentos, tudo isso com o objetivo de favorecer interpretações realistas e adaptativas. • Treinar os pacientes na resolução de problemas. • Modelar e reproduzir as atuações reais, a regulação das emo- ções e as habilidades de confronto próprias do autocontrole. • Modelar e reproduzir as atuações reais, a regulação das emo- ções e as habilidades de confronto próprias do autocontrole. 89 • Ensinar aos pacientes a maneira de utilizar respostas desa- daptativas como sinais para colocar em prática seus repertó- rios de confronto. • Ensinar aos pacientes a maneira de utilizar respostas desa- daptativas como sinais para colocar em prática seus repertó- rios de confronto. S U M Á R I O • Oferecer práticas de ensaio imaginado e ao vivo para fomentar a confiança do paciente e a utilização de seus repertórios de confronto. • Oferecer práticas de ensaio imaginado e ao vivo para fomentar a confiança do paciente e a utilização de seus repertórios de confronto. • Ajudar os pacientes a adquirir conhecimentos suficientes para facilitar maneiras de abordar situações estressantes inespera- das (A MENTE É MARAVILHOSA, 2020)2. 2 A MENTE É MARAVILHOSA. Como o treinamento de inoculação de estresse pode nos aju- dar. Online, 29 ago. 2020. Disponível em: https://amenteemaravilhosa.com.br/treinamento- -de-inoculacao-de-estresse/. Acesso em: 18 fev. 2022. 3 WECAREON. O que é a Intervenção na Crise? Online, 23 dez. 2021. Disponível em: https://wecareon.com/blog/intervencao-crise/. Acesso em: 18 fev. 2022. O que significa estar em crise? Uma crise é um estado ou condição temporária em que um in- divíduo não encontra a capacidade de lidar com um tipo específico de problema e uma situação experiencial emocional e psicológica. Como resultado da resolução bem-sucedida das crises da vida, observar-se-á no sujeito uma personalidade mais segura e altamente integrada, com maior capacidade de lidar com crises futuras. De fato, o estado atual de saúde mental de uma pessoa pode ser entendido como “o produto da maneira como ela resolveu uma série de crises no passado” (ELLIOT; EISDORFER, 1982). S U M Á R I O Intervenção em crise, então, será o processo de ajudar a supor- tar um evento traumático, visando ajudar uma pessoa, família ou grupo, de modo que a probabilidade de continuidade dos efei- tos negativos (físicos ou emocionais) do evento seja diminuída, e a aumenta a probabilidade de crescimento do sujeito (novas habilidades para resolver seus problemas), com o menor custo pessoal e social possível (WECAREON, 2021)3. 90 EXISTEM QUATRO FASES EM UMA CRISE S U M Á R I O 1. Elevação inicial do nível de tensão emocional do sujeito devido ao impacto de um ou vários eventos externos, que o levam a ter a sensação de “ter que fazer algo naquele exato momento para resolver o problema”. Essa elevação inicial da tensão emocional faz com que esses eventos sejam enfrentados de acordo com os comportamentos habituais do sujeito e orientações para re- solver seus problemas em outros momentos. 2. Ao não ter sucesso na resolução de problemas, a tensão au- menta e surgem sentimentos de ineficiência, incapacidade e desordem, principalmente se o impacto continuar. 3. Quando a tensão aumenta, outros recursos são colocados em prática para resolver os problemas. A partir daí, a crise pode ser desviada, seja para a redução da ameaça externa (real ou não) pelo sucesso das estratégias de enfrentamento utilizadas, para uma redefinição do problema ou seu significado, ou para a renúncia de ter fixado e objetivos coerentes (reagindo pontual- mente aos aspectos específicos do problema, mas de forma globalmente desorganizada). S U M Á R I O Quanto menos o problema global for controlado, devido à ine- ficácia das estratégias postas em prática, maior será o aumento da tensão (que chegará a um ponto crucial, que varia de pessoa para pes- soa) e maior será a desorganização. crescer o estado emocional do indivíduo, que pode ser maior na probabilidade de perda de controle. Durante esse processo temporal e psicológico, o sujeito passa a vivenciar paralelamente uma série de experiências que devem ser conhecidas mais de perto e levadas em conta na hora de agir: 91 Durante a primeira fase, o sujeito tenta enquadrar o problema percebido, mas não consegue no tempo esperado. Ele percebe seu fracasso inicial e permanece insatisfeito, experimentando tensão e an- siedade progressivas, com sentimentos desagradáveis e somatizantes. S U M Á R I O Durante a segunda fase, a tensão continua a aumentar e cer- to grau de desorganização começa a ocorrer. O sujeito experimenta sentimentos mal definidos de culpa, medo, ansiedade, etc. Diante da sensação de fracasso, podem tentar fugir, inibir-se ou tentar repetida- mente tentativas e erros. Até este ponto, os mecanismos usuais para restaurar o equilíbrio atuam. Na terceira fase, à medida em que a tensão e a desorganização aumentam, o sujeito pode ser arrastado por seus impulsos ou por ou- tras influências, ativa ou passivamente. EXISTEM QUATRO FASES EM UMA CRISE O sujeito pode tentar redefinir o problema total ou parcialmente, buscar novas soluções. Se isso for eficaz, a crise será superada e isso permitirá o crescimento. Se essa redefinição cortar a crise, mas for mal adaptada, será posto em movi- mento um quadro neurótico que deixará sequelas. Se ele não conse- guir resolver a situação, ele passará para uma quarta fase. Na quarta fase, a desorganização aumenta e chega-se a um ponto de ruptura em que podem ocorrer episódios psicóticos e comportamentos indesejá- veis, de forma ilógica e imprevisível. S U M Á R I O No entanto, existem certas diferenças importantes entre o que podemos considerar uma crise e o que pode ser considerado uma situação de angústia, ansiedade ou estresse agudo. Assim, entre a ansiedade, o medo, etc., e a crise, embora sejam muitas vezes di- fíceis de distinguir na prática, existem algumas diferenças que são fundamentais, pois requerem diferentes formas de intervenção. Dife- renciadores que são importantes: 1. Os padrões de enfrentamento são diferentes: durante a crise, a abordagem é mais pessoal, utilizando menos apoio familiar e núcleos cerca de formais (vizinhos, amigos, etc.). 92 2. Durante a crise, o indivíduo tende a ser menos defensivo e mais aberto a sugestões, influências e apoio, o que é caracteristica- mente revertido em situações estressantes. S U M Á R I O 3. O resultado da crise pode ser o enfraquecimento ou o crescimen- to diante dos problemas da vida cotidiana. Enquanto o estresse é comumente relacionado à patologia, seu resultado é a adaptação à deterioração e sofrimento causados pelas circunstâncias da vida. 4. A crise está relacionada a um incidente súbito e influencia o su- jeito por um tempo limitado e relativamente curto (de minutos a, no máximo, seis semanas). O estresse vem de um processo crônico, que se desenvolveu ao longo do tempo e leva tempo para desempenhar um papel autolimitante. 4. A crise está relacionada a um incidente súbito e influencia o su- jeito por um tempo limitado e relativamente curto (de minutos a, no máximo, seis semanas). O estresse vem de um processo crônico, que se desenvolveu ao longo do tempo e leva tempo para desempenhar um papel autolimitante. EXISTEM QUATRO FASES EM UMA CRISE Em linhas gerais, então, podemos considerar que uma crise é a si- tuação em que uma pessoa se encontra como consequência do fracasso em resolver um problema importante para ela (entendendo o problema como “a consequência do fracasso na busca de soluções”. O grande obstáculo é que, como dissemos antes, uma vez conhecido o problema, sabemos que enfatizar suas dimensões e/ou importância só aumenta a angústia, ao mesmo tempo em que diminui a capacidade de encontrar soluções e que a tendência natural da pessoa que é motivada por seu pro- blema é geralmente concentrar-se em sua gravidade e consequências. S U M Á R I O Intervenção em situações de crise é sinônimo de ajuda às pes- soas que se encontram nesse tipo de dificuldade. E essa ajuda, tendo em vista o que já foi dito em relação ao que é uma crise, como uma pessoa se sente nessa situação e o que a causa, deve ser não apenas respeitosa das pessoas e útil a elas, mas, acima de tudo, que não é prejudicial para eles no momento em que ocorre, ou no futuro. Assim, a primeira coisa a considerar é que a eficácia de um ser- viço de intervenção em crise é função da sua proximidade ao evento desencadeante. Ou seja, a velocidade com que ela é fornecida. 93 Podemos resumir as características da intervenção em situa- ções de crise em função do tempo, do local, das pessoas que a pres- tam e dos objetivos que se estabelecem. S U M Á R I O • Tempo: entre minutos e horas. Devemos lembrar que a eficácia de um serviço de intervenção em crise aumenta ou diminui em função da sua proximidade no tempo ou no espaço, ao local do evento desencadeante; • Onde: no local do primeiro contato e em outros locais que, de- vido ao desenrolar dos acontecimentos, sejam visitados no pe- ríodo imediatamente subsequente; Onde: no local do primeiro contato e em outros locais que, de- vido ao desenrolar dos acontecimentos, sejam visitados no pe- ríodo imediatamente subsequente; • Quem: quem atua no primeiro momento, na primeira linha, ou assume as primeiras responsabilidades; • Objetivo: dar suporte, manter os canais de comunicação abertos, reduzir a mortalidade, vincular recursos de ajuda de segunda li- nha e prevenir complicações e/ou novas situações semelhantes. EXISTEM QUATRO FASES EM UMA CRISE Portanto, os objetivos de uma intervenção de crise podem ser resumidos, em geral, em quatro grandes blocos: 1. Sobrevivência física de todos os envolvidos às consequências que esta situação acarreta, de forma integral; S U M Á R I O 2. Controle e domínio da consciência e da razão da experiência que esta situação supõe, de forma completa; 3. Expressão e liberação (dos sentimentos ligados à crise, que são diferentes daqueles relacionados às causas da crise), da forma mais livre, dentro da qual seja possível admitir; 4. Planejamento de curto prazo das mudanças e adaptações inter- pessoais necessárias para o futuro imediato. 94 Mas, em geral, a intervenção em crise se move fundamental- mente em três grandes grupos de objetivos: S U M Á R I O 1. Estabelecer e facilitar a comunicação (seja entre as pessoas di- retamente envolvidas, seja entre essas pessoas e outros perso- nagens da situação, seja com as instituições, etc.); 2. Ajudar o indivíduo e/ou a família a perceber corretamente a si- tuação (focando a atenção em eventos específicos, seus reais significados, seus possíveis resultados, etc.); 3. Ajudar os envolvidos a gerenciar seus sentimentos e emoções. Assim, e do ponto de vista teórico, podem distinguir-se cinco fases sucessivas no processo de intervenção em crise: 1. Contato com pessoas; 2. Contato com problemas; 3. Redução dos problemas à sua essência; 4. Detecção dos recursos disponíveis para as pessoas envolvidas. 4. Detecção dos recursos disponíveis para as pessoas envolvidas. Determine a melhor maneira possível de aplicar os recursos dis- poníveis aos problemas. S U M Á R I O Mas, do ponto de vista prático, como lidar com uma crise? A abordagem genérica para qualquer situação de crise pode ser siste- matizada em sete etapas ou fases: 1. Defina os problemas concretos; 2. Aborde os sentimentos presentes; 3. Tome decisões ou previna soluções; 4. Determine os aspectos do problema levantado, os conflitos sub- jacentes representados na crise, ou reativados por ela; 95 5. Identifique e resolva, pelo menos parcialmente, os conflitos sub- jacentes representados na crise, ou por ela reativados; S U M Á R I O 6. Defina os recursos individuais internos e/ou externos disponí- veis, para a solução de crises; 7. Reduza ao máximo os efeitos desagradáveis ou desconfortáveis relacionados à crise. EXISTEM QUATRO FASES EM UMA CRISE Trata-se de garantir que o sujeito consiga desenvolver aquelas características de comportamento que suponham uma forma de en- frentamento eficaz das crises, que em todo caso devem ser aquelas possuídas pela pessoa que deve prestar a assistência e que, em seu tempo, você deve tentar induzir em quem sofre a situação. Estes comportamentos seriam: 1. Explorar ativamente a situação e os reais resultados ou conse- quências buscando as informações precisas para poder tratá-la de forma eficaz; 2. Expressão, sem reservas, de sentimentos positivos e/ou negati- vos, enfrentando a frustração; 3. Solicitar ajuda de terceiros, se for considerado necessário; S U M Á R I O 4. Decompor os problemas em seus elementos constituintes, tor- nando-os gerenciáveis e tratáveis; 5. Trabalhar a cada momento com apenas um aspecto específi- co ou diferenciado do problema. Estar ciente da fadiga e das tendências ou impulsos que permitem a desorganização das diferentes áreas de funcionamento equilibrado, mantendo o controle e o esforço, tanto quanto possível; 6. Administrar os sentimentos na medida do possível, aceitan- do-os quando possível, sendo flexível e disposto a mudar se for necessário. 96 A verdadeira dificuldade é saber avaliar em todos os momentos como essas fases da intervenção podem ser operacionalizadas. Guar- dadas as circunstâncias, possibilidades e disponibilidades particulares de cada caso e de cada momento, há uma série de etapas genéricas pelas quais o recomendado seria: S U M Á R I O 1. Estabeleça um contato com o assunto que seja o mais tranquiliza- dor possível para ele. Dê-lhe apoio emocional (não comece repri- mindo a expressão de suas emoções, mas canalizando-as). Alcan- ce um contato psicológico e empático, mas sem perder a distância; 2. Examine as dimensões do problema. Obtenha todas as informa- ções possíveis sobre o que está acontecendo com o sujeito, dê a ele a oportunidade de “descarregar” e expresse seu interesse nele como pessoa; 3. Faça uma formulação explicativa do que acontece com o indiví- duo, colocando nela os fatores precipitantes ou desencadean- tes. Explore soluções possíveis; 4. Apresente ao sujeito, se possível, sua teoria. Procure saber a opi- nião deles sobre isso e, assim, leve-os a ver seu problema de outra perspectiva e da forma mais objetiva (ou porque a teoria que propomos é adequada, ou porque o sujeito não pensa assim e tem que corrigi-la). Tente não expressá-lo de forma em que o su- jeito se sinta culpado. EXISTEM QUATRO FASES EM UMA CRISE Seja seletivo ao focar a questão nas ques- tões realmente cruciais, mas deixá-lo divagar um pouco quando atinge um nível de tensão excessiva e depois voltar ao nó; S U M Á R I O 5. Explore, junto com o sujeito, possibilidades de soluções alterna- tivas, diferentes daquela que ele está utilizando. Veja que ajuda você pode precisar para usar outras soluções melhores do que as que você está tentando atualmente. Ajudá-lo a tomar as deci- sões necessárias e iniciar as ações concretas necessárias; 5. Explore, junto com o sujeito, possibilidades de soluções alterna- tivas, diferentes daquela que ele está utilizando. Veja que ajuda você pode precisar para usar outras soluções melhores do que as que você está tentando atualmente. Ajudá-lo a tomar as deci- sões necessárias e iniciar as ações concretas necessárias; 5. 97 6. Comprometa-se com o sujeito, na medida do possível, a tentar outras soluções em troca de facilitar o que realmente está em nossas mãos (compromisso, pacto, etc.). Verifique o início da realização das decisões adotadas. Então, vamos ver o que deve ser feito em situações de crise: 6. S U M Á R I O Então, vamos ver o que deve ser feito em situações de crise: FASE DE CONTATO a. Você deve: i. Ouvir com atenção; ii. Refletir fatos e sentimentos; iii. Comunicar a aceitação. b. Você não deve: i. Contar a “sua própria história”; ii. Ignorar fatos ou sentimentos; iii. Julgar ou tomar partido. S U M Á R I O FASE DE CALIBRAÇÃO DO PROBLEMA a. Você deve: i. Fazer perguntas claras e abertas. Pergunte às pessoas por es- pecificidades; ii. Avaliar todos os riscos físicos e morais existentes. b. Você não deve: i. Responder sim/não ou perguntas alternativas; a. Você deve: i. Fazer perguntas claras e abertas. Pergunte às pessoas por es- pecificidades; i. Fazer perguntas claras e abertas. Pergunte às pessoas por es- pecificidades; ii. Avaliar todos os riscos físicos e morais existentes. i. Responder sim/não ou perguntas alternativas; 98 ii. Permitir abstrações ou divagações contínuas; ii. Permitir abstrações ou divagações contínuas; S U M Á R I O iii. Ignore qualquer sinal de “perigo”. iii. Ignore qualquer sinal de “perigo”. FASE DE BUSCA DE SOLUÇÃO a. Você deve: i. Estimular a inspiração repentina do interessado. Enfrentar os obstáculos diretamente. sem fugir; ii. Estabelecer prioridades. b. Você não deve: i. Permitir visão de túnel; ii. Deixar obstáculos inexploráveis ou evitar núcleos de conflito; iii. Tolerar misturas de problemas, necessidades ou conflitos. a. Você deve: i. Estimular a inspiração repentina do interessado. Enfrentar os obstáculos diretamente. sem fugir; FASE DE REALIZAÇÃO S U M Á R I O a. Deve fazer: i. Faça apenas uma medição de cada vez; ii. Estabeleça metas específicas a curto prazo; iii. Revise as metas quando necessário; iv. Seja gestor se (e somente se) for essencial. b. Não se deve: i. Tentar resolver tudo agora e/ou ao mesmo tempo; ii. Tomar decisões a longo prazo de forma obrigatória; iii. Ser tímido ou mostrar sinais de insegurança (passividade) ou domínio (agressividade); 99 FASE DE MONITORAMENTO S U M Á R I O a. Você deve: i. Assumir o compromisso de estabelecer contato novamente (com o mesmo ou com outro profissional, conforme o caso); ii. Avaliar e planejar com as partes interessadas as várias etapas da ação de resolução de problemas. b. Você não deve: i. Deixar no ar detalhes fundamentais ou pressupor prematuramente ii. Não realizar a avaliação ou planejamento das fases imediata- mente futuras para sair da crise. c. Tente: i. Escutar cuidadosamente; ii. Refletir fatos e sentimentos; iii. Comunicar-se internamente com você mesmo; iv. Fazer perguntas claras; v. Fazer uma coisa de cada vez; vi. Definir metas; vii. Estabelecer compromisso; viii. Avaliar cada problema; ix. Não fugir dos obstáculos; x. Assumir responsabilidade. d. Procure: i. Ignorar o que está acontecendo; ii. Julgar a si mesmo; iii. Culpar-se por tudo; iv. Deixar tudo sem acabar; S U M Á R I O 100 v. Tentar resolver tudo ao mesmo momento; vi. Assumir responsabilidades que não são suas; vii. Mostrar insegurança; viii. Permitir que o medo o domine. v. Tentar resolver tudo ao mesmo momento; vi. Assumir responsabilidades que não são suas; vii. Mostrar insegurança; viii. Permitir que o medo o domine. S U M Á R I O viii. Permitir que o medo o domine. RESOLUÇÃO DE CONFLITOS 1. Evitação: a pessoa não pode admitir a existência de um conflito e sua reação, portanto, pode ser evitá-lo ou negá-lo. É usado quando: • É uma questão trivial ou há problemas mais importantes que precisam ser resolvidos; • É uma questão trivial ou há problemas mais importantes que precisam ser resolvidos; • A pessoa toma consciência de que não será capaz de satisfazer seus desejos; • É mais provável que surjam novos problemas; • Você tem que fazer com que as pessoas se acalmem e recupe- rem a objetividade; S U M Á R I O • Outros podem resolver o conflito de forma mais satisfatória. 2. Adequação: As partes opostas querem acalmar seu oponen- te e estão dispostas a colocar os interesses dele acima dos seus. É usado quando: 2. Adequação: As partes opostas querem acalmar seu oponen- te e estão dispostas a colocar os interesses dele acima dos seus. É usado quando: • A pessoa descobre que está errada; • A pessoa descobre que está errada; • As questões discutidas são mais importantes para os outros do que para si mesmo; • Destina-se à obtenção de crédito social em edições posteriores; 101 • Devemos minimizar a perda porque a maioria não compartilha nossas opiniões; S U M Á R I O • Você quer estabelecer um clima de harmonia e estabilidade. 3. Compromisso: ocorre quando as partes são obrigadas a abrir mão de algo e optar pela negociação, o que leva a um compromisso, em que não há vencedores nem vencidos. É usado quando: • As metas são importantes, mas não justificam o esforço; • Os oponentes têm o mesmo poder e perseguem objetivos mu- tuamente exclusivos; • O objetivo é alcançar acordos temporários em situações com- plexas; • Você deseja obter soluções adequadas na falta de tempo; • A colaboração ou competição não for conveniente. Com base em duas dimensões básicas (cooperação e assertivi- dade), podem ser distinguidas cinco orientações diferentes de controle de conflitos. Elas serão: S U M Á R I O 1. Competição: a pessoa tenta atingir seus objetivos sem se impor- tar com as consequências que isso tem para os outros. É usado quando: a. A ação imediata e decisiva é vital; a. A ação imediata e decisiva é vital; b. São questões importantes, em que se faz necessário tomar me- didas impopulares; c. RESOLUÇÃO DE CONFLITOS São questões vitais para o bem da organização (se você tem certeza de que estamos certos); d. Há pessoas que estão se aproveitando do comportamento não competitivo. 102 2. Colaboração: as partes desejam satisfazer os desejos ou inte- resses da outra, por meio da cooperação e da busca de um resultado que beneficie a todos. É usado quando: S U M Á R I O a. Uma solução integradora deve ser encontrada; O objetivo é aprender; b. O objetivo é aprender; c. Você tem que aproveitar as ideias de pessoas com pontos de vista diferentes; d. A aceitação deve ser alcançada e o consenso alcançado; e. Você tem que resolver os sentimentos que atrapalharam o relacionamento; f. Por meio da negociação, pretende-se chegar a um acordo com alguém sobre algo de interesse comum. Negociar é uma medi- da fundamental para conseguir o que se deseja e requer uma comunicação efetiva de mão dupla para chegar a um acordo. Apesar da negociação ser uma coisa cotidiana em nossas vidas (negociamos com nosso parceiro, com nossos pais e amigos para dividir as tarefas domésticas, para chegar a um acordo sobre o filme que vamos ver, para poder chegar em casa mais tarde, etc.), muitas vezes cometemos erros frequentes e não chegamos a acordos justos e equitativos. Às vezes, as estratégias usadas na negociação deixam as pessoas insatisfeitas, cansadas ou alienadas. S U M Á R I O Desejamos evitar a aparência de confrontos pessoais entre as par- tes e, portanto, decidimos fazer concessões imediatas para solucionar o conflito. O que se busca é chegar a uma solução amigável, porém, mui- tas vezes, o negociador que adota essa posição acaba sendo explorado. O sucesso da psicoterapia emocional é alcançado através do estabelecimento de um bom controle emocional. As emoções negati- vas inapropriadas são aquelas que aumentam as condições adversas 103 e o desconforto, impedindo que o problema seja resolvido. Geralmente estão ligados a velhas situações não resolvidas, que podem levar ao desenvolvimento de ansiedade, depressão, raiva ou culpa. S U M Á R I O Enquanto as emoções negativas apropriadas são aquelas que ocorrem quando os desejos, necessidades e gostos da pessoa são bloqueados e frustrados. Este último pode levar à tristeza, raiva ou arrependimento. Para um correto controle emocional é necessário co- nhecer os aspectos que caracterizam as emoções humanas. RESOLUÇÃO DE CONFLITOS O exercício é altamente tera- pêutico, pois gera sensações agradáveis que afetam o humor; • Encontre pessoas que queiram ajudá-lo ou ouvi-lo, evitando aquelas que destacam suas frustrações e promovem sua queda; • Não faça nada que faça você se sentir pior; • Seja seu próprio amigo, de modo que, se você se valoriza, os outros também. Não pense que sentir-se solitário e triste é uma fraqueza, pois faz parte do ser humano. 105 • Esteja ciente das causas de seus próprios sentimentos de so- lidão, aprendendo a identificar sua causa raiz antes que eles ocorram. Apenas estar ciente desse processo o ajudará a ge- renciá-lo emocionalmente; • Desenvolva descargas construtivas e criativas fazendo qualquer coisa que estimule seu pensamento e permita que você se con- centre em algo fora de si mesmo; • Tente ver as coisas positivas em sua própria vida, porque você precisa tê-las; • Cultive a capacidade de aproveitar a vida cotidiana. Tire um tem- po para apreciar e desfrutar da grande variedade de paisagens, sons e situações que o cercam (passeie por um parque, ouça um concerto, desenvolva atividades com seus amigos, etc.); • Envolva-se em algo emocionante e construtivo, evitando cair na sensação de vazio e medo do futuro; • Identifique e dê as perspectivas adequadas a cada medo, por- que a incerteza é o inimigo oculto mais perigoso; • Aprenda a se recompensar, pois tendemos a ser generosos com os outros, mas não com nós mesmos; • Envolva-se em atividades físicas. O exercício é altamente tera- pêutico, pois gera sensações agradáveis que afetam o humor; • Encontre pessoas que queiram ajudá-lo ou ouvi-lo, evitando aquelas que destacam suas frustrações e promovem sua queda; • Não faça nada que faça você se sentir pior; • Seja seu próprio amigo, de modo que, se você se valoriza, os outros também. Não pense que sentir-se solitário e triste é uma fraqueza, pois faz parte do ser humano. • Esteja ciente das causas de seus próprios sentimentos de so- lidão, aprendendo a identificar sua causa raiz antes que eles ocorram. RESOLUÇÃO DE CONFLITOS Eles são: • Uma situação concreta; • Uma série de reações ou sensações fisiológicas específicas (aceleração do pulso e da respiração, tensão muscular, etc.); • Uma série de reações ou sensações fisiológicas específicas (aceleração do pulso e da respiração, tensão muscular, etc.); • Certos pensamentos; • Certos pensamentos; • Um tipo de respostas concretas apropriadas a essa situação. Estabeleça uma série de medidas para controlar o estresse tanto no nível individual, no nível da equipe e no nível da instituição-organização: Conheça as principais formas de prevenção do estresse laboral; S U M Á R I O • Estude as formas de proceder perante uma intervenção em si- tuações de crise; • Relacione o estresse no trabalho com a resolução de conflitos Para prevenir e gerir o estresse a nível pessoal, existem dife- rentes estratégias que podemos desenvolver a nível íntimo, para nos encontrarmos, tais como: • Evite gastar muito tempo ruminando problemas pessoais, am- pliando-os e caindo em um ciclo de autopiedade, auto-degra- dação e improdutividade pessoal; • Evite gastar muito tempo ruminando problemas pessoais, am- pliando-os e caindo em um ciclo de autopiedade, auto-degra- dação e improdutividade pessoal; 104 • Esteja ciente das causas de seus próprios sentimentos de so- lidão, aprendendo a identificar sua causa raiz antes que eles ocorram. Apenas estar ciente desse processo o ajudará a ge- renciá-lo emocionalmente; • Desenvolva descargas construtivas e criativas fazendo qualquer coisa que estimule seu pensamento e permita que você se con- centre em algo fora de si mesmo; • Tente ver as coisas positivas em sua própria vida, porque você precisa tê-las; • Cultive a capacidade de aproveitar a vida cotidiana. Tire um tem- po para apreciar e desfrutar da grande variedade de paisagens, sons e situações que o cercam (passeie por um parque, ouça um concerto, desenvolva atividades com seus amigos, etc.); • Envolva-se em algo emocionante e construtivo, evitando cair na sensação de vazio e medo do futuro; • Identifique e dê as perspectivas adequadas a cada medo, por- que a incerteza é o inimigo oculto mais perigoso; • Aprenda a se recompensar, pois tendemos a ser generosos com os outros, mas não com nós mesmos; • Envolva-se em atividades físicas. RESOLUÇÃO DE CONFLITOS Apenas estar ciente desse processo o ajudará a ge- renciá-lo emocionalmente; S U M Á R I O Desenvolva descargas construtivas e criativas fazendo qualquer coisa que estimule seu pensamento e permita que você se con- centre em algo fora de si mesmo; Desenvolva descargas construtivas e criativas fazendo qualquer coisa que estimule seu pensamento e permita que você se con- centre em algo fora de si mesmo; • Tente ver as coisas positivas em sua própria vida, porque você precisa tê-las; • Cultive a capacidade de aproveitar a vida cotidiana. Tire um tem- po para apreciar e desfrutar da grande variedade de paisagens, sons e situações que o cercam (passeie por um parque, ouça um concerto, desenvolva atividades com seus amigos, etc.); • Envolva-se em algo emocionante e construtivo, evitando cair na sensação de vazio e medo do futuro; Envolva-se em algo emocionante e construtivo, evitando cair na sensação de vazio e medo do futuro; • Identifique e dê as perspectivas adequadas a cada medo, por- que a incerteza é o inimigo oculto mais perigoso; • Aprenda a se recompensar, pois tendemos a ser generosos com os outros, mas não com nós mesmos; S U M Á R I O • Envolva-se em atividades físicas. O exercício é altamente tera- pêutico, pois gera sensações agradáveis que afetam o humor; • Encontre pessoas que queiram ajudá-lo ou ouvi-lo, evitando aquelas que destacam suas frustrações e promovem sua queda; • Não faça nada que faça você se sentir pior; • Seja seu próprio amigo, de modo que, se você se valoriza, os outros também. Não pense que sentir-se solitário e triste é uma fraqueza, pois faz parte do ser humano. 105 Uma forma eficaz de prevenção e intervenção a nível pessoal é mudar a nossa forma de encarar a vida. O trabalho deve nos satisfazer e ter objetivos viáveis, se possível, dentro da área mais próxima das preferências pessoais. RESOLUÇÃO DE CONFLITOS S U M Á R I O Para realizar esta mudança de abordagem que estamos suge- rindo, é muito importante levar em conta algumas recomendações: • Não seja um perfeccionista, porque a perfeição é impossível; • Não seja um perfeccionista, porque a perfeição é impossível; • Lembre-se de que somos todos diferentes e que o que funciona para os outros não necessariamente funciona para si mesmo; • Pergunte-se se nossos problemas são realmente críticos, pois muitas vezes, depois de analisá-los, percebemos que não são grande coisa; • Pense positivamente, pois é mais saudável contemplar o lado po- sitivo da vida, mesmo que sinta que está falhando na tentativa; • Não adie a resolução de problemas ou conflitos irritantes, pois o fato de estar atento gera mais ansiedade; • Não afunde ou desça por não atingir plenamente os seus objetivos. • Não afunde ou desça por não atingir plenamente os seus objetivo S U M Á R I O No entanto, nem sempre é fácil fazer com que esse tipo de estra- tégia funcione, pois, em muitas ocasiões, é difícil nos conscientizarmos de que há um problema e de que precisamos fazer alguma mudança. Quando nos encontramos numa situação de estresse, mais concretamente de estresse laboral, pode ser necessário outro tipo de intervenção, externa à própria pessoa. A ideia principal é tentar iden- tificar os agentes que causam esse estresse, conscientizar a pessoa sobre eles, mostrar as possibilidades de resolução da situação e ge- renciamento inteligente do estresse. 106 As técnicas de atenção, que consistem em métodos para ajudar as pessoas a resolver suas reações fisiológicas e psicológicas, com es- tratégias para reduzir o estresse no ambiente de trabalho, são muito úteis. Estes consistem em exercícios de relaxamento, autotreinamento, exercí- cios de respiração, autoestima, meditação, etc. De forma complementar, são também ensinadas estratégias de gestão do tempo, priorização de problemas, desenvolvimento da capacidade de planeamento, técnicas de negociação, capacidade de decisão e resolução de conflitos. S U M Á R I O Para a prevenção do estresse através de uma intervenção no indivíduo, é necessário saber em que “pontos” o problema pode ori- ginar-se e, portanto, em que aspetos temos de intervir. Basicamente, as dificuldades podem ter origem em um ou vários aspectos. RESOLUÇÃO DE CONFLITOS A seguir, descreveremos alguns deles: Devido ao processamento cognitivo defeituoso, uma situação pode ser percebida de forma diferente por diferentes sujeitos, de modo que para alguns pode ser ameaçadora, e para outros, motivadora. Essas diferenças ocorrem porque, diante de uma mesma situação, nem todos coletam as mesmas informações, pois nem todos prestam atenção nas mesmas coisas. A relevância dessa situação ou como ela me afeta ou meu futuro também é percebida de forma diferente. Um indivíduo também pode ter dificuldades devido ao fato de valorizar inadequadamente suas capacidades de resposta, o que acarretará a escolha de um ou outro tipo de métodos de enfrentamento. S U M Á R I O Estratégias para modificar essa situação buscam: • A adaptação do indivíduo em relação a que ou quanta informa- ção coletar e como coletá-la; • A adaptação do indivíduo em relação a que ou quanta informa- ção coletar e como coletá-la; • A rápida identificação de aspectos que podem ser relevantes e a concentração nesses aspectos; • A rápida identificação de aspectos que podem ser relevantes e a concentração nesses aspectos; 107 • O foco da atenção do indivíduo em informações úteis para re- solver o problema e não focar sua atenção em seu próprio estado emocional; S U M Á R I O • A modificação dos esquemas errôneos sob os quais percebe e sob os quais atua (atenção seletiva, atribuições generalizantes, interpretação polarizada, etc.); A modificação dos esquemas errôneos sob os quais percebe e sob os quais atua (atenção seletiva, atribuições generalizantes, interpretação polarizada, etc.); • Avaliação adequada de suas próprias capacidades. • Avaliação adequada de suas próprias capacidades. Avaliação adequada de suas próprias capacidades. Em suma, busca-se que a pessoa tenha uma perspectiva mais ampla e saiba valorizar a informação obtida em sua justa medida. De- vido às demandas excessivas do ambiente, por falta de comportamen- tos de enfrentamento ou uso inadequado deles. Em algumas situações, o que é exigido de nós em nosso tra- balho é excessivo. Desta forma nós nos deparamos com problemas que são muito difíceis para lidarmos. Pode-se dizer que para combater essas situações existe uma recomendação genérica de que a organi- zação deve ajustar as demandas de seus trabalhadores, levando em consideração suas capacidades, necessidades e expectativas. No en- tanto, embora este seja um problema que “vem de fora”, a pessoa tem certa margem de manobra para modificar essas condições. Às vezes ele não sabe como enfrentar a situação, o que pode ser por quê: S U M Á R I O • Ele percebeu adequadamente o que é exigido dele e sabe quais comportamentos deve emitir, mas não os possui; • Não sabe selecionar a resposta correta entre todos os compor- tamentos que poderiam ser realizados; • Ele tem comportamentos adequados, mas não é capaz de emi- ti-los no momento preciso, seja porque não sabe discriminar quando devem ser realizados, seja porque certas situações ge- ram respostas emocionais (medo, ansiedade, etc.) de tal mag- nitude que o impedem de responder. • Ele tem comportamentos adequados, mas não é capaz de emi- ti-los no momento preciso, seja porque não sabe discriminar quando devem ser realizados, seja porque certas situações ge- ram respostas emocionais (medo, ansiedade, etc.) de tal mag- nitude que o impedem de responder. 108 Aparecimento de respostas fisiológicas e emocionais inadequadas S U M Á R I O Diante de uma situação estressante, a pessoa desenvolve uma série de reações fisiológicas e emocionais inadequadas (tensão físi- ca e emocional, ansiedade, fadiga, etc.). A presença dessas reações, além de serem os primeiros sintomas de estresse, interferem em outras áreas do indivíduo. Estas respostas inadequadas produzem grandes distorções no nível cognitivo (o modo de perceber a situação) e com- portamental (o modo de controlar a situação). A intervenção neste nível é direcionada para controlar essas respostas emocionais. O conceito de estresse no trabalho é muitas vezes confundido com desafio, mas são teorias diferentes. O desafio nos revigora psico- logicamente e fisicamente e nos motiva a aprender novas habilidades e dominar nossos trabalhos. Quando enfrentamos um desafio, nos senti- mos relaxados e satisfeitos. Portanto, o desafio é uma parte importante do trabalho saudável e produtivo e é a esse conceito que nos referimos quando dizemos que “um pouco de estresse faz bem”. Em nível técnico S U M Á R I O As pessoas costumam falar de “prevenção de conflitos” para se referir à necessidade de agir antes que eles explodam (crise) e se ma- nifestem em sua forma mais dura. Tudo isso nos leva a não poder, nem querer, falar sobre prevenção de conflitos. Se considerarmos o conflito como um conflito de interesses, é inútil falar em prevenção. O processo de intervenção diante da crise nos leva a: O processo de intervenção diante da crise nos leva a: • uma explicação adequada do conflito, incluindo a sua dimen- são humana; • uma explicação adequada do conflito, incluindo a sua dimen- são humana; 109 • uma compreensão das mudanças estruturais necessárias para eliminar suas causas; S U M Á R I O • promoção de condições que criem um clima adequado e favo- reçam relações cooperativas que diminuam o risco de novos surtos, aprendendo a lidar e resolver contradições antes que se tornem antagonismos. Promova uma boa comunicação A boa comunicação é fundamental no processo de aprender a resolver conflitos de forma não violenta, pois o diálogo é uma de suas principais ferramentas. Coloque em prática jogos e dinâmicas que nos permitam desenvolver uma comunicação eficaz, que realmente nos ensine a dialogar e a ouvir o outro de forma ativa e empática. Trabalharemos os diferentes canais de comunicação e sua im- portância. Por um lado, temos o canal verbal. É importante trabalhar e observar como, apesar de ser o canal mais utilizado e mais preciso para uma das seções de comunicação, a transmissão da mensagem, no entanto, pode gerar muitas confusões e mal-entendidos. Aprenda a estabelecer um código comum, não tomando nada como garanti- do, mas verificando se realmente estamos nos entendendo e falando sobre a mesma coisa e/ou entendendo as palavras com as quais estamos lidando da mesma maneira. Tudo isso será especialmente relevante em situações de conflito. S U M Á R I O Apesar da importância da comunicação verbal, não devemos esquecer os canais não verbais e como a comunicação não tem ape- nas o aspecto da informação (a mensagem), mas também o aspec- to relacional que faz a mesma mensagem pode ser interpretado de maneiras muito diferentes. São canais que nos permitirão transmitir 110 melhor emoções, sentimentos, etc. que muitas vezes estão por trás das posições ou posturas que tomamos em muitos conflitos. S U M Á R I O Aprender a ter coerência entre os dois aspectos da comunica- ção (informativa e relacional) e entre o que alguns canais transmitem e outros, será essencial. Se as palavras dizem uma coisa e os canais não verbais dizem outra, perde-se credibilidade e confiança. A mensagem não passa e mais conflito e confusão são criados. Também será muito importante trabalhar a escuta ativa. Não se trata apenas de ouvir, mas também de fazer a outra pessoa sentir que me importo com o que ela diz, que ela está sendo ouvida. Podemos fazer isso tanto verbalmente (parafraseando empaticamente, verifican- do, fazendo perguntas esclarecedoras.Trabalhar aspectos da comuni- cação envolve muito mais coisas: aprender a tomar e usar a palavra, a se expressar, a respeitar quando outra pessoa a tem, a colocar em prática técnicas que permitam uma distribuição justa da palavra, etc. Tomada de decisão por consenso Tudo o que foi dito acima deve ser colocado em prática apren- dendo a tomar decisões consensuais, de forma igualitária, participativa e não sexista. Vá além da votação e das maiorias, e aprenda a tomar decisões em que todos tenham tido a oportunidade de se expressar e sintam que sua opinião foi levada em consideração na decisão final. Para aprender isso, é preciso colocar em prática, o que significa abrir mão de parcelas de responsabilidade/poder e dar espaços para tomar decisões. Como sempre com a ideia de processo. Começaremos dan- do a oportunidade de tomar decisões sobre questões simples, para gradualmente decidir sobre coisas cada vez mais são importantes. S U M Á R I O Mude suas atitudes pessoais em relação à profissão e ao trabalho! 111 Um dos aspectos mais importantes quando se trata de preve- nir o burnout (ou superá-lo, se já estivermos em uma de suas fases), é o componente de atitudes. E, para mudá-los, você tem que mudar seus pensamentos. S U M Á R I O Vamos fazer a seguinte reflexão: seus pensamentos são a forma como você interpreta a realidade, o prisma pelo qual você a vê. Se esse prisma tende a ser negativo, muitas vezes você verá as coisas mais desfavoráveis do que realmente são. Cada vez que algo der errado, você interpretará como uma ratificação de sua opinião, de tal forma que as situações que você vê como adversas serão progressivamente mais frequentes, até que finalmente você interprete tudo ou pratica- mente tudo como negativo. É daí que nascem suas atitudes, que são as que marcarão o humor com que você encara suas experiências e a maneira como você se comporta em relação a elas. Uma situação objetivamente desfavorável no trabalho pode ser abordada de vários ângulos: • Perceba tudo como negativo, estressante e, assim, corra um certo risco de se esgotar; • Escolha ver tanto o lado positivo quanto o lado negativo da reali- dade, analisando o que você pode mudar e como, bem como o que você tem que se resignar a aceitar, tentando assim minimi- zar os danos pessoais que a situação pode-lhe causar; S U M Á R I O Finalmente, você pode avaliar se esse trabalho não vale o preço que você está pagando por ele, tanto física quanto mentalmen- te, e decidir deixá-lo. Tomada de decisão por consenso Mas seria desejável que você o fizesse a partir de uma visão realista de como as coisas são, e não de uma interpretação distorcida que o leve a ver o que está aconte- cendo ao seu redor pior do que é. 112 Atitudes frente ao conflito S U M Á R I O Competição (ganhar/perder). Encontramo-nos numa situação em que conseguir o que quero, afirmar os meus objetivos, as minhas metas, é o mais importante, por mais que tenha de passar por cima de meus medos. Eu não me importo com o relacionamento. No modelo da competição levada até as últimas consequências, o importante é que eu ganhe e para isso o mais fácil é que os outros percam. Vemos isso cons- tantemente nos esportes, por exemplo. Essa perda, às vezes, traduz-se não apenas no fato de o outro não atingir seus objetivos, mas em ser eliminado ou destruído (discriminação, exclusão, expulsão, anulação). Aceitação (perder/ganhar). Contanto que eu não confronte a ou- tra parte, não afirmo ou mesmo declaro meus objetivos. É um modelo tão difundido ou mais do que a concorrência. Muitas vezes, confundi- mos respeito, boa educação, com não fazer valer nossos direitos por- que isso pode causar tensão ou desconforto. Nós aguentamos até não aguentarmos mais e então destruímos a nós mesmos ou a outra parte. Evasão (perder/perder). Nem os objetivos nem o relacionamen- to vão bem, nenhum deles é alcançado. S U M Á R I O Cooperação (eu ganho/você ganha). Nesse modelo, atingir os objetivos é muito importante, mas o relacionamento também. Tem mui- to a ver com algo muito intrínseco à filosofia “não-violenta”: o fim e os meios têm que ser consistentes. É o modelo para o qual vamos tentar direcionar o processo educacional. É um modelo em que só funcionam as soluções ganho-ganha. Negociação. Alcançar a cooperação plena é muito difícil, razão pela qual se propõe outro modelo em que ambas as partes ganham fundamentalmente, uma vez que não podem chegar a 100%. Há pes- soas que, quando falam em negociação, estão na verdade pensando 113 em uma mera tática do modelo de competição. Se uma das duas par- tes não sair com a sensação de que o fundamental venceu, não esta- mos nesse modelo, mas no de competição ou acomodação. S U M Á R I O É muito fácil confundir cooperar com o que chamamos de ser uma pessoa boa ou acomodada. Cooperar não é acomodar, não pode ser abrir mão do que é fundamental para nós. Sem ceder, você pode ne- gociar, na verdade, você não deve ceder fundamentalmente. Outra coisa diferente é que você pode comprometer o que é menos importante. Atitudes frente ao conflito O próprio diagrama dá algumas pistas sobre isso. Em circuns- tâncias em que o que está em jogo não é importante para nós e quem está em jogo é alguém que mal conhecemos e com quem quase não temos relacionamento, provavelmente a melhor opção é algo que a princípio pode parecer muito negativo, como evitar conflitos. No entanto, este esquema também nos diz algo muito importan- te, que quanto mais importantes forem os objetivos e o relacionamen- to, mais importante será aprender a cooperar. Nessas circunstâncias, os modelos perde-ganha e ganha-perde não funcionarão no médio e curto prazo e nos levarão a uma situação em que todos perdemos. Um exemplo poderia ser a tomada de decisões por maiorias estreitas em uma faculdade. O grupo que perder a votação não sairá particularmente feliz. Se isso acontece com frequência, o que vai acabar acontecendo é que eles colocarão obstáculos para que a decisão seja realizada ou que gradualmente se inibam das res- ponsabilidades e tarefas do centro. O que pode parecer a melhor estratégia individual es a estratégia coletiva, mas a pior estratégia individual quando estamos interagindo com outras pessoas e isso é algo que não entendemos completamente. S U M Á R I O O grande desafio é começar a fazer com que as partes vejam que cooperar para construir é melhor, não só do ponto de vista ético, mas também do ponto de vista da eficiência. Em todo conflito, de 114 alguma forma, as partes cooperam, mas para destruir uma à outra. Se concordassem em cooperar para construir, alcançariam soluções muito melhores e mutuamente satisfatórias. S U M Á R I O Mas, sem dúvida, a melhor solução seria não ter que detectar que alguém está em situação crítica, pois a prevenção seria possível e eficaz. Como é lógico, a prevenção exige tanto a detecção precoce dos sinais que indicam o perigo da situação, quanto o conhecimento do processo de desenvolvimento de uma crise e quais são as fases pelas quais um indivíduo passa de uma situação normal até atingir aquela situação crítica. Sabemos que a passagem da normalidade para a situação de crise ocorre, em linhas gerais, em quatro fases: 1. Elevação inicial do nível de tensão emocional do sujeito devido ao impacto de um ou vários eventos externos, que o levam a ter a sensação de “ter que fazer algo naquele exato momento para resolver o problema”. Atitudes frente ao conflito Essa elevação inicial da tensão emocional faz com que esses eventos sejam enfrentados de acordo com os comportamentos habituais do sujeito e orientações para re- solver seus problemas em outros momentos; S U M Á R I O 2. Ao não ter sucesso na resolução de problemas, a tensão au- menta e surgem sentimentos de ineficiência, inadequação e de- sordem, principalmente se o impacto continuar; 3. Quando a tensão aumenta, outros recursos destinados à solução são lançados. A partir daí, a crise pode ser desviada, seja para a redução da ameaça externa (real ou não) pelo sucesso das estratégias de enfrentamento utilizadas, para uma redefinição do problema ou seu significado, ou para a renúncia de ter fixado e objetivos coerentes (reagindo pontualmente aos aspectos espe- cíficos do problema, mas de forma globalmente desorganizada); 115 4. Quanto menos o problema global for controlado, devido à inefi- cácia das estratégias postas em prática, maior será o aumento da tensão (que aumentará a um ponto crucial, que varia de uma pessoa para outra) e maior será a desorganização emocional é a probabilidade de perda de controle. S U M Á R I O Durante esse processo temporal e psicológico, o sujeito vive si- multaneamente uma série de experiências que convém conhecer com mais cuidado e levar em conta ao agir: 1. Na primeira fase, o sujeito tenta enquadrar o problema per- cebido, mas não consegue no tempo esperado. Ele percebe seu fracasso inicial e permanece insatisfeito, experimentando tensão e ansiedade progressivas, com sentimentos desagra- dáveis e somatizantes; 2. A segunda fase, a tensão continua a aumentar e um certo grau de desorganização começa a ocorrer. O sujeito experimenta sentimentos mal definidos de culpa, medo, ansiedade; 3. Na terceira fase, à medida em que a tensão e a desorganização aumentam, o sujeito pode ser arrastado por seus impulsos ou por outras influências, ativa ou passivamente. O sujeito pode tentar redefinir o problema total ou parcialmente, para encontrar novas soluções. Se isso for eficaz, a crise será superada e isso permitirá o crescimento. Se essa redefinição cortar a crise, mas for mal adaptada, será lançado um quadro neurótico que deixa- rá sequelas. Se ele não conseguir resolver a situação, ele passa- rá para uma quarta fase. A desorganização aumenta e atinge um ponto de ruptura no qual podem ocorrer episódios psicóticos e comportamentos indesejáveis, de forma lógica e imprevisível. 3. Atitudes frente ao conflito Na terceira fase, à medida em que a tensão e a desorganização aumentam, o sujeito pode ser arrastado por seus impulsos ou por outras influências, ativa ou passivamente. O sujeito pode tentar redefinir o problema total ou parcialmente, para encontrar novas soluções. Se isso for eficaz, a crise será superada e isso permitirá o crescimento. Se essa redefinição cortar a crise, mas for mal adaptada, será lançado um quadro neurótico que deixa- rá sequelas. Se ele não conseguir resolver a situação, ele passa- rá para uma quarta fase. A desorganização aumenta e atinge um ponto de ruptura no qual podem ocorrer episódios psicóticos e comportamentos indesejáveis, de forma lógica e imprevisível. S U M Á R I O No entanto, existem algumas diferenças importantes entre o que podemos considerar uma situação de crise e o que pode ser conside- rado uma situação de angústia, ansiedade ou estresse agudo. Assim, 116 entre a ansiedade, o medo, etc., e a crise, embora muitas vezes sejam difíceis de distinguir na prática, existem algumas diferenças que são fundamentais, pois requerem diferentes formas de intervenção. S U M Á R I O S U M Á R I O 117 117 SOBRE O AUTOR S U M Á R I O Robson Marins de Abreu Estudante de psicologia, blogueiro, pensador. Marins Escreve de uma forma metódica, seus livros de poemas, pensamentos, reflexões, da realidade que vive e sofre e também das experiências de pessoas próximas com quem convive e compartilha experiências de todos os tipos. Atualmente combina a escrita e publicação de suas obras com estudos em filosofia, psicologia. Em 2019 foi selecionado para participar na III Antologia de Poesia Hispano-A- mericana Contemporânea Y lo demás es silencio, realizado na Universidade Autónoma de Madrid. CONSIDERAÇÕES FINAIS S U M Á R I O A todos que vivem em uma guerra interna, com conflitos inter- nos, todos temos direito à vida. Não importa a classe social ou raça, há prazer em viver, e muitas vezes viver significa aprender a ganhar, perder, chorar e rir. Pare de rotular as pessoas que falam sobre seus problemas quando na verdade elas estão apenas procurando alguém para ouvi-las. A vida é um presente que deve ser desfrutado, não se permita viver a história dos outros, aprenda com a sua e ensine aos outros que você também faz parte desse grande universo e que não existem pessoas especiais. S U M Á R I O 118 É autor dos livros: S U M Á R I O Superar a paixão enganosa - (2017) As armadilhas do amor – (2018) Afogando-se em silêncio – (2020) O armário secreto - (2021) Eu não te perdôo, quando o perdoar é um conflito (2021) Sua pagina web: https://marinsbreu.com Superar a paixão enganosa - (2017) As armadilhas do amor – (2018) Afogando-se em silêncio – (2020) O armário secreto - (2021) Eu não te perdôo, quando o perdoar é um conflito (2021) Sua pagina web: https://marinsbreu.com Afogando-se em silêncio – (2020) Eu não te perdôo, quando o perdoar é um conflito (2021) Sua pagina web: https://marinsbreu.com 119 119
https://openalex.org/W2963118843	https://europepmc.org/articles/pmc6820803?pdf=render	English	null	<i>Streptococcus mutans</i> isolated from a 4‐year‐old girl diagnosed with infective endocarditis	Clinical and experimental dental research	2,019	cc-by	5,151	K E Y W O R D S biofilm, infective endocarditis, Streptococcus mutans O R I G I N A L A R T I C L E O R I G I N A L A R T I C L E Streptococcus mutans isolated from a 4-year-old girl diagnosed with infective endocarditis Yoshio Kondo1 \| Tomonori Hoshino1,2 \| Midori Ogawa3 \| Kiyoshi Hidaka1 \| Tomoyuki Hasuwa4 \| Hiroyuki Moriuchi4 \| Taku Fujiwara1 Correspondence Results: The isolated strain was S. mutans serotype e. However, its morphology was observed to be short chains, unlike that of the NCTC 10449 reference strain. Fibrino- gen adhesion and biofilm formation were more apparent than in NCTC 10449. The fingerprinting pattern showed that HM came from the patient's saliva. Conclusions: HM differs from NCTC 10449 in its higher fibrinogen affinity. HM was also found to be derived from the oral cavity. These results highlight the importance of good oral hygiene for the prevention of IE in children. K E Y W O R D S biofilm, infective endocarditis, Streptococcus mutans Yoshio Kondo1 \| Tomonori Hoshino1,2 \| Midori Ogawa3 \| Kiyoshi Hidaka1 \| Tomoyuki Hasuwa4 \| Hiroyuki Moriuchi4 \| Taku Fujiwara1 1Department of Paediatric Dentistry, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan 2Department of Paediatric Dentistry, Meikai University School of Dentistry, Saitama, Japan 3Department of Microbiology, School of Medicine, University of Occupational and Environmental Health Japan, Kitakyushu, Japan 4Department of Paediatrics, Nagasaki University Graduate School of Biochemical Sciences, Nagasaki, Japan Correspondence Taku Fujiwara, Department of Paediatric Dentistry, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki, Japan. Email: takufuji@nagasaki-u.ac.jp Funding information Japan Society for the Promotion of Science, Grant/Award Numbers: 16K11808, 19K10388 1Department of Paediatric Dentistry, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan 2Department of Paediatric Dentistry, Meikai University School of Dentistry, Saitama, Japan 3Department of Microbiology, School of Medicine, University of Occupational and Environmental Health Japan, Kitakyushu, Japan 4Department of Paediatrics, Nagasaki University Graduate School of Biochemical Sciences, Nagasaki, Japan Correspondence Taku Fujiwara, Department of Paediatric Dentistry, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki, Japan. Email: takufuji@nagasaki-u.ac.jp Funding information Japan Society for the Promotion of Science, Grant/Award Numbers: 16K11808, 19K10388 Abstract Objectives: Infective endocarditis (IE) has an extremely high fatality rate. In this study, we isolated a strain of Streptococcus mutans, which we called HM, from the blood drawn from a 4-year-old girl diagnosed with IE. We aimed to fully type the HM strain and investigate its biological properties, including its virulence with respect to IE. Material and methods: A 16S rRNA phylogenetic tree and glucosyltransferase gene sequences were used to type HM. Serotyping was performed using the Ouchterlony method. Morphological observations were made using phase contrast and electron microscopy. Fibrinogen adhesion and biofilm formation were investigated to examine the tissue colonization properties of HM, whereas its bodily origin was determined from its fingerprinting pattern. Received: 12 February 2019 Revised: 3 June 2019 Accepted: 10 June 2019 Received: 12 February 2019 Revised: 3 June 2019 Accepted: 10 June 2019 Received: 12 February 2019 Revised: 3 June 2019 Accepted: 10 June 2019 DOI: 10.1002/cre2.220 wileyonlinelibrary.com/journal/cre2 5\34 2.2 \| Bacterial strains The bacterial strain isolated from the patient's blood sample, desig- nated here as strain HM, was stored at −70C until use. S. mutans NCTC 10449 was used as the reference strain (Hardie & Genus Strep- tococcus, 1986; Smibert et al., 1994). Both strains were grown anaer- obically in brain heart infusion (BHI) broth (Difco Laboratories) and on trypticase soy agar plates (Difco Laboratories) supplemented with defibrinated sheep blood (5% vol/vol). 2.3 \| Determining the species isolated from the specimen IE is most frequently caused by staphylococci or streptococci bac- teria (Hall-Stoodley, Costerton, & Stoodley, 2004; Moreillon & Que, 2004; Murdoch et al., 2009). Population-based cohort studies show that viridans group streptococci are the most common IE-causing organisms, followed by Staphylococcus aureus (Tleyjeh et al., 2005). Viridans group streptococci constitute the largest group among the streptococci, which are also known to be the most prevalent bacterial group in the oral cavity. Within the viridans streptococcal group, Streptococcus sanguinis and Streptococcus oralis are the pathogens most frequently isolated from patients with IE. Streptococcus mutans, a well-known cariogenic bacterium, also belongs to the viridans strep- tococcal group. Although this bacterium has also been reported as an IE-causing pathogen, its detection frequency is very low (Douglas, Heath, Hampton, & Preston, 1993). Therefore, we analyzed the genetic and biological characteristics of the bacterial strain we isolated from a 4-year-old girl diagnosed with IE and investigated its relation- ship with IE. A phylogenetic tree based on 16S rRNA was constructed using MEGA (ver. 7.0) by the neighbor-joining method. The 16S rRNA DNA sequences from the data published for oral streptococci that were used in this study are as follows: Streptococcus mitis NCTC 12261 (GenBank accession D38482.1), Streptococcus pneumoniae NCTC 7465 (X58312.1), S. oralis CCUG 24891 (DQ303190.1), Streptococcus gordonii NCTC 7865 (D38483.1), S. sanguinis JCM 5708 (AB596946.1), Staphylococcus intermedius SK54 (JN787124.1), Strep- tococcus constellatus ATCC 27823 (Z69041.1), Streptococcus anginosus ATCC 33397 (Z69038.1), Streptococcus pyogenesis ATCC 12344 (AB002521.1), Streptococcus bovis ATCC 33317 (M58835.1), Strepto- coccus vestibularis CCRI 17387 (FJ154805.1), Streptococcus salivarius CCUG 7215 (FJ154803.1), Streptococcus sobrinus ATCC 33478 (AY188349.1), S. mutans NCTC 10449 (X58303.1), and our own previ- ously determined database sequence for the S. mutans HM strain (NZ_BDOS00000000.1; Kondo et al., 2017). made a recommendation to not use preventative antibiotics in all patients, including those in the high-risk group (Excellence NIfHaC, 2008). diagnosed with IE according to the Duke diagnostic criteria. For treat- ment, based on the sensitivity profile revealed by the antibiotic test results, ampicillin was selected and infused intravenously. An antipy- retic effect was observed the next day, and antibiotic therapy was administered for a total of 4 weeks. Blood culture tests after the anti- microbial therapy were negative, and the patient remained apyrexial. After discharge, she visited pediatric dentistry; a few dental caries were observed, and poor oral hygiene was evident. diagnosed with IE according to the Duke diagnostic criteria. For treat- ment, based on the sensitivity profile revealed by the antibiotic test results, ampicillin was selected and infused intravenously. An antipy- retic effect was observed the next day, and antibiotic therapy was administered for a total of 4 weeks. Blood culture tests after the anti- microbial therapy were negative, and the patient remained apyrexial. After discharge, she visited pediatric dentistry; a few dental caries were observed, and poor oral hygiene was evident. After changing the guidelines, the number of preventative antibi- otic administrations has dropped sharply in the United Kingdom (Dayer et al., 2015; Thornhill et al., 2011). A slight but statistically sig- nificant increase in the number of IE episodes was observed after 5 years (Dayer et al., 2015). Regarding the U.S. and European coun- tries except the United Kingdom, there are various reports about the increase or decrease in the incidence of IE (Bikdeli et al., 2013; Cahill et al., 2017; DeSimone et al., 2015; Duval et al., 2012; Erichsen, Gislason, & Bruun, 2016; Keller et al., 2017; Mackie, Liu, Savu, Marelli, & Kaul, 2016; Pant, Deshmukh, & Mehta, 2015; Toyoda et al., 2017). Among these reports, the number of IE episodes was reported to have increased due to oral streptococci, suggesting a causal relationship between preventive medication and dental treatment (Pant et al., 2015). Although much controversy about preventive administration at the time of dental treatment remains, IE has serious consequences once it occurs and has a great impact on patients' lives (Franklin et al., 2016). Therefore, it is important to prevent IE, and in addition to pro- phylaxis with antibiotics, education of patients in the maintenance of oral hygiene is necessary. 1 \| INTRODUCTION of dental treatment has been conducted in Western countries (Danchin, Duval, & Leport, 2005; Excellence NIfHaC, 2008; Habib et al., 2009; Wilson et al., 2007). In the guidelines of France in 2002 (Danchin et al., 2005), those of the United States in 2007 (Wilson et al., 2007), and those of Europe in 2009 (Habib et al., 2009), the admin- istration of antibiotics for moderate-risk patients was not rec- ommended, whereas the administration of antibiotics for high-risk patients was recommended. Moreover, the National Institute for Health and Care Excellence guidelines of the United Kingdom in 2008 License, which permits use, distribution and reproduction in any medium, Dental antibiotic prophylaxis for prevention of infective endocarditis (IE) has been recommended by some guidelines since the 1950s. However, there is no strong evidence to support this practice. In addi- tion, concerns regarding the development of allergies and the emer- gence of resistant bacteria caused by the administration of antimicrobial agents have also been expressed. Based on these issues, since the 2000s, a review of antimicrobial administration at the time Clin Exp Dent Res. 2019;5:534–540. KONDO ET AL. 535 536 2.5 \| Fibrinogen-binding and biofilm formation assays The bacterial strain isolated from the blood drawn from the 4-year- old girl who was hospitalized for IE was designated as HM. To identify the bacterial species, a phylogenetic tree based on 16S rRNA was constructed, and it showed that the HM strain displayed high sequence homology with S. mutans NCTC 10449 (Figure 1a). How- ever, the optical microscopic observations revealed that HM displayed a shorter chain-like structure than that of the NCTC 10449 strain. After further examination with an electron microscope, the individual cells of the HM strain were found to be elongated, similar to bacteria in the Corynebacterium genus, and they were also club shaped (Figure 1b). The fibrinogen-binding properties of the S. mutans strains were evalu- ated according to the methods described previously (O'Toole, 2011), with some modifications. Tissue culture plates (48-well, Becton Dick- inson) were coated with fibrinogen (Sigma-Aldrich Co.) prepared in carbonate–bicarbonate buffer (0.05-M Na2CO3, pH 9.6) and then incubated overnight at 4C. The plates were then washed three times with PBS and blocked for 1.5 hr with bovine serum albumin (Sigma- Aldrich Co.) in PBS at 37C. BHI broth (1 ml) and 1 μl of bacterial cells from an overnight culture of S. mutans were added to each well. After 24 hr of incubation at 37C, the adherent cells were washed three times with PBS, stained with 100 μl of 0.1% crystal violet in water for 10 min, and washed three times with PBS. The dye was dissolved by the addition of 30% acetic acid (100 μl) before the optical density for each strain at 570 nm was determined. The data are expressed as the mean and standard deviation of six independent experiments with four wells per sample. Platelet aggregation, which can occur after infection with patho- genic bacteria, is thought to be one of the most important factors affecting the pathogenesis of IE. Hence, in this study, we also investi- gated fibrinogen adhesion and biofilm formation. In both respects, strain HM exhibited higher levels than NCTC 10449 (Figure 2). FIGURE 1 Bacterial identification and microscopic observations. (a) The phylogenetic tree, which was based on the 16S rRNA gene, was constructed by the neighbor-joining (NJ) method. (b) Scanning electron microscopy observations of Streptococcus mutans HM and NCTC 10449 strains FIGURE 1 Bacterial identification and microscopic observations. 2.1 \| Subject S. mutans strains were precultured on BHI agar for 24 hr. Bacterial cells were suspended in phosphate buffered saline (PBS). They were fixed with 2.5% glutaraldehyde in PBS overnight at 4C, washed with PBS, postfixed with 1% osmium tetroxide in PBS for 2 hr at room temperature, and washed with double distilled water. The fixed speci- mens were immersed in t-butyl alcohol after dehydration via a graded series of acetone and then freeze dried. They were coated with osmium and observed using a Hitachi S-4500 scanning electron microscope. The patient was a 4-year-old girl with a ventricular septal defect (with no history of surgery) who visited Nagasaki University Hospital, Japan, with a fever of unknown origin. The fever appeared approximately 2 months before her visit. She had received clarithromycin and cefcapene pivoxil as oral antimicrobial drugs, which had an antipyretic effect. However, when the oral dosing was interrupted, the pyrexia returned. The echocardiography examination showed vegetation near the apex of the tricuspid valve. Gram-positive cocci were detected by blood culture testing. On the basis of these findings, she was KONDO ET AL. 4 \| DISCUSSION (Nakano et al., 2007). It was also reported that strains of serotypes e and f can invade primary human coronary artery endothelial cells (Abranches et al., 2011). Invasive strains were also found to be signifi- cantly more virulent than noninvasive strains in the Galleria mellonella (greater wax worm) model of systemic disease (Abranches et al., 2011). This is a report of S. mutans isolated from a 4-year-old girl diagnosed with IE in Japan. To characterize the bacterium responsible for the IE, we conducted a phylogenetic tree analysis based on 16S rRNA sequences for HM and various database strains. The phylogenetic tree showed that the HM strain shared high sequence homology with S. mutans. Although 16S rRNA sequence analysis is a widely used method for typing bacteria, we also used an additional sequence data analysis for discrimination purposes because the sequence homology among oral streptococcal species is relatively high (Bentley, Leigh, & Collins, 1991). Further analysis using the glycosyltransferase gene sequence, as previously reported (Hoshino et al., 2004), also identified the HM strain as an S. mutans member (data not shown). Also, we clas- sified the serotype of HM, which involved extracting the cell surface polysaccharide molecules and observing their reaction with a specific antibody. The results showed that HM was serotype e. S. mutans is classified into three serotypes, namely, c, e, and f, based on the struc- tures of the cell wall-associated polysaccharides (Okahashi, Koga, Akada, & Hamada, 1983). In general, the major serotype found in the oral cavity is serotype c (approximately 70–80% of isolates), followed by serotype e (approximately 20%), whereas the prevalence of sero- type f is low (Fitzgerald, Fitzgerald, Adams, & Morhart, 1983; Hamada, Masuda, & Kotani, 1980; Hirasawa & Takada, 2003; Shibata et al., 2003). Nonserotype c strains of S. mutans, namely, serotypes e and f, have been detected at high frequencies in specimens from patients who underwent surgery for the removal of atheromatous plaques and heart valve replacement (Nakano et al., 2007). It has been speculated that nonserotype c strains are isolated at higher frequencies than other strains because of their prolonged persistence in the blood Our observation of the HM strain revealed that the chain struc- ture of this bacterium is shorter than that of NCTC 10449. KONDO ET AL. 4 \| DISCUSSION (Nakano et al 2007) It was also reported that strains of serotypes e FIGURE 2 Adhesion to fibrinogen and biofilm formation in the Streptococcus mutans HM strain. (a) Bacterial cells were grown for 24 hr in brain heart infusion broth in a 48-well plate containing immobilized fibrinogen, and biofilm formation was assessed using a crystal violet-based assay. (b) Bars represent the average absorbance values at 570 nm, with four wells used for each bacterial strain. The assay was performed six times. “Blank” represents the experiment when no bacterial culture was added FIGURE 2 Adhesion to fibrinogen and biofilm formation in the Streptococcus mutans HM strain. (a) Bacterial cells were grown for 24 hr in brain heart infusion broth in a 48-well plate containing immobilized fibrinogen, and biofilm formation was assessed using a crystal violet-based assay. (b) Bars represent the average absorbance values at 570 nm, with four wells used for each bacterial strain. The assay was performed six times. “Blank” represents the experiment when no bacterial culture was added FIGURE 2 Adhesion to fibrinogen and biofilm formation in the Streptococcus mutans HM strain. (a) Bacterial cells were grown for 24 hr in brain heart infusion broth in a 48-well plate containing immobilized fibrinogen, and biofilm formation was assessed using a crystal violet-based assay. (b) Bars represent the average absorbance values at 570 nm, with four wells used for each bacterial strain. The assay was performed six times. “ ” 2.5 \| Fibrinogen-binding and biofilm formation assays (a) The phylogenetic tree, which was based on the 16S rRNA gene, was constructed by the neighbor-joining (NJ) method. (b) Scanning electron microscopy observations of Streptococcus mutans HM and NCTC 10449 strains FIGURE 1 Bacterial identification and microscopic observations. (a) The phylogenetic tree, which was based on the 16S rRNA gene, was constructed by the neighbor-joining (NJ) method. (b) Scanning electron microscopy observations of Streptococcus mutans HM and NCTC 10449 strains 537 REFERENCES Abranches, J., Miller, J. H., Martinez, A. R., Simpson-Haidaris, P. J., Burne, R. A., & Lemos, J. A. (2011). The collagen-binding protein Cnm is required for Streptococcus mutans adherence to and intracellular inva- sion of human coronary artery endothelial cells. Infection and Immunity, 79, 2277–2284. https://doi.org/10.1128/IAI.00767-10 Bedran, T. B., Azelmat, J., Spolidorio, D. P., & Grenier, D. (2013). Fibrino- gen-induced Streptococcus mutans biofilm formation and adherence to endothelial cells. BioMed Research International, 2013, 431465. Bentley, R. W., Leigh, J. A., & Collins, M. D. (1991). Intrageneric structure of Streptococcus based on comparative analysis of small-subunit rRNA sequences. International Journal of Systematic Bacteriology, 41, 487– 494. https://doi.org/10.1099/00207713-41-4-487 Bikdeli, B., Wang, Y., Kim, N., Desai, M. 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Heart, 103, 937– 944. https://doi.org/10.1136/heartjnl-2015-309102 • In this study, it was concluded that minor oral mucosal damage due to everyday life activities, such as teeth brushing, was the cause of IE for the following reasons: (a) The causative microorganisms were oral streptococci, (b) no dental treatment occurred before the onset of IE, and (c) the oral cavity was in an unsanitary condition. • In this study, it was concluded that minor oral mucosal damage due to everyday life activities, such as teeth brushing, was the cause of IE for the following reasons: (a) The causative microorganisms were oral streptococci, (b) no dental treatment occurred before the onset of IE, and (c) the oral cavity was in an unsanitary condition. Danchin, N., Duval, X., & Leport, C. (2005). Prophylaxis of infective endo- carditis: French recommendations 2002. Heart, 91, 715–718. https:// doi.org/10.1136/hrt.2003.033183 Dayer, M. J., Jones, S., Prendergast, B., Baddour, L. M., Lockhart, P. 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Therefore, in this study, to clarify the pathway by which HM invaded the bloodstream, the strains present in the dental plaque specimen from this patient were analyzed. The fingerprinting patterns of the S. mutans resident in the oral cavity of this patient and strain HM are concordant (data not shown), making it likely that the HM strain originated from the oral cavity. It is also known that severe tooth decay may lead to a continuous focus of infectious disease, whereby pathogenic bacteria may reach pathogenic lesions. However, recent studies suggest that bacteremia occurs even with routine daily tooth brushing and dental flossing procedures (Forner, Larsen, Kilian, & Holmstrup, 2006; Guntheroth, 1984; Sonbol, Spratt, Roberts, & Lucas, 2009). Additionally, in this case, because severe dental caries were not observed, the origin of HM is likely associated with everyday life, with the IE seemingly having developed from bacteremia. Recently, we have completed the draft genome sequence for the HM strain and deposited it in the DDBJ/EMBL/GenBank database under accession no. BDOS00000000 (Kondo et al., 2017). Having access to the complete genomic sequence and the findings from the present study will be useful for clarifying the pathogenesis of S. mutans- related IE. 4 \| DISCUSSION Some studies have predicted that the conditions that may affect cellular chain length may also affect adhesion (Murchison, Larrimore, Hull, & Curtiss, 1982) and aggregation (Murchison et al., 1982; Nakano, Fujita, Nishimura, Nomura, & Ooshima, 2005), both of which can contribute to virulence. Streptococcal cellular chain length and morphology are influenced by several factors. Previous studies have suggested that cell wall components such as peptidoglycan, lipoteichoic acids, and cell wall-anchored proteins can greatly affect the morphology of the cells (Thibodeau & Ford, 1991). How- ever, the factors affecting the morphology of the HM strain await elucidation. Platelet aggregation, which occurs after infection with pathogenic bacteria, is thought to be one of the most important factors affecting the pathogenesis of IE. The mechanisms by which endocarditis-caus- ing bacterial species induce platelet aggregation have also been stud- ied (Douglas, Brown, & Preston, 1990; Herzberg et al., 2005). These studies have reported that bacterial extracellular matrix binding pro- teins are potential factors affecting platelet aggregation. Regarding S. mutans, extracellular matrix binding protein also mediates platelet aggregation and biofilm formation (Bedran, Azelmat, Spolidorio, & Grenier, 2013; Nomura et al., 2014), and the results for HM are con- sistent with these findings. Contribution of the interaction of Streptococcus mutans serotype k strains with fibrinogen to the pathogenicity of infective endocarditis. 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How to cite this article: KondoY, HoshinoT, Ogawa M, et al. Streptococcus mutans isolated from a 4-year-old girl diagnosed with infective endocarditis. Clin Exp Dent Res. 2019;5:534–540 https://doi.org/10.1002/cre2.220 540.
https://openalex.org/W2096575615	https://europepmc.org/articles/pmc2783265?pdf=render	English	null	Relative roles of TGF‐β1 and Wnt in the systemic regulation and aging of satellite cell responses	Aging Cell	2,009	cc-by	13,908	Summary y Muscle stem (satellite) cells are relatively resistant to cell- autonomous aging. Instead, their endogenous signaling proﬁle and regenerative capacity is strongly inﬂuenced by the aged P-Smad3, differentiated niche, and by the aged circulation. With respect to muscle ﬁbers, we previ- ously established that a shift from active Notch to exces- sive transforming growth factor-beta (TGF-b) induces CDK inhibitors in satellite cells, thereby interfering with pro- ductive myogenic responses. In contrast, the systemic inhibitor of muscle repair, elevated in old sera, was sug- gested to be Wnt. Here, we examined the age-dependent myogenic activity of sera TGF-b1, and its potential cross- talk with systemic Wnt. We found that sera TGF-b1 becomes elevated within aged humans and mice, while systemic Wnt remained undetectable in these species. Wnt also failed to inhibit satellite cell myogenicity, while TGF-b1 suppressed regenerative potential in a biphasic fashion. Intriguingly, young levels of TGF-b1 were inhibi- tory and young sera suppressed myogenesis if TGF-b1 was activated. Our data suggest that platelet-derived sera TGF-b1 levels, or endocrine TGF-b1 levels, do not explain the age-dependent inhibition of muscle regeneration by this cytokine. In vivo, TGF-b neutralizing antibody, or a soluble decoy, failed to reduce systemic TGF-b1 and rescue myogenesis in old mice. However, muscle regeneration Correspondence Morgan Carlson, Department of Bioengineering, UC Berkeley, B104 Stanley Hall, Berkeley, CA 94720, USA. Tel.: +1 (510) 666-2793; fax: +1 (510) 642-5835; e-mail: mcarlson@berkeley.edu Accepted for publication 24 August 2009 Re-use of this article is permitted in accordance with the Terms and Conditions set out at http://www3.interscience.wiley.com/authorresources/onlineopen.html 676 Journal compilation Aging Cell Muscle stem (satellite) cells are relatively resistant to cell- autonomous aging. Instead, their endogenous signaling proﬁle and regenerative capacity is strongly inﬂuenced by the aged P-Smad3, differentiated niche, and by the aged circulation. With respect to muscle ﬁbers, we previ- ously established that a shift from active Notch to exces- sive transforming growth factor-beta (TGF-b) induces CDK inhibitors in satellite cells, thereby interfering with pro- ductive myogenic responses. In contrast, the systemic inhibitor of muscle repair, elevated in old sera, was sug- gested to be Wnt. Here, we examined the age-dependent myogenic activity of sera TGF-b1, and its potential cross- talk with systemic Wnt. We found that sera TGF-b1 becomes elevated within aged humans and mice, while systemic Wnt remained undetectable in these species. Wnt also failed to inhibit satellite cell myogenicity, while TGF-b1 suppressed regenerative potential in a biphasic fashion. Doi: 10.1111/j.1474-9726.2009.00517.x Doi: 10.1111/j.1474-9726.2009.00517.x Aging Cell (2009) 8, pp676–689 Introduction Neuroscience Institute, University of California, Berkeley, CA, USA Adult skeletal muscle robustly regenerates throughout an organism’s life but, with advancing age, its ability to repair diminishes and ultimately fails (Grounds, 1998; Renault et al., 2002). Muscle satellite cells play a crucial role in this regenerative process, and are physically situated beneath the basal lamina of myoﬁbers (i.e. differentiated muscle cells) (Mauro, 1961). Upon myoﬁber damage, quiescent satellite cells become activated and begin proliferating as myogenic progenitor cells, which later dif- ferentiate into fusion-competent myoblasts and de novo multi- nucleated myoﬁbers (Collins et al., 2005; Wagers & Conboy, 2005). Although diminished satellite cell numbers have been implicated in the age-related decline of muscle tissue regenera- tion, several reports identiﬁed key molecular changes within Notch and P-Smad3 signaling in the aged endogenous popula- tions (Conboy et al., 2003; Carlson & Conboy, 2007b; Carlson et al., 2008). In this regard, aging of the satellite cell micro-niche was found to promote underproduction of Notch ligand Delta, and overproduction of transforming growth factor-beta (TGF-b). Consequently, satellite cells residing in old muscle ﬁbers express excessive levels of CDK inhibitors, and thus fail to break quies- cence and proliferate in response to injury. However, the regen- erative responses of aged satellite cells can be rescued, either by forced activation of Notch, or by shRNA targeting of Smad3 (Schultz & Lipton, 1982; Conboy et al., 2003; Carlson et al., 2008). The regenerative capacity of aged muscle can also be enhanced by exposing old satellite cells to young systemic factors – such as when young and old mice are physically connected to share blood circulation (parabiosis), or when aged satellite cells are cultured in the presence of young sera (Conboy et al., 2005; Carlson & Conboy, 2007a). This improved regener- ation of aged muscle is due to the enhanced activity of endo- genous aged satellite cells, and not from physical contributions of young cells within the common circulation (Conboy et al., 2005). Therefore, in addition to local tissue niches, systemic factors in sera also regulate the regenerative responses of organ stem cells and importantly, the aged circulation inhibits the regenerative potential of even young satellite cells (Carlson & Relative roles of TGF-b1 and Wnt in the systemic regulation and aging of satellite cell responses Morgan E. Carlson,1 Michael J. Conboy,1 Michael Hsu,1 Laurel Barchas,1 Jaemin Jeong,1 Anshu Agrawal,2 Amanda J. Mikels,3 Smita Agrawal,4 David V. Schaffer4 and Irina M. Conboy1 Morgan E. Carlson,1 Michael J. Conboy,1 was improved by the systemic delivery of a TGF-b receptor kinase inhibitor, which attenuated TGF-b signaling in skel- etal muscle. Summarily, these ﬁndings argue against the endocrine path of a TGF-b1-dependent block on muscle regeneration, identify physiological modalities of age- imposed changes in TGF-b1, and introduce new thera- peutic strategies for the broad restoration of aged organ repair. was improved by the systemic delivery of a TGF-b receptor kinase inhibitor, which attenuated TGF-b signaling in skel- etal muscle. Summarily, these ﬁndings argue against the endocrine path of a TGF-b1-dependent block on muscle regeneration, identify physiological modalities of age- imposed changes in TGF-b1, and introduce new thera- peutic strategies for the broad restoration of aged organ repair. 1Department of Bioengineering, University of California Berkeley, Berkeley, CA, USA 1Department of Bioengineering, University of California Berkeley, Berkeley, CA, USA 2Division of Basic and Clinical Immunology, University of California, Irvine, CA, USA 3Department of Developmental Biology, Stanford University, Stanford, CA, USA Key words: aging; anti-aging; cytokines; skeletal muscle. 4Department of Chemical Engineering and The Helen Wills Correspondence Morgan Carlson, Department of Bioengineering, UC Berkeley, B104 Stanley Hall, Berkeley, CA 94720, USA. Tel.: +1 (510) 666-2793; fax: +1 (510) 642-5835; e-mail: mcarlson@berkeley.edu Accepted for publication 24 August 2009 Re-use of this article is permitted in accordance with the Terms and Conditions set out at http://www3.interscience.wiley.com/authorresources/onlineopen.html ª 2009 The Authors Journal compilation ª Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2009 Deﬁning the inhibitory range of systemic TGF-b1 The TGF-b family is composed of roughly 35 different ligands. In addition to other cytokines (e.g. IGF-1, TNF-a, IL-6, etc.) many of these have been implicated in the process of aging, and with respect to muscle regeneration (Grounds, 2002; Moresi et al., 2008; Dorshkind et al., 2009). Here, we focused on ﬁrst determining whether the aged circulation is inhibitory to adult myogenesis through the activity of one particular family member – TGF-b1. We speciﬁcally postulated that TGF-b1 levels of young sera are benign or positive to myogenic regulation, while TGF-b1 levels found in aged circulation are inhibitory. To explore this, we ﬁrst depleted TGF-b1 from young and old mouse serum, fol- lowed by re-addition at a range of tested concentrations. Muscle stem cells, derived from young and old mice, were then cultured in the presence of young and old TGF-b1-depleted, or modiﬁed, mouse serum. The regenerative responses of these cells were assayed, in vitro. Sera was depleted of TGF-b1 by incubation with a TGF-b1-speciﬁc antibody (or isotype-matched control IgG), followed by removal of the TGF-b1–antibody complexes (or control IgG antibody complexes) using protein G-coated aga- rose beads. The success of TGF-b1 depletion was also conﬁrmed by ELISA (not shown). Muscle stem cell myogenic regenerative potential was quantiﬁed, based on their ability to generate de-novo myogenic lineages – i.e. BrdU-incorporating desmin+ myoblasts (myogenic proliferation) that differentiate into post- mitotic eMyHC+ myotubes (myogenic differentiation) (Conboy et al., 2003, 2005; Sherwood et al., 2004). The time course of such de-novo in vitro myogenesis recapitulates in vivo muscle repair (Conboy & Rando, 2002; Conboy et al., 2005), and is shown in Supporting Fig. S1A–D. In agreement with previous ﬁndings (Carlson & Conboy, 2007a), myogenic proliferation (24–48 h of culture) and myogenic differentiation (48–72 h of culture) were consistently higher for young satellite cells and in young serum at all time points, compared to old (Figs 1 and S1A–D). Nonmyogenic, desmin) proliferating ﬁbroblasts were less numerous in cultures with experimentally calibrated levels of TGF-b1 (Fig. 1), and also declined in cultures with exogenous Wnt3A addition (Fig. 3, below). These cells are known to contaminate satellite cell preparations at 2–5%, and to expand in the presence of old serum or high levels of TGF-b (Carlson et al., 2008). Transforming growth factor-beta proteins are multifunctional cytokines, secreted by numerous cell types. Summary Intriguingly, young levels of TGF-b1 were inhibi- tory and young sera suppressed myogenesis if TGF-b1 was activated. Our data suggest that platelet-derived sera TGF-b1 levels, or endocrine TGF-b1 levels, do not explain the age-dependent inhibition of muscle regeneration by this cytokine. In vivo, TGF-b neutralizing antibody, or a soluble decoy, failed to reduce systemic TGF-b1 and rescue myogenesis in old mice. However, muscle regeneration Aging Cell Re-use of this article is permitted in accordance with the Terms and Conditions set out at http://www3.interscience.wiley.com/authorresources/onlineopen.html 676 ª 2009 The Authors Journal compilation ª Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2009 Age-related systemic control of muscle repair, M. E. Carlson et al. 6 677 Faulkner, 1989; Brack & Rando, 2007; Carlson & Conboy, 2007a). This evidence suggests, in the case of hetero- chronic parabiosis, the inhibitory factors introduced into shared circulation by old partners were continuously removed, or func- tionally neutralized, by the young partners. collection, inhibits satellite cell responses in vitro. These ﬁndings also suggest that young sera may contain a functional and natural decoy of TGF-b1, or a competitor of TGF-b1 signaling pathway (either endocrine or released by platelets). Lastly, our results demonstrate that Wnt antagonizes, rather than synergiz- es with TGF-b1-mediated satellite cell response inhibition. It was reported that the aged circulation inhibits satellite cell responses by acting through the Wnt pathway (Brack et al., 2007). These ﬁndings raised particular interest, as Wnt proteins are not known to be endocrine in nature, and Wnt signaling was recently demonstrated to enhance myogenic proliferation and differentiation in both embryonic and adult development (Ikeya & Takada, 1998; Marom et al., 1999; Wagner et al., 2000; Shi et al., 2002; Brack et al., 2008; Le Grand et al., 2009). A recent study from our laboratory implicated the TGF-b family in the local inhibition of aged satellite cell responses (Carlson et al., 2008). We therefore decided to explore a poten- tial age-dependent endocrine activity of TGF-b, and its potential cross-talk with Wnt. Moreover, we extrapolated our previous ﬁndings by narrowing down the anti-myogenic inhibitory activity to one TGF-b family member, namely TGF-b1 (Li et al., 2004; Shen et al., 2008). ª 2009 The Authors Journal compilation ª Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2009 ª 2009 The Authors Deﬁning the inhibitory range of systemic TGF-b1 They are capable of signaling to virtually every cell type and broadly control cell pro- liferation, differentiation, apoptosis, inﬂammation and scarring in various tissues (Massague, 1998; Massague & Chen, 2000; Derynck & Zhang, 2003). TGF-b ligands are synthesized by cells and secreted as precursor complexes that are composed of mature dimeric C-terminal polypeptides; speciﬁcally, the latency associated propeptide and the latent TGF-b binding protein (LTBP) (Annes et al., 2003; Hyytiainen et al., 2004). These com- plexes can be transported in the blood, and are normally tar- geted to tissue extracellular matrix by the appropriate LTBP, where the mature polypeptide is cleaved. These activated ligands are then capable of binding to their speciﬁc receptors, thereby initiating TGF-b ⁄ P-Smad signal transduction. Several validated small molecule inhibitors, which speciﬁcally interfere with TGF-b receptor I kinase activity, are capable of blocking Smad phosphorylation upon receptor I ligand binding (Singh et al., 2003; Gellibert et al., 2004). Similarly, an exogenous dominant-negative form of TGF-b receptor II has been shown to effectively prevent TGF-b signaling in several cell types (Brand et al., 1993; Tang et al., 1999). Both active and inactive TGF-b proteins are found in sera (Assoian et al., 1983; Massague, 1987), whereby inactive TGF-b, transported in blood plasma, is available for activation locally in tissues. In this regard, blood cells (mostly platelets and CD4+ T lymphocytes) are known to produce and release high levels of total and biologically-active TGF-b ligands (Assoian et al., 1983). Considering the strong morphogenic properties of TGF-b, changes in the levels of either total (latent plus active) or active form alone, are likely to inﬂu- ence immune responses, tissue maintenance and regeneration. Very interestingly, the productive myogenic proliferation of young and old muscle stem cells was robust in TGF-b1-depleted serum, only when low levels of recombinant TGF-b1 were intro- The results shown here argue against the notion of systemic TGF-b1 endocrine activity and strongly suggest that TGF-b, released by the known process of platelet activation during sera Age-related systemic control of muscle repair, M. E. Carlson et al. 678 678 (A) (B) (C) Fig. 1 Old sera inhibits satellite cell responses by transforming growth factor (TGF)-b-dependent mechanism. Deﬁning the inhibitory range of systemic TGF-b1 Young (A) and old (B) myoﬁber- associated myogenic progenitor cells were isolated 3 days postinjury and cultured overnight in Opti- MEM containing either 10% young serum (YS), 10% old (OS), TGF-b1 antibody depleted serum alone, or with ﬁxed amounts of recombinant TGF-b1 in the culture system. Cells were cultured with their speciﬁc sera for 24 h, and transferred to differentiation medium for additional 48 h (Fig S2). BrdU was added for the last 2 h to measure proliferation. Cells were then ﬁxed and immunostained for desmin (green) and BrdU (red), with Hoechst (blue) marking all nuclei (as shown in C), and scored in multiple random ﬁelds from the above assays. Results are displayed as the mean percent of Desmin+ ⁄ BrdU+ ⁄ total cells, ±SD. P < 0.05 for isochronic Y+YS ⁄ O+OS 0 ng mL)1, compared to 0.5 ng mL)1, and 5.0 ng mL)1 compared to 0.5 ng mL)1; n = 3. (A) (C) (B) (D) Fig. 2 Transforming growth factor (TGF)-b1 levels become elevated in old sera. (A) Mouse sera TGF-b1 becomes elevated with age. Mean serum levels, ±SEM, of TGF-b1 in young (Ym) or old (Om, n = 12 for each) mice as determined by sandwich ELISA; P < 0.0001. (B) Human TGF-b1 serum levels become elevated with age. TGF-b1 levels in the serum of aged humans (AG: 65–90 years old) vs. young (YG: 20–35 year old) were determined by ELISA. Error bars (SEM) represent the mean of 53 different subjects in each group; P < 0.0001. Similar age-related elevations in TGF-b1 were detected by bioactivity assay (Fig. S3). Dot plots represent separate animals (A) and separate individuals (D) of indicated ages. (C) Young and old myoﬁber explants were isolated at 3 days postinjury, and cultured overnight in Opti-MEM containing either young or old serum alone (control), or serum that had undergone activation of total TGF-b alone, in combination with neutralizing antibody to TGF-b1, or mixed with control serum. BrdU was added for the last 2 h to measure proliferation. Cells were ﬁxed and immunostained for desmin (green) and BrdU (red), with Hoechst (blue) marking all nuclei (as shown in C, quantiﬁed in D). ª 2009 The Authors Journal compilation ª Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2009 Fig. 1 Old sera inhibits satellite cell responses by transforming growth factor (TGF)-b-dependent mechanism. Young (A) and old (B) myoﬁber- associated myogenic progenitor cells were isolated 3 days postinjury and cultured overnight in Opti- MEM containing either 10% young serum (YS), 10% old (OS), TGF-b1 antibody depleted serum alone, or with ﬁxed amounts of recombinant TGF-b1 in the culture system. Cells were cultured with their speciﬁc sera for 24 h, and transferred to differentiation medium for additional 48 h (Fig S2). BrdU was added for the last 2 h to measure proliferation. Cells were then ﬁxed and immunostained for desmin (green) and BrdU (red), with Hoechst (blue) marking all nuclei (as shown in C), and scored in multiple random ﬁelds from the above assays. Results are displayed as the mean percent of Desmin+ ⁄ BrdU+ ⁄ total cells, ±SD. P < 0.05 for isochronic Y+YS ⁄ O+OS 0 ng mL)1, compared to 0.5 ng mL)1, and 5.0 ng mL)1 compared to 0.5 ng mL)1; n = 3. Deﬁning the inhibitory range of systemic TGF-b1 Cells were scored in multiple random ﬁelds and results are displayed th t f D i + ⁄ B dU+ ll SD P 0 05 f ti t d d t t l ti t d tib d d t ti t d d t l Age related systemic control of muscle repair, M. E. Carlson et al. 678 (A) (B) (C) Fig. 1 Old sera inhibits satellite cell response transforming growth factor (TGF)-b-depende mechanism. Young (A) and old (B) myoﬁber- associated myogenic progenitor cells were iso 3 days postinjury and cultured overnight in O MEM containing either 10% young serum (Y 10% old (OS), TGF-b1 antibody depleted seru alone, or with ﬁxed amounts of recombinant TGF-b1 in the culture system. Cells were cultu with their speciﬁc sera for 24 h, and transferr differentiation medium for additional 48 h (F BrdU was added for the last 2 h to measure proliferation. Cells were then ﬁxed and immunostained for desmin (green) and BrdU with Hoechst (blue) marking all nuclei (as sho C), and scored in multiple random ﬁelds from above assays. Results are displayed as the me percent of Desmin+ ⁄ BrdU+ ⁄ total cells, ±SD. P < 0.05 for isochronic Y+YS ⁄ O+OS 0 ng m compared to 0.5 ng mL)1, and 5.0 ng mL)1 compared to 0.5 ng mL)1; n = 3. (A) (B) (C) (A) (C) (B) (D) Fig. 2 Transforming growth factor (TGF)-b1 levels become elevated in old sera. (A) Mouse sera TGF-b1 becomes elevated with age. Mean serum levels, ±SEM, of TGF-b1 in young (Ym) or old (Om, n = 12 for each) mice as determined by sandwich ELISA; P < 0.0001. (B) Human TGF-b1 serum levels become elevated with age. TGF-b1 levels in the serum of aged humans (AG: 65–90 years old) vs. young (YG: 20–35 year old) were determined by ELISA. Error bars (SEM) represent the mean of 53 different subjects in each group; P < 0.0001. Similar age-related elevations in TGF-b1 were detected by bioactivity assay (Fig. S3). Dot plots represent separate animals (A) and separate individuals (D) of indicated ages. (C) Young and old myoﬁber explants were isolated at 3 days postinjury, and cultured overnight in Opti-MEM containing either young or old serum alone (control), or serum that had undergone activation of total TGF-b alone, in combination with neutralizing antibody to TGF-b1, or mixed with control serum. Deﬁning the inhibitory range of systemic TGF-b1 Raw ﬂuorescence values for young sera were marginally higher compared to old sera, but both were not signiﬁcantly different from the negative control (no Wnt3a samples). Inset panel shows 0.1–10 ng mL)1 Wnt3a range in greater detail. Myoﬁber-associated myogenic progenitor cells were isolated 3 days postinjury and cultured overnight in Opti-MEM containing either (C) 10% young serum (YS); YS+Wnt3A; YS+Wnt3A+TGF-b; or (E) 10% old (OS), OS+FRP3, OS+FRP3 + anti-TGF-b. BrdU was added for the last 2 h to measure proliferation. Cells were then ﬁxed and immunostained for desmin (green) and BrdU (red), with Hoechst (blue) marking all nuclei. TGF-b addition reduced myoblast and myotube production in young serum, TGF-b neutralization resulted in a much improved myogenic proliferation in old serum, and some cells even formed de novo myotubes. In contrast, exogenous Wnt3A did not decrease myogenic responses in young serum and FRP3 did not rescue myogenic responses in old serum. No synergy in regulation of myogenesis was detected between Wnt and TGF-b. (D) Quantiﬁcation of C. Cells were scored in multiple random ﬁelds from the above assays and the results displayed as the mean percent of BrdU+, desmin+ ⁄ total cells, ±SD. P < 0.05 between young untreated or +Wnt3a vs. +TGF-b or +TGF-b ⁄ Wnt3a; P < 0.05 for old ﬁbers, as described above for young. n = 3 for each set. (F) Quantiﬁcation of E. Cells were scored and displayed as in E. P < 0.05 between young ﬁbers + OS untreated or +FRP3 vs. +anti-TGF-b or +anti-TGF-b ⁄ FRP3; *P < 0.05 for old ﬁbers as described for young ﬁbers; n = 3 for each set. duced (Fig. 1A–C). At 1–5 ng mL)1 (and higher), TGF-b1 alone sufﬁced for the inhibition of satellite cell responses, while myo- genesis was positively regulated at 0.2 ng mL)1 (Fig. 1A–C). Similarly, myogenic differentiation responses from young cells also peaked in TGF-b1-depleted serum, which received low lev- els of exogenous recombinant TGF-b1 (Supporting Fig. S2A). In contrast, old cell differentiation was improved by TGF-b1 deple- tion from serum alone, as well as in a low range of recombinant TGF-b1 addition (Supporting Fig. S2B). The overall differentia- tion response from old cells was also diminished, compared to young cells (Supporting Fig. S2A,B). Deﬁning the inhibitory range of systemic TGF-b1 Human serum was collected from three young (20–25 years) and three old (65–75 years) individuals. Mouse serum was four young and four aged mice. Levels of biologically active Wnt were analyzed, using a Wnt-reporter expressing cell line. As compared to Wnt3A, no detectable Wnt activity was found in either young or old serum. Raw ﬂuorescence values for young sera were marginally higher d sera, but both were not signiﬁcantly different from the negative control (no Wnt3a samples). Inset panel shows 0.1–10 ng mL)1 Wnt3a range in Myoﬁber-associated myogenic progenitor cells were isolated 3 days postinjury and cultured overnight in Opti-MEM containing either (C) 10% YS); YS+Wnt3A; YS+Wnt3A+TGF-b; or (E) 10% old (OS), OS+FRP3, OS+FRP3 + anti-TGF-b. BrdU was added for the last 2 h to measure ells were then ﬁxed and immunostained for desmin (green) and BrdU (red), with Hoechst (blue) marking all nuclei. TGF-b addition reduced myotube production in young serum, TGF-b neutralization resulted in a much improved myogenic proliferation in old serum, and some cells even o myotubes. In contrast, exogenous Wnt3A did not decrease myogenic responses in young serum and FRP3 did not rescue myogenic responses in ynergy in regulation of myogenesis was detected between Wnt and TGF-b. (D) Quantiﬁcation of C. Cells were scored in multiple random ﬁelds assays and the results displayed as the mean percent of BrdU+, desmin+ ⁄ total cells, ±SD. P < 0.05 between young untreated or +Wnt3a vs. F-b ⁄ Wnt3a; P < 0.05 for old ﬁbers, as described above for young. n = 3 for each set. (F) Quantiﬁcation of E. Cells were scored and displayed as between young ﬁbers + OS untreated or +FRP3 vs. +anti-TGF-b or +anti-TGF-b ⁄ FRP3; P < 0.05 for old ﬁbers as described for young ﬁbers; set. (A) (B) (C) (D) (E) (F) (F) (F) Fig. 3 Old serum inhibits satellite cell responses by a Wnt-independent mechanism. Bioactive human (A) and mouse (B) Wnt is undetectable in human or mouse sera, and is not elevated with age. Human serum was collected from three young (20–25 years) and three old (65–75 years) individuals. Mouse serum was collected from four young and four aged mice. Levels of biologically active Wnt were analyzed, using a Wnt-reporter expressing cell line. As compared to recombinant Wnt3A, no detectable Wnt activity was found in either young or old serum. Deﬁning the inhibitory range of systemic TGF-b1 BrdU was added for the last 2 h to measure proliferation. Cells were ﬁxed and immunostained for desmin (green) and BrdU (red), with Hoechst (blue) marking all nuclei (as shown in C, quantiﬁed in D). Cells were scored in multiple random ﬁelds and results are displayed as the mean percent of Desmin+ ⁄ BrdU+ cells, ±SD. P < 0.05 for activated compared to control, activated + antibody compared to activated, and control compared to activate + control. n = 3. (A) (C) (B) (D) (A) (B) (C) (D) Fig. 2 Transforming growth factor (TGF)-b1 levels become elevated in old sera. (A) Mouse sera TGF-b1 becomes elevated with age. Mean serum levels, ±SEM, of TGF-b1 in young (Ym) or old (Om, n = 12 for each) mice as determined by sandwich ELISA; P < 0.0001. (B) Human TGF-b1 serum levels become elevated with age. TGF-b1 levels in the serum of aged humans (AG: 65–90 years old) vs. young (YG: 20–35 year old) were determined by ELISA. Error bars (SEM) represent the mean of 53 different subjects in each group; P < 0.0001. Similar age-related elevations in TGF-b1 were detected by bioactivity assay (Fig. S3). Dot plots represent separate animals (A) and separate individuals (D) of indicated ages. (C) Young and old myoﬁber explants were isolated at 3 days postinjury, and cultured overnight in Opti-MEM containing either young or old serum alone (control), or serum that had undergone activation of total TGF-b alone, in combination with neutralizing antibody to TGF-b1, or mixed with control serum. BrdU was added for the last 2 h to measure proliferation. Cells were ﬁxed and immunostained for desmin (green) and BrdU (red), with Hoechst (blue) marking all nuclei (as shown in C, quantiﬁed in D). Cells were scored in multiple random ﬁelds and results are displayed as the mean percent of Desmin+ ⁄ BrdU+ cells, ±SD. P < 0.05 for activated compared to control, activated + antibody compared to activated, and control compared to activate + control. n = 3. ª 2009 The Authors Journal compilation ª Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2009 Age-related systemic control of muscle repair, M. E. Carlson et al. 679 679 (A) (B) (C) (D) (E) (F) um inhibits satellite cell responses by a Wnt-independent mechanism. Bioactive human (A) and mouse (B) Wnt is undetectable in human or mouse elevated with age. ª 2009 The Authors Journal compilation ª Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2009 ª 2009 The Authors Correlation between systemic levels and myogenic effects of Wnt Using a luciferase-reporter assay (Kaykas et al., 2004; Brack et al., 2007), we also measured the systemic levels of biologi- cally active Wnt, since an age-dependent increase of circulating Wnt (from 10 ng mL)1 in young to 20 ng mL)1 in old sera) was recently reported (Brack et al., 2007). In our experiments, Wnt was undetectable in young and old serum from both humans and mice (Fig. 3A,B). Measurable levels of Wnt in all samples were substantially < 5 ng mL)1 (based on a recombi- nant Wnt standard curve). Interestingly, the relative luciferase activity values were actually higher for young mouse serum, compared to old (Fig. 3B). Curiously, TGF-b1 levels in young mouse sera (100 ng mL)1) are predicted to be inhibitory to satellite cell responses (data shown in Fig. 1), even if used at 5–10%, in vitro. Therefore, despite apparent pro-myogenic differences between young and old sera, physiological TGF-b1 levels alone do not explain the inhibitory affects of aged sera on myogenic responses. To deter- mine whether these inhibitory affects correlated with biologically active TGF-b1 levels, a bio-activity assay was performed on non- activated serum samples, using the plasminogen activator inhibi- tor-1 (PAI-1) promoter luciferase-reporter (Danielpour et al., 1989; Abe et al., 1994), and by ELISA (Supporting Fig. S3C,D). These data clearly demonstrated that while bioactive TGF-b1 was higher in old mouse serum, compared to young, the levels in either sera were minute (> 4 ng mL)1) and unlikely to be inhibitory in the 5–10% range examined (Figs S3C,D and 1). A rescue of satellite cell responses, in the presence of old serum, was reported to be achieved by the inhibition of Wnt via FRPs. In addition, exogenous Wnt3A was reported to inhibit satellite cell myogenic responses, in favor of ﬁbroblast trans- differentiation (Brack et al., 2007). We thus investigated whether Wnt was potentially synergistic with the TGF-b1- dependent attenuation of myogenic potential, and if Wnt sera activity might be detected using satellite cell myogenicity assays. To do so, the myogenic potential of young and old satellite cells was examined in the presence of (i) young serum with exo- genous Wnt3A (by itself, or simultaneously with exogenous TGF-b1), or (ii) old serum with exogenous FRP3 (by itself, or simultaneously with TGF-b neutralization). As shown in Fig. 3C– F, exogenous FRP inhibition of Wnt did not rescue myogenic responses in old serum. Deﬁning the inhibitory range of systemic TGF-b1 As myogenic differentiation was assayed at 48–72 h of culture, and aged satellite cells have elevated TGF-b1 production (Carlson et al., 2008), these data suggest that endogenous TGF-b1 production by old cells, in vi- tro, counteracts the pro-myogenic effect of TGF-b1 depletion from mouse serum (Figs 1, S1A–D and S2A,B). Together, these data demonstrate that sera-derived TGF-b1 inhibits satellite cell responses, and that speciﬁc levels of TGF-b1 are required for productive myogenic responses. Even with com- plete TGF-b1 depletion, old serum remained less myogenic than Age-related systemic control of muscle repair, M. E. Carlson et al. 680 young (by 10–20%), suggesting that while TGF-b1 is a main inhibitor of satellite cell responses, it is not the only suppressor of regeneration present in old sera (Figs 1A–C, S2A,B). mixed with standard young serum, forced-activated young serum was dominant in myogenic inhibition – suggesting that the presence of negative regulators, rather than the removal of positive regulators, accounts for such affects. These data uncover that the strong inhibitory inﬂuence of aged sera (as opposed to the pro-myogenic properties of young sera) are not solely explained by age-related differences in TGF-b1 levels. Correlating the inhibitory range of TGF-b1 with physiological levels found in young and old sera To further substantiate these conclusions, we correlated the inhibitory range of TGF-b1 with its levels found in young vs. aged sera. Speciﬁcally, we analyzed TGF-b1 levels as a function of age in mice and in humans (Fig. 2A,B). In mice, TGF-b1 levels sharply increased between 12 months (early postreproductive age, analogous to 5th–6th decades in humans) and 24 months (analogous to 8th–9th decades in humans), Fig. 2A. Moreover, this systemic age-related rise was found to be conserved in humans (Fig. 2B), where TGF-b1 plateaus at its highest systemic levels between the 6th and 9th decades of life (i.e. roughly at the onset and progression of age-imposed regenerative decline). Sera TGF-b1 for both species thus becomes elevated from the end of the reproductive period, to the end of lifespan. ª 2009 The Authors Journal compilation ª Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2009 Correlation between systemic levels and myogenic effects of Wnt The successful down-modulation of TGF-b signaling by DN RII virus provided restoration of satellite cell potential in cells exposed to old serum, as evidenced by large number of de-novo proliferating desmin+ myoblasts that form multinucleated (A) (B) (C) Fig. 4 Down-modulation of transforming growth factor (TGF)-b signaling intensity by dominant negative TGF-b RII attenuates old sera-imposed satellite cell regenerative potential inhibition. (A) Young and old mice were injured with CTX, as described; bulk myoﬁbers with activated satellite cells were then explanted and transiently transduced with a TGF-b RII DN-expressing lentivirus, or control virus, in vitro. Following transduction, myoﬁber-derived cells were cultured for 48 h in OPTI-MEM + 5% young (YS) or old (OS) mouse serum. Cells were ﬁxed and analyzed by immunoﬂuorescence for the expression of desmin (green), and levels of BrdU incorporation (red). Hoechst (blue) labels all nuclei. The successful down-modulation of TGF-b signaling by DN RII virus provided restoration of satellite cell regenerative potential in cells exposed to old serum, as evidenced by large number of de-novo proliferating desmin+ myoblasts that form multinucleated myotubes. Bar = 50 um. (B) Quantiﬁcation of regenerative potential shown in A, The percent of desmin+ ⁄ BrdU+ cells is shown as means with standard deviations. () indicates P < 0.05, compared to treatment with young serum and control virus (Y serum + cntl.) () indicates P < 0.05, as compared to treatment with old serum and control virus (O serum + cntl.). (C) Levels of nuclear P-Smad3 were diminished in old satellite cells transduced with DN RII-expressing lentivirus, in vitro. Old satellite cells, exposed to old serum (OS) plus control virus, or old serum plus DN RII-expressing virus (as indicated), were immunostained for P-Smad3 (green). Hoechst (blue) labels nuclei. As expected, levels of satellite cell nuclear P-Smad3 were diminished in cells transduced with DN RII-expressing virus, as compared to control (white arrow). Bar = 25 lm. results further clarify the respective roles of Wnt and TGF-b1 in adult myogenesis, demonstrate that Wnt activity is not present in sera (determined by bioactivity and satellite cell myogenicity assays), and also demonstrate that Wnt does not contribute to the TGF-b1-dependent inhibition of satellite cell myogenic potential. sufﬁcient to restore myogenic responses in old cultures. TGF-b receptor levels in satellite cells increase approximately three to fourfold with age (Supporting Fig. Correlation between systemic levels and myogenic effects of Wnt S6A), suggesting a com- pounding effect on signaling from an age-dependent elevation of ligand (Carlson et al., 2008) and receptor. In this regard, we compared the intensity of TGF-b signaling, and the efﬁciency of myogenic responses, in satellite cells expressing a TGF-b domi- nant-negative receptor II (TGF-b DN RII), compared to control vector (Tang et al., 1999; Yu & Schaffer, 2006). Young and old satellite cells were activated by muscle injury, followed by pro- genitor cell isolation and retroviral transduction with TGF-b DN RII or control vector, ex vivo. The dominant-negative TGF-b receptor is an inactive kinase, which effectively inhibits signaling by active TGF-b ligands (Tang et al., 1999). As shown in Fig- s 4A,B and S6B, while having no effect on satellite cells cultured Correlation between systemic levels and myogenic effects of Wnt Moreover, exogenous Wnt3A did not reduce myogenic potential in young serum. Consistent with a reported pro-myogenic activity of Wnt (Brack et al., 2008), Wnt3A also enhanced myotube formation (Supporting Fig. S4A). However, in contrast to the induction of a ﬁbroblastic cell fate (Brack et al., 2007), the presence of nonmyogenic ﬁbro- blastic cells (e.g. desmin) ⁄ BrdU+) was not increased, but reduced in the presence of exogenous Wnt3A, thereby further validating the pro-myogenic activity of Wnt. Wnt modulation also failed to synergize with the TGF-b1-promoted inhibition of myogenic responses. In contrast, Wnt3A antagonized TGF-b1 and enhanced the generation of satellite cell-derived, fusion- competent proliferating myoblasts (Fig. 4C–F). Additionally, young satellite cells cultured with young serum had less active GSK3b and more inactive GSK3b (compared to old satellite cells cultured with old serum), suggesting that Wnt signaling is stron- ger in young conditions (Supporting Fig. S4B,C). These ﬁndings were also examined and conﬁrmed using single myoﬁber assays (Brack et al., 2007 and Supporting Fig. S5). Combined, these Based on published literature, and in agreement with our results, sera and plasma contain little-to-no active TGF-b1. How- ever, the total TGF-b1 released by platelets during sera collection becomes activated (e.g. by cells in culture), evoking an interplay between positive and negative regulators that determines the intracellular signaling strength. The platelet source of TGF-b1 in our serum samples was corroborated, as expected (Supporting Fig. S3E,F). We further postulated that, in the case of young sera, TGF-b1 activity is attenuated and may therefore render young sera pro-myogenic. To test this, we compared satellite cell myogenic responses in cultures containing serum with (i) endogenous TGF-b activity, (ii) forced-activated endogenous TGF-b and (iii) forced-activated endogenous TGF-b, with subse- quent TGF-b1-speciﬁc antibody neutralization. As shown in Fig. 2C,D, young serum became inhibitory to satellite cell responses (and similar in this regard to old sera) when endo- genous young levels of TGF-b were forced-activated. Impor- tantly, inhibition of myogenic activity was dependent on TGF-b1 and not related to sera-activation, as TGF-b1-speciﬁc neutraliza- tion restored myogenicity to these cultures. Additionally, when ª 2009 The Authors Journal compilation ª Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2009 (A) (B) (C) Fig. 4 Down-modulation of transforming growth factor (TGF)-b signaling intensity by dominant negative TGF-b RII attenuates old sera-imposed satellite cell regenerative potential inhibition. ª 2009 The Authors l l ª 2009 The Authors Journal compilation ª Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2009 Correlation between systemic levels and myogenic effects of Wnt (A) Young and old mice were injured with CTX, as described; bulk myoﬁbers with activated satellite cells were then explanted and transiently transduced with a TGF-b RII DN-expressing lentivirus, or control virus, in vitro. Following transduction, myoﬁber-derived cells were cultured for 48 h in OPTI-MEM + 5% young (YS) or old (OS) mouse serum. Cells were ﬁxed and analyzed by immunoﬂuorescence for the expression of desmin (green), and levels of BrdU incorporation (red). Hoechst (blue) labels all nuclei. The successful down-modulation of TGF-b signaling by DN RII virus provided restoration of satellite cell regenerative potential in cells exposed to old serum, as evidenced by large number of de-novo proliferating desmin+ myoblasts that form multinucleated myotubes. Bar = 50 um. (B) Quantiﬁcation of regenerative potential shown in A, The percent of desmin+ ⁄ BrdU+ cells is shown as means with standard deviations. () indicates P < 0.05, compared to treatment with young serum and control virus (Y serum + cntl.) (*) indicates P < 0.05, as compared to treatment with old serum and control virus (O serum + cntl.). (C) Levels of nuclear P-Smad3 were diminished in old satellite cells transduced with DN RII-expressing lentivirus, in vitro. Old satellite cells, exposed to old serum (OS) plus control virus, or old serum plus DN RII-expressing virus (as indicated), were immunostained for P-Smad3 (green). Hoechst (blue) labels nuclei. As expected, levels of satellite cell nuclear P-Smad3 were diminished in cells transduced with DN RII-expressing virus, as compared to control (white arrow). Bar = 25 lm. Age-related systemic control of muscle repair, M. E. Carlson et al. 6 Age-related systemic control of muscle repair, M. E. Carlson et al. 681 681 (A) (B) (C) n-modulation of transforming growth factor (TGF)-b signaling intensity by dominant negative TGF-b RII attenuates old sera-imposed satellite cell potential inhibition. (A) Young and old mice were injured with CTX, as described; bulk myoﬁbers with activated satellite cells were then explanted tly transduced with a TGF-b RII DN-expressing lentivirus, or control virus, in vitro. Following transduction, myoﬁber-derived cells were cultured for 48 h M + 5% young (YS) or old (OS) mouse serum. Cells were ﬁxed and analyzed by immunoﬂuorescence for the expression of desmin (green), and levels of oration (red). Hoechst (blue) labels all nuclei. Systemic delivery of a TGF-b receptor inhibitor (but not anti-TGF-b antibody or soluble TGF-b receptor) restores old muscle repair, in vivo Examination of our data suggested that sera-derived TGF-b1 was released by platelets during collection procedures. Nevertheless, it remained possible that plasma-based endocrine TGF-b1 could play a role in the inhibition of satellite cells. To examine potential age-dependent endocrine affects of TGF-b1, and to distinguish them from its local inﬂuences, we sought to rejuvenate aged muscle repair by attenuating the TGF-b pathway through sys- temic pharmacological intervention, in vivo. Three independent attenuators were used in these experiments: (i) a small molecule inhibitor of TGF-b RI kinase (Singh et al., 2003; Gellibert et al., 2004), (ii) TGF-b neutralizing antibody and (iii) a decoy composed of the extracellular portion of TGF-b receptor II. These molecules were systemically administered for 10 days, followed by cardio- toxin (CTX) muscle injury. Samples were harvested at 5 days postinjury, and examined histologically. The efﬁciency of adult myogenesis, and age-related changes in this process, were reli- ably determined by the detection and quantiﬁcation of newly formed myoﬁbers. Such de novo ﬁbers are characteristically small, express eMyHC and have centrally located BrdU+ nuclei – indicating that these cells were recently generated by proliferat- ing, fusion-competent myoblasts (Wagers & Conboy, 2005; Carlson & Conboy, 2007a; Carlson et al., 2008). Newly formed muscle ﬁbers are typically numerous and visible in young regen- erating muscle, but less numerous in old muscle, which instead shows more mononucleated cells and scar formation (Fig. 5A). The rejuvenating effects produced by TGF-b RI kinase inhibitor systemic delivery are best explained by the attenuation of local TGF-b signaling in all tissues, including skeletal muscle. As shown in Fig. 6B,C, this hypothesis is directly substantiated by our experimental data. Namely, the high levels of P-Smad3 and TGF-b1, typical of old muscle (Carlson et al., 2008), are signiﬁ- cantly reduced in old mice receiving systemically administered TGF-b RI kinase inhibitor, but not in old mice receiving anti- TGF-b antibody or soluble receptor. Importantly, osmotic pump delivery TGF-b RI kinase inhibitor not only reduced P-Smad3 levels, but also diminished levels of TGF-b1 production by muscle cells, suggesting positive feed-back regulation in the TGF-b pathway (Fig. 6B,C). Together, these data establish that systemically administered TGF-b RI kinase inhibitor acts locally to attenuate TGF-b1 ⁄ P-Smad3 signaling in satellite cells, and restores muscle repair, in vivo. These ﬁndings are also in agreement with our previous report (Carlson et al., 2008). Expression of a dominant-negative TGF-b receptor restores productive myogenic responses to satellite cells exposed to aged serum After justifying our experimental focus on TGF-b1 as a myogenic inhibitor, we decided to conﬁrm that (i) young sera, compared to old, down-modulates TGF-b1 signaling in satellite cells, and that (ii) the attenuation of TGF-b receptor engagement is ª 2009 The Authors Journal compilation ª Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2009 Age-related systemic control of muscle repair, M. E. Carlson et al. 682 682 we also employed osmotic subcutaneous pumps for the constit- utive systemic delivery of these molecules. Under these condi- tions, the anti-TGF-b neutralizing antibody still failed to signiﬁcantly enhance repair of old muscle, while TGF-b RI kinase inhibitor robustly promoted muscle repair (Fig. 5B). These key data suggest that endocrine TGF-b1 does not play a major role in the age-related decline of muscle regeneration, in vivo. Furthermore, ELISA assays on systemic TGF-b1 levels not only corroborated, but strengthened this conclusion (Fig. 6A). These data demonstrated that TGF-b1 neutralizing antibody, or soluble TGF-b receptor II, failed to reduce circulating levels of TGF-b1 in old mice (Fig. 6A). In agreement with other reports, our ﬁndings suggest that bio-active TGF-b1 is not present in the circulation, which would be recognizable by the herein tested antibody or cognate receptor (Annes et al., 2003; Hyytiainen et al., 2004; Feng & Derynck, 2005). with young serum, TGF-b DN RII expression rescued myogenic proliferation and differentiation in the presence of old serum. The age-related differences in TGF-b signaling strength, and the attenuation of TGF-b signaling DN RII expression, were con- ﬁrmed by nuclear P-Smad3 analysis in muscle cells (Fig. 4C). Ectopic expression of truncated TGF-b DN RII was conﬁrmed by Western blot (Supporting Fig. S6C). Systemic delivery of a TGF-b receptor inhibitor (but not anti-TGF-b antibody or soluble TGF-b receptor) restores old muscle repair, in vivo In contrast, systemic levels and intra-muscular TGF-b1 signaling strength are not down- modulated, and old muscle repair is not enhanced by circulating neutralizing antibody or soluble receptor (which do not access endocrine or tissue TGF-b when administered systemically). Thus, while TGF-b1 is released by platelets during sera collec- tion, and does exhibit an inhibitory and age-dependent inﬂu- ence on satellite cells, in vitro (Figs 1, 2 and S2), there is no evidence for TGF-b1 endocrine anti-myogenic activity, in vivo. Muscle regeneration from mice treated by systemic adminis- tration of the TGF-b RI kinase inhibitor was signiﬁcantly improved, and similar to the regeneration seen in young control animals (Fig. 5A,B), or young animals receiving TGF-b RI kinase inhibitor treatment (Supporting Fig. S7A–D). In contrast, sys- temic administration of either TGF-b neutralizing antibody, or soluble TGF-b receptor II, failed to improve myogenic regenera- tive capacity in old mice (Fig. 5A,B). To control for the biological activity of the anti-TGF-b neutralizing antibody, we performed intra-muscular injections [which enhanced old muscle repair as expected (Supporting Fig. S7E,F)]. In agreement with the data shown in Figs 3 and S5, experimental attenuation of Wnt (by FRP) did not improve the regeneration of old muscle, in vivo. Fur- ther, Wnt attenuation did not synergize with intra-muscular attenuation of TGF-b, which alone sufﬁced for the rescue of old muscle repair (Supporting Fig. S7E,F). ª 2009 The Authors Journal compilation ª Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2009 Discussion TGF-b proteins have been shown to behave as morphogens which, depending on concentration, induce various gene subsets and promote differ- ent effects (McPherron et al., 1997; Itoh et al., 2003; Kamanga- Sollo et al., 2003; Muller et al., 2003; Wharton et al., 2004). The morphogenic nature of the TGF-b family agrees with our conclusion that, at speciﬁc concentrations, TGF-b1 is permissive or nonpermissive to productive myogenic regenerative responses (Figs 1, 2 and S1, S2). Consequently, pronounced down-modulation of TGF-b1 causes immune disorders, inﬂam- mation and organ dysfunction. This complicates the use of TGF-b1 attenuators, such as antibodies and small molecules (delivered by pumps, or otherwise), or the use of genetically altered mice, which do not survive into old age if they harbor TGF-b pathway insufﬁciencies. For example, genetic deﬁciencies in TGF-b1,2,3, TGF-b RI,II,III, Smad2, Smad4 and others (Oshima et al., 1996; Bonyadi et al., 1997; Larsson et al., 2001; Brionne et al., 2003; Andrews et al., 2006) all cause embryonic or were prescreened for confounding medical conditions. Despite this, none of the aged individuals (‡ 60 years) had less than measurable levels of TGF-b1, as opposed to many of the young individuals (£ 30 years) enrolled in these studies. Considering that TGF-b1 is broadly produced, and signals to a variety of cells, the age-related and evolutionarily conserved elevation of its functional levels in old sera may affect stem cell responses in var- ious other tissues. Such a ﬁnding may provide a preliminary explanation for the general organ stem cell regenerative poten- tial decline of mice and humans. were prescreened for confounding medical conditions. Despite this, none of the aged individuals (‡ 60 years) had less than measurable levels of TGF-b1, as opposed to many of the young individuals (£ 30 years) enrolled in these studies. Considering that TGF-b1 is broadly produced, and signals to a variety of cells, the age-related and evolutionarily conserved elevation of its functional levels in old sera may affect stem cell responses in var- ious other tissues. Such a ﬁnding may provide a preliminary explanation for the general organ stem cell regenerative poten- tial decline of mice and humans. With respect to satellite cell activation, TGF-b1 operates in a threshold fashion (Fig. 1), and appears to function through the TGF-b II receptor and P-Smad signaling (Figs 4 and 6). ª 2009 The Authors Journal compilation ª Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2009 Discussion The data presented in this work establish the anti-regenerative properties and inhibitory range of TGF-b1. These data also dem- onstrate that the age-related increase in TGF-b1 levels is con- served between mouse and human sera (Figs 1 and 2), and may therefore be universal to the aging process of mammals. In our studies, there was less variation in measurable TGF-b1 levels within mice (when compared with humans), likely owing to their environmental and genetic homogeneity. It is also important to note that our reported human TGF-b1 levels are representative of the healthy aging population, as individuals for these studies To control for potential half-life differences of the anti-TGF-b neutralizing antibody, compared to TGF-b RI kinase inhibitor, Age-related systemic control of muscle repair, M. E. Carlson et al. 683 683 (A) (B) f (A) Fig. 5 Systemic pharmacological intervention lowers sera transforming growth factor (TGF)-b levels and improves regeneration in old animals. Old or young mice were injected (subcutaneous) with a small molecule inhibitor of the TGF-b RI kinase (Ki), for 2 weeks. Five days before the end of treatment, muscle was injured as described. At the end of treatment, animals were sacriﬁced and muscle was collected. BrdU was injected (intraperitoneal) at 3 days postinjury to label proliferating, fusion-competent myoblasts. (A) Cryosections (10 lm) were performed in young and old muscle (receiving vehicle alone), old + Ki and old + RII (extracellular portion of TGF-b receptor II). Sections were analyzed by hematoxylin ⁄ eosin (H&E) staining and immunostaining for both embryonic myosin heavy chain (eMyHC, shown in green) and BrdU incorporation (shown in red). Hoechst stains nuclei (blue). As shown, the regenerative outcome of old and old + RII was worse than young (judged by scar tissue formation, de novo myoﬁber size ⁄ scarcity and reduction of BrdU+ nuclei within eMyHC ﬁbers). Scale bar = 100 lm. In contrast, muscle repair was improved by treatment with TGF-b RI kinase (Ki) small molecule inhibitor (similar to young with respect to large ⁄ dense eMyHC+ myoﬁbers with centrally located BrdU+ nuclei). (B) Regeneration was quantiﬁed from muscle sections, and is presented as mean percent of newly regenerated myoﬁbers per square millimeter of injury site. Error bars indicate SD, n = 3, P = 0.01 between young or old +Ki, and old. (B) ing is needed for efﬁcient immune cell and wound clearance responses (Dunker & Krieglstein, 2000). ª 2009 The Authors Discussion Speciﬁcally, if all endogenous young sera TGF-b1 were active, it would readily suppress satellite cell responses, in vitro (Figs 1 and 2). Further, neutralization of circulatory TGF-b1 is not achievable by experi- mental ligand traps, in vivo (Figs 5 and 6). These data suggest the presence of a natural TGF-b1 modiﬁer ⁄ decoy (predictably more active in the young), which is likely endocrine in nature, or released during platelet activation (Figs 1 and 2). Future work on the characterization of such natural systemic antagonist(s) of TGF-b1 may also explain the regenerative phenotypes observed in hetereochronic parabiosis (Conboy et al., 2005). This work further conﬁrms and extrapolates the auto- crine ⁄ paracrine mode of TGF-b age-dependent inhibition of organ stem cell responses (Carlson et al., 2008). Speciﬁcally, old tissue repair was improved only when P-Smad3 levels were attenuated locally in muscle (e.g. through small molecule systemic delivery), which is also expected to attenuate TGF-b signaling broadly in all tissues (Figs 5 and 6). In contrast, systemi- cally administered neutralizing antibody, or soluble receptor, failed to restore old muscle repair. These molecules did not attenuate endocrine or local TGF-b1, nor diminish P-Smad3 levels in satellite cells, in vivo (Figs 5 and 6). These data also predict the presence of a positive feed-back loop, where a certain TGF-b signaling strength is required to maintain TGF-b1 ligand expression (Fig. 6), which was additionally suggested by TGF-b level reduction in muscle treated with RNAi to Smad3 (Carlson et al., 2008). It remains to be determined whether there is a broad TGF-b1 increase within old tissues (in addition to skeletal muscle ﬁbers), or if a narrow subset of cells in aged organisms overproduce TGF-b1. (C) (C) While the involvement of additional TGF-b family members (or other age-dependent cytokines involved with muscle repair decline) is certainly not ruled out, TGF-b1 was identiﬁed here as a key, age-related inhibitor of myogenic responses. Additionally, myostatin was previously reported to not locally increase in old muscle tissue (Carlson et al., 2008), thus implicating the ubiqui- tous TGF-b1, rather than tissue-speciﬁc family members in these age-related phenotypes. While other circulatory molecules may contribute to the aging of muscle stem cell responses, our data suggest that Wnt is not a systemic age-dependent attenuator of myogenicity (Figs 2, 3, S2 and S4). These data are in apparent disagreement with those reported by Brack et al. Discussion Our ﬁnd- ings suggest that certain levels of TGF-b1 are actually required for normal satellite cell responses (Fig. 1). Experimental down- modulation of this pathway is therefore expected to be typically harmful, unless it is transient (Figs 1, 4––6) or performed to pre- cise ‘beneﬁcial’ levels. These conclusions ﬁt well with studies, reporting that among other important functions, TGF-b1 signal- ª 2009 The Authors Journal compilation ª Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2009 Age-related systemic control of muscle repair, M. E. Carlson et al. 684 684 neonatal lethality, due to the abnormal development of various organ systems. Furthermore, Smad3 knockout animals survive after birth, but die before old age due to chronic infections, accelerated rate of cancers and T-cell dysfunction (Datto et al., 1999; Yang et al., 1999). TGF-b1 heterozygous mice also sur- vive after birth, but have increased neuronal abnormalities, inﬂammation and ﬁbrosis of blood vessels (Wyss-Coray et al., 1997, 2001; Brionne et al., 2003). Combined, these data agree with our conclusion that prolonged, or sustained, experimental down-modulation of TGF-b1 levels will perturb cellular function. (B) (A) (C) Fig. 6 Systemic pharmacological intervention lowers sera transforming growth factor (TGF)-b levels and improves regeneration in old animals. (A) Myoﬁbers were isolated from young, old and old mice with indicated treatments for TGF-b systemic down-modulation. Cell lysates were then analyzed by Western blot for levels of secretory TGF-b and P-Smad3 signaling strength. Actin immunodetection was used as a loading control. As expected, higher levels of TGF-b and P-Smad3 were detected in old samples, as compared to young. O + Ki pump showed reduction in both TGF-b and P-Smad3, whereas O + Ki injection only reduced P-Smad3 signaling strength. (B) Results of multiple Western blot assays were quantiﬁed (normalization of TGF-b and P-Smad3 pixel density by actin-speciﬁc pixel density) and are depicted by relative pixel intensity, as shown; () indicates P < 0.01, O Ki compared to old and young. Error bars indicate SD. n = 3–4. (C) TGF-b levels in serum for each animal in young, old or old + experimental treatments were determined by ELISA. Shown are mean values with standard deviations (B) (A) Our work reveals that platelet-derived sera TGF-b1 levels, or endocrine TGF-b1 levels, do not explain the age-dependent inhi- bition of tissue regeneration by this cytokine. ª 2009 The Authors Journal compilation ª Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2009 ª 2009 The Authors Journal compilation ª Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2009 Discussion (2008); and further study will resolve whether Wnt is present in the circula- tion, is upregulated with aging and becomes inhibitory to myo- genic responses at some age-speciﬁc level. Of note, in reports examining Wnt in young mice only, it is suggested that Wnt Fig. 6 Systemic pharmacological intervention lowers sera transforming growth factor (TGF)-b levels and improves regeneration in old animals. (A) Myoﬁbers were isolated from young, old and old mice with indicated treatments for TGF-b systemic down-modulation. Cell lysates were then analyzed by Western blot for levels of secretory TGF-b and P-Smad3 signaling strength. Actin immunodetection was used as a loading control. As expected, higher levels of TGF-b and P-Smad3 were detected in old samples, as compared to young. O + Ki pump showed reduction in both TGF-b and P-Smad3, whereas O + Ki injection only reduced P-Smad3 signaling strength. (B) Results of multiple Western blot assays were quantiﬁed (normalization of TGF-b and P-Smad3 pixel density by actin-speciﬁc pixel density) and are depicted by relative pixel intensity, as shown; () indicates P < 0.01, O Ki compared to old and young. Error bars indicate SD. n = 3–4. (C) TGF-b levels in serum for each animal in young, old or old + experimental treatments were determined by ELISA. Shown are mean values with standard deviations (n = 3–12, P £ 0.05 between O + Ki pump and O + anti-TGFb pump, and O + Ki ⁄ O + RII injection. Age-related systemic control of muscle repair, M. E. Carlson et al. 685 promotes satellite cell myogenicity for both differentiation of myoblasts to myotubes, and with respect to satellite cell self- renewal (Brack et al., 2008; Le Grand et al., 2009). changed 12 hours after transfection. Forty-eight hours after transfection, the cell culture supernatant, containing the virus, was collected and puriﬁed by ultra centrifugation through 20% sucrose in phosphate-buffered saline (PBS) and resuspended in PBS. The virus also encoded a gene that rendered it resistant against puromycin, and was titered on 293T cells using WST1 reagent (Roche, Mannheim, Germany) following the manufac- turer’s protocol. Comprehensively, these studies identify physiological sources of TGF-b1 that are responsible for age-related muscle repair inhibition, and point toward novel therapeutic strategies for the enhancement of organ regenerative potential. In proof of princi- ple research, intra-muscular attenuation of Smad3, by RNAi, rescued aged muscle repair (Carlson et al., 2008). Retrovirus production and transfection pMSCV-TbRIIDN retroviral vector was a kind gift from Dr Chung Lee (Kundu et al., 2000; Shah et al., 2002). TbRIIDN was excised from the vector using EcoRI ⁄ BamHI, blunted with Klenow (NEB) and inserted into the MoMLV retroviral vector pCLPCGFP (Ignowski & Schaffer, 2004). pCLPCGFP was ﬁrst digested with BamHI, following which it was also blunted with Klenow and ligated with the TbRIIDN fragment. The resulting pCLPC TbRIIDN vector was sequenced to conﬁrm its sequence and directionality of insertion. A plasmid made by ligating the blunt ends of the pCLPC vector was used as a control. To produce retrovirus, HEK 293T cells (cultured in Iscove’s modiﬁed Dulbecco’s medium (Hyclone, Logan, UT, USA) with 10% fetal bovine serum (Invitro- gen) and 1% penicillin–streptomycin (Gibco, Carlsbad, CA, USA) and maintained at 37 C and 5% CO2) were co-transfect- ed with the retroviral vector (pCLPC TbRIIDN or control plasmid) and the helper plasmids pCMV-VSVG, a vector encoding the viral envelope protein from the vesicular stomatitis virus and pCMV-gagpol, a vector encoding the enzymatic and structural retroviral proteins, using the calcium phosphate transfection method as described (Yu & Schaffer, 2006). Medium was Antibodies and reagents Antibody against embryonic myosin heavy chain (develop- mental) was purchased from Vector Laboratories (Burlingame, CA, USA) and the Developmental Studies Hybridoma Bank. Phosphorylated-Smad2 ⁄ 3 antibody [1:200 WB (Western blot), 1:100 IF (immunoﬂuorescence) was obtained from Chemicon (Temecula, CA, USA) and Santa Cruz Biotechnologies (Santa Cruz, CA, USA; sc-11769)]. Bioactivity neutralizing antibody against TGF-b1 (MAB240), rTGF-b1 (240-B) rWnt3a (1324- WN) sFRP3 (592-FR) and huTGF-b RIII (242-R3) were purchased from R&D Systems. Delta (9120) and TGF-beta receptor I (SC 9048) (1:100 WB) antibodies were obtained from Santa Cruz Biotechnologies. Activated Notch (1:500 WB) (ab8925) and BrdU (1:100 IF) (ab6326) antibody were obtained from Abcam (Cambridge, MA, USA). Desmin (1:25 IF) (clone DE- U-10, #D1033) and actin (1:250 WB, A5060) antibodies were purchased from Sigma. Cell culture Young (2–4 month) and old (20–24 month) C57 ⁄ Bl6 male mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA) and NIA (Bethesda, MD, USA) respectively. Myoﬁber explant cultures and primary myogenic progenitor cells were generated from C57 ⁄ Bl6 mice as described previously (Conboy et al., 2003). Myoblasts were maintained in growth media (Ham’s F-10, 20% FBS, 5 ng mL)1 basic-FGF and penicil- lin–streptomycin). Opti-MEM was used to culture myoﬁber explants in 10% young, 10% old or 10% young plus old mouse serum (5% each). Differentiation media consisted of DMEM and 5% horse sera. Basic-FGF was obtained from Sigma (St Louis, MO, USA) and R&D systems (Minneapolis, MN, USA), Opti-MEM was obtained from Invitrogen (Carlsbad, CA, USA), and all other cell culture reagents were obtained from Cellgro (Herndon, VA, USA). Discussion Such method- ology, however, is not easily applicable to an organism-wide restoration of old organ repair. In contrast, our herein reported systemic delivery of a TGF-b receptor kinase inhibitor allows one to reach all tissues, to recalibrate TGF-b1 ligand levels to their youthful states and to potentially rejuvenate their regenerative function. Viral transduction was performed on bulk myoﬁber ex- plants with activated satellite cells, isolated 3 days postinjury from CTX-injured young and old animals. Cells were infected with TbRIIDN (2 · 106 TU mL)1), or control viruses (1 · 107 TU mL)1), at assay-dependent MOIs, and cultured for 48 hours in the presence of OPTI-MEM, +5% young or old mouse serum prior to ﬁxation and analysis. No tetracycline was added in these experiments. ª 2009 The Authors Journal compilation ª Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2009 ª 2009 The Authors Depletion of TGF-b from culture media Opti-MEM containing either 10% young or old mouse serum was incubated in the presence of 5 lg mL)1 TGF-b antibody (R&D Systems) overnight at 4 C. Protein-G agarose was then added to the media ⁄ antibody mix and incubated for 4 h at 4 C. The mixture was then centrifuged at 10 000 RCF for 5 min. Supernatant was collected and centrifugation was repeated. TGF-b depleted supernatant was used for culture. Muscle injury Isoﬂurane was used to anesthetize the animal during the muscle injury procedure. For bulk myoﬁber satellite cell activation, tibial- is anterior and gastrocnemius muscles were injected with CTX I (Sigma) dissolved at 100 lg mL)1 in PBS, at 2–5 sites for each muscle for approximately 50 lL g)1 muscle. Muscles were har- vested 3 days later. For focal injury, to assay regeneration in vivo, 2–3 lL of 1 mg mL)1 CTX was injected at one site to the middle of the tibialis anterior, or two sites at the gastrocnemius, and muscle harvested 5 days later. In some experiments, FRP3 (500 ng), anti-TGF-b (10 ng), FRP3 ⁄ anti-TGF-b or IgG (10 ng) were injected intramuscularly into injury sites, 24 h following ª 2009 The Authors Journal compilation ª Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2009 Age-related systemic control of muscle repair, M. E. Carlson et al. 686 initial injury. In these studies, BrdU (50 mg kg)1) was injected IP at 2 days following injury. Serum TGF-b was activated by mixing 100 lL of serum with 100 lL 10 M Urea ⁄ 2.5 M acetic acid, followed by incubation for 10 min at room temperature. Acid treatment was neutral- ized with 2.7 M sodium hydroxide ⁄ 1 M HEPES, and salts were removed via ultraﬁltration (Vivaspin 500, 3 kDa MWCO; GE Healthcare, Piscataway, NJ, USA). Measurement of bioactive levels of WNT in serum L cells, stably harboring SuperTopFlash (Kaykas et al., 2004) and pEF1-LacZ constructs (‘LSL cells’), were plated into 96-well microtiter dishes at approximately 80–90% conﬂuency. After 6 h postplating, media was removed and LSL cells were trea- ted with serum or Wnt protein diluted into complete media (DMEM ⁄ 10% FBS plus antibiotics) for approximately 16 h at 37 C luciferase activity was measured in a Lumat LB 9507 lumi- nometer (Berthold) using the Dual light TROPIX kit according to manufacturer’s instructions. All assays were performed in tripli- cate, and the relative luciferase units were normalized to LacZ readings. Immunoﬂuorescence and histological analysis All muscle tissue was dissected, ﬂash frozen in OCT compound (Tissue Tek; Sakura Finetek, Zoeterwoude, The Netherlands) and cryo-sectioned at 8–10 lm, as previously described (Conboy et al., 2003). Muscle sections were stained with aqueous hema- toxylin and eosin (H&E), as per the manufacturer’s instructions (Sigma-Aldrich). Regeneration and myogenic potential was quantiﬁed by examining injury sites from representative sections along the muscle (spanning the volume of injury), then measur- ing the injured ⁄ regenerating area using Adobe Photoshop Elements. In all experiments, regenerating tissue was obtained from both tibialis anterior and gastrocnemius muscles. Cryo- sectioning was performed through the entire volume of muscle (typically 50–70 sections total, done at 200 lm intervals), thereby serially reconstituting the entire tissue, ex vivo. Myoﬁber regeneration was quantiﬁed by counting total newly regener- ated ﬁbers at injury epicenters, per volume of injury (typically 1 mm2) and dividing ﬁbers per area. Immunostaining was performed as described (Conboy et al., 2003). Brieﬂy, after permeabilization in PBS + 1% FBS + 0.25% Triton-X-100, tissues and cells were incubated with primary antibodies in staining buffer (PBS + 1% FBS) for 1 h at room temperature, followed by 1 h incubation ﬂuorochrome-labeled secondary antibodies (ALEXA; Molecular Probes, Carlsbad, CA, USA at 1:1000). P-Smad2 ⁄ 3 and BrdU-speciﬁc immunostaining required an extra step of 2 M HCl treatment (denaturation of DNA) before permeablization. The index of myogenic potential was evaluated, based on BrdU ⁄ eMyHC costaining, as previously described (Carlson & Conboy, 2007a; Carlson et al., 2008). Measurement of bioactive levels of TGF-b in serum Serum was isolated as previously described (Conboy et al., 2003). Brieﬂy, blood cells were coagulated at 37C for 15 min and centrifuged to isolate serum. In the luciferase-reporter trans-activation assay, the biological activity of TGF-b was measured via induction of the PAI-1 promoter in mink lung epithelial cells stably expressing a PAI promoter-luciferase con- struct (Abe et al., 1994). Brieﬂy, 3 · 104 transfected cells were plated in 96-well plates in 50 lL of Dulbecco’s modiﬁed eagle medium (DMEM) with 0.5% fetal bovine serum and incubated for 3 h at 37 C in 5% CO2. Experimental sample (50 lL of DMEM with young or old mouse serum at 1:2 and 1:10 dilutions) was added to triplicate wells and incubated for 16 h. Cells were then washed with PBS and lysed. The lucifer- ase activity in the lysate was measured in an EG&G Berthold Microlumat LB 96P (Oak Ridge, TN, USA) for 10 s, immedi- ately following auto-injection of 20 lL of luciferase substrate and recorded as relative light units integrated over time. A TGF-b standard curve was performed to determine the total amount of active TGF-b. Neutralizing antibodies speciﬁc for individual TGF-b (R&D Systems) were added to demonstrate speciﬁcity. ª 2009 The Authors Journal compilation ª Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2009 ª 2009 The Authors Western blot analysis Western blotting was performed according to standard proto- cols. Typically, 30 lg of protein extract from myoﬁbers, or satellite cells, were run on SDS–PAGE gels (Bio-Rad, Hercules, CA, USA). Primary antibodies were diluted in 5% nonfat milk in 1· PBS-T at indicated dilutions (see above). HRP-conjugated secondary antibodies (Santa Cruz Biotech) were diluted 1:1000 in 1· PBS, 2%BSA, and 0.5% Tween-20. Nitrocellu- lose membranes were treated with secondary antibodies for 1 h at room temperature, developed using ECL reagent (Amersham, Sunnyvale, CA, USA), and imaged on a Bio-Rad Chemidoc XRS. Measurement of serum TGF-b levels For plasma preparation, < 100 lL was collected from the tail into 10 lL of 100 mM EDTA. Red blood cells were removed by sedimentation, twice at 150 g for 10 min, followed by platelet sedimentation for 10 min at 1500 g. Platelet-poor plasma was re-cleared by an additional sedimentation for 10 min at 1500 g. Platelets were washed once with 0.08 M sodium cit- rate, pH 6 ⁄ 0.9 g L)1 sodium chloride, sedimented again at 1500 g, and resuspended in cold water before ﬂash freezing. Mouse blood was collected upon euthanasia, and serum was prepared as above. Mouse serum levels were determined by sandwich ELISA, as per the manufacturer’s protocol (R&D Sys- tems #DY1679), with the recommended pretreatment of sam- ple with acid ⁄ urea, followed by neutralization. To avoid dilution effects on measurement, peak readings calculated from the corrected dilution curve were used for comparison among samples. Serum from aged and young human subjects ª 2009 The Authors Journal compilation ª Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2009 Age-related systemic control of muscle repair, M. E. Carlson et al. 6 687 was collected and stored in aliquots at )70 C until used. Young donors were between 20 and 35 years of age and elderly donors were between 65 and 90 years of age. Elderly subjects belonged to middle-class socio-economic status, and were living independently. This study was approved by the Institutional Review Board of the University of California, Irvine. Human TGF-b levels were determined using a speciﬁc ELISA kit (BD Biosciences, San Jose, CA, USA) with the recom- mended pretreatment of sample with acid, followed by neu- tralization with the base. Author contributions MEC provided Figs 1, 2C,D, 3C–F, 4, 5, 6, S1, S2, S3E,F, S4, S5, S6B and S7A,B, D–F and wrote the manuscript; MJC provided Figs 2A, 3B, 5, 6 S3D and S7C,D, and wrote the manuscript; MH provided preliminary data for Fig. 1; JJ provided Fig. S6A; LB provided Fig. S3C; AA provided Figs 2B and S3B and DVF pro- vided the TGFb DN RII expressing retrovirus and experimental design in Fig. 4; AJM provided data for Fig. 3A,B; IMC designed and coordinated the study, interpreted the data and wrote the manuscript. Statistical analysis Brack AS, Conboy MJ, Roy S, Lee M, Kuo CJ, Keller C, Rando TA (2007) Increased Wnt signaling during aging alters muscle stem cell fate and increases ﬁbrosis. Science (New York, NY) 317, 807–810. A minimum of three replicates were performed for each experi- mental condition. Quantiﬁed data are presented as means and standard deviations. Analysis of variance was used to compare data from different experimental groups. P-values of < 0.05 were considered statistically signiﬁcant. Brack AS, Conboy IM, Conboy MJ, Shen J, Rando TA (2008) A tempo- ral switch from notch to Wnt signaling in muscle stem cells is neces- sary for normal adult myogenesis. Cell Stem Cell 2, 50–59. Brand T, MacLellan WR, Schneider MD (1993) A dominant-negative receptor for type beta transforming growth factors created by dele- tion of the kinase domain. J. Biol. Chem. 268, 11500–11503. References Abe M, Harpel JG, Metz CN, Nunes I, Loskutoff DJ, Rifkin DB (1994) An assay for transforming growth factor-beta using cells transfected with a plasminogen activator inhibitor-1 promoter-luciferase con- struct. Anal. Biochem. 216, 276–284. Andrews ZB, Zhao H, Frugier T, Meguro R, Grattan DR, Koishi K, McLennan IS (2006) Transforming growth factor beta2 haploinsufﬁ- cient mice develop age-related nigrostriatal dopamine deﬁcits. Neu- robiol. Dis. 21, 568–575. Pharmacological intervention For modulating TGF-b levels systemically, the TGF-b RI kinase inhibitor (Calbiochem ⁄ EMD Chemicals Gibbstown, NJ, USA; catalog # 616452) was delivered at 10 nmole over 2 weeks, by injecting twice daily for 4 weeks (subcutaneous) with 100 lL of a 1:10 dilution of 3.4 mM inhibitor (dissolved in dim- ethylsulfoxide), diluted in PBS with diluted DMSO as control. Five days before the end of treatment, muscle was injured as described. At the end of treatment, animals were sacriﬁced and muscles were collected. BrdU was injected (intra- peritoneal) at 3 days postinjury to label proliferating, fusion- competent myoblasts. Annes JP, Munger JS, Rifkin DB (2003) Making sense of latent TGF- beta activation. J. Cell Sci. 116, 217–224. Assoian RK, Komoriya A, Meyers CA, Miller DM, Sporn MB (1983) Transforming growth factor-beta in human platelets. Identiﬁcation of a major storage site, puriﬁcation, and characterization. J. Biol. 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This work was supported by NIH RO1 AG027252, CIRM RN10083R, Ellison’s Medical Foundation and Glen Research Foundation grants to IMC, and a CIRM training grant to MEC. ª 2009 The Authors Journal compilation ª Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2009 ª 2009 The Authors Journal compilation ª Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2009 ª 2009 The Authors Acknowledgments Brionne TC, Tesseur I, Masliah E, Wyss-Coray T (2003) Loss of TGF- beta 1 leads to increased neuronal cell death and microgliosis in mouse brain. Neuron 40, 1133–1145. We would like to thank Rik Derynck for very helpful discussions and Mary-Helen Barcellos-Hoff for advice and help with bio- active TGF-b assays, and we also thank Roel Nusse for his expert advice on the Wnt assays. We thank Margaret A. Weitzman and Rajiv Desai for technical assistance, and Wendy Cousin for FRP bioactivity controls. 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https://openalex.org/W4387392425	https://hal.science/hal-04260438/document	English	null	The tip protein PAAR is required for the function of the type VI secretion system	Microbiology spectrum	2,023	cc-by	17,317	The tip protein PAAR is required for the function of the type VI secretion system Solène G Beauvois, Nicolas Flaugnatti, Marianne Ilbert, Marie Boyer, Esther Gavello-Fernandez, Rémi Fronzes, Dukas Jurėnas, Laure Journet To cite this version: Solène G Beauvois, Nicolas Flaugnatti, Marianne Ilbert, Marie Boyer, Esther Gavello-Fernandez, et al.. The tip protein PAAR is required for the function of the type VI secretion system. Microbiology Spectrum, 2023, 10.1128/spectrum.01478-23. hal-04260438 Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-04260438 https://hal.science/hal-04260438v1 Submitted on 26 Oct 2023 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 The tip protein PAAR is required for the function of the type VI secretion system. 1 2 Solène G. Beauvoisa, Nicolas Flaugnattia,e, Marianne Ilbertb, Marie Boyera, Esther Gavello- 3 Fernandezc,d, Rémi Fronzesc,d, Dukas Jurėnasa,f and Laure Journeta# 4 5 aLaboratoire d’Ingénierie des Systèmes Macromoléculaires, Institut de Microbiologie, 6 Bioénergies et Biotechnologie, Institut de Microbiologie de la Méditerranée, Aix-Marseille 7 Université - CNRS UMR7255, Marseille, France 8 bLaboratoire de Bioénergétique et Ingénierie des Protéines, Institut de Microbiologie, 9 Bioénergies et Biotechnologie, Institut de Microbiologie de la Méditerranée, Aix-Marseille 10 Université—CNRS UMR7281, Marseille, France. 11 cInstitut Européen de Chimie et Biologie, University of Bordeaux, Pessac, France / 12 dCNRS UMR 5234 Microbiologie Fondamentale et Pathogénicité, Bordeaux, France 13 14 #Address correspondence to Laure Journet, ljournet@imm.cnrs.fr 15 ePresent address: Nicolas Flaugnatti, Laboratory of Molecular Microbiology, School of Life 16 Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland. 17 fPresent address: Dukas Jurėnas, Laboratoire de Génétique et Physiologie Bactérienne, 18 Département de Biologie Moléculaire Université Libre de Bruxelles, Gosselies, Belgium. 19 20 Running title: The type VI secretion system PAAR tip protein 21 22 The tip protein PAAR is required for the function of the type VI secretion system. 1 2 Solène G. Beauvoisa, Nicolas Flaugnattia,e, Marianne Ilbertb, Marie Boyera, Esther Gavello- 3 Fernandezc,d, Rémi Fronzesc,d, Dukas Jurėnasa,f and Laure Journeta# 4 5 aLaboratoire d’Ingénierie des Systèmes Macromoléculaires, Institut de Microbiologie, 6 Bioénergies et Biotechnologie, Institut de Microbiologie de la Méditerranée, Aix-Marseille 7 Université - CNRS UMR7255, Marseille, France 8 bLaboratoire de Bioénergétique et Ingénierie des Protéines, Institut de Microbiologie, 9 Bioénergies et Biotechnologie, Institut de Microbiologie de la Méditerranée, Aix-Marseille 10 Université—CNRS UMR7281, Marseille, France. 11 cInstitut Européen de Chimie et Biologie, University of Bordeaux, Pessac, France / 12 dCNRS UMR 5234 Microbiologie Fondamentale et Pathogénicité, Bordeaux, France 13 aLaboratoire d’Ingénierie des Systèmes Macromoléculaires, Institut de Microbiologie, 6 Bioénergies et Biotechnologie, Institut de Microbiologie de la Méditerranée, Aix-Marseille 7 Université - CNRS UMR7255, Marseille, France 8 bLaboratoire de Bioénergétique et Ingénierie des Protéines, Institut de Microbiologie, 9 Bioénergies et Biotechnologie, Institut de Microbiologie de la Méditerranée, Aix-Marseille 10 Université—CNRS UMR7281, Marseille, France. 11 bLaboratoire de Bioénergétique et Ingénierie des Protéines, Institut de Microbiologie, 9 Bioénergies et Biotechnologie, Institut de Microbiologie de la Méditerranée, Aix-Marseille 10 Université—CNRS UMR7281, Marseille, France. 11 bLaboratoire de Bioénergétique et Ingénierie des Protéines, Institut de Microbiologie, 9 Bioénergies et Biotechnologie, Institut de Microbiologie de la Méditerranée, Aix-Marseille 10 Université—CNRS UMR7281, Marseille, France. 11 cInstitut Européen de Chimie et Biologie, University of Bordeaux, Pessac, France / 12 #Address correspondence to Laure Journet, ljournet@imm.cnrs.fr 15 25 ABSTRACT 26 Bacteria are constantly competing to colonize crowded ecological niches, such as the human 27 gut. The Type VI secretion system (T6SS) is a critical bacterial weapon in this warfare. It 28 resembles a crossbow with a poisoned arrow allowing bacteria to inject toxic effectors 29 directly into target cells. This machinery is formed by an envelope-spanning complex which 30 recruits the baseplate, an assembly platform allowing the polymerization of a contractile 31 structure. The tail consists of a tube surrounded by a sheath and topped by the needle complex 32 composed of the VgrG and PAAR proteins. In the enteric pathogen enteroaggregative 33 Escherichia coli (EAEC), the Tle1 phospholipase toxin ensures the antibacterial activity of 34 the T6SS-1. For its transport, Tle1 interacts directly with the trimeric spike protein VgrG. 35 However, the importance and the function of the tip protein PAAR in the T6SS remains 36 unclear. Here we characterized the PAAR protein of EAEC using biochemical, fluorescence 37 microscopy and antibacterial competition approaches. Using pull-down assays and cryo-EM 38 analysis of the (VgrG)3-(Tle1)3-PAAR complex we show that PAAR tops and closes the β- 39 prism structure of the VgrG spike. The PAAR protein structure is further tightened by the 40 zinc atom coordinated via conserved residues essential for its function. We provide evidence 41 that PAAR is necessary for T6SS-1 mediated killing due to its requirement for proper T6SS 42 baseplate assembly and further sheath polymerization. Our results suggest that the PAAR 43 protein is an essential component of T6SS. 44 50 51 IMPORTANCE 52 The Type VI secretion system (T6SS) is a bacterial contractile injection system involved in 53 bacterial competition by the delivery of antibacterial toxins. The T6SS consists of an 54 envelope-spanning complex that recruits the baseplate, allowing the polymerization of a 55 contractile tail structure. The tail is a tube wrapped by a sheath and topped by the tip of the 56 system, the VgrG spike/PAAR complex. Effectors loaded onto the puncturing tip or into the 57 tube are propelled in the target cells upon sheath contraction. The PAAR protein tips and 58 sharpens the VgrG spike. However, the importance and the function of this protein remain 59 unclear. Here we provide evidence for association of PAAR at the tip of the VgrG spike. We 60 also found that the PAAR protein is a T6SS critical component required for baseplate and 61 sheath assembly. 62 Keywords: Type VI secretion system; cryo-electron microscopy; bacterial competition; 64 protein secretion; VgrG; bacterial toxin; zinc 65 75 76 INTRODUCTION 78 Bacteria constantly compete for colonization of the ecological niches granting the access to 79 nutrient sources. The best armed bacteria will have greater chances to win this warfare and 80 plenty of weapons are deployed to succeed in this competition. The type VI secretion system 81 is one of the crucial weapons allowing bacteria to inject toxic effectors directly into the target 82 cells. There are two categories of T6SS toxins. T6SS periplasmic-acting toxins can degrade 83 peptidoglycan, membrane phospholipids or form pores in the inner membrane. T6SS 84 cytoplasmic effectors target DNA, deplete energy resources, inhibit cell division or inhibit 85 translation (1-4). T6SS can also target eukaryotic cells, employing effectors mainly targeting 86 the eukaryotic cytoskeleton and membranes (1, 2, 5). 87 The T6SS machinery is composed of 13-14 core components arranged in gene clusters (6, 7). 88 The architecture can be subdivided into three subcomplexes: the membrane complex, the 89 baseplate and the tail tube/sheath complex (TTC). The membrane complex is a 1.7 MDa 90 complex that anchors the system to the bacterial envelope (8, 9). It is composed of outer 91 membrane lipoprotein TssJ and inner membrane proteins TssM and TssL (in a 15:10:10 92 stoichiometry). The membrane complex recruits the baseplate which serves as an assembly 93 platform required for the polymerization of the contractile phage tail-like tubular structure 94 (10-15). The baseplate is assembled from six wedges composed of Tss-K, -E, -F, -G proteins 95 that arrange around a central hub, formed by the trimeric spike protein VgrG (14,15). With 96 the help of TssA protein, the baseplate directs the polymerization of the contractile TTC 97 structure (11,16). The TTC is composed of an internal tube made of Hcp protein hexamers, 98 surrounded by a contractile sheath, made of TssB and TssC subunits. This cytoplasmic tail is 99 about 1μm long and assembles within roughly 40 seconds through the TssA protein- 100 coordinated polymerization of Hcp and TssB/C components (10, 16, 17). Upon contact with 101 prey, the sheath contracts and expels the tube surmounted by the spike, together with the 102 effectors into the target cell. Indeed, the effectors are fused or interact with these structural 103 components (1, 3, 4, 18, 19). The enteroaggregative Escherichia coli T6SS cluster 1 (EAEC 104 T6SS-1) delivers the phospholipase toxin Tle1 (20). INTRODUCTION 78 Three Tle1 cargo effectors are loaded on 105 the sides of the VgrG trimer via direct protein-protein interactions (20, 21). The N-terminal β- 106 strand of Tle1 interacts with the C-terminal transthyretin (TTR) extension of VgrG through 107 fold complementation. VgrG-Tle1 interaction is further stabilized by additional zones of 108 contact on the sides and at the base of the gp5-like domain of VgrG. The Tle1 phospholipase 109 A1 (PLA1) activity is inhibited by the interaction with the VgrG protein (21). Almost entire 110 T6SS, except the membrane complex, structurally resembles contractile bacteriophages, such 111 as T4 (22-25). The T6SS spike protein VgrG is structurally homologous to the T4 puncturing 112 device composed of gp27 and gp5 protein trimers (22, 23). Precisely, the triangular base of 113 VgrG also known as a hub domain is homologous to gp27. It is followed by an OB-fold 114 domain which is homologous to gp5- domain, and extended by a β-helical gp5C-like 115 domain (21-23, 26, 27). The blunt end of the VgrG β-helical prism is covered by a single 116 PAAR protein that knots the trimeric structure into a uniform sharp tip (28). PAAR, named 117 after proline-alanine-alanine-arginine repeats, is structurally homologous to the gp5.4 protein 118 of bacteriophage T4, that similarly caps the central spike complex formed by gp5C (12, 28, 119 29). 120 PAAR was shown to be either important or absolutely required for T6SS secretion or killing 121 PAAR was shown to be either important or absolutely required for T6SS secretion or killing 121 activity in different systems (28, 30-32). Its role remains to be determined, although some 122 hypotheses proposed that it stabilizes the trimeric VgrG complex or sharpens the T6SS for 123 target cell puncturing. 124 Here we report structural and functional analysis of EC042_4537 gene of the EAEC T6SS-1 125 that is predicted to code for a PAAR protein. Single particle cryo-EM analysis of the VgrG- 126 Tle1-PAAR complex from EAEC revealed that PAAR caps the blunt end of the VgrG needle. 127 ICP-OES analysis of purified PAAR protein shows that a zinc atom is coordinated by 128 conserved histidines and cysteines (H14, H46, C41, C74). Bacterial competition assays 129 indicate that PAAR protein and its metal coordinating residues H46, C41 and C74 are 130 essential for T6SS-1 antibacterial activity. INTRODUCTION 78 Finally, using fluorescence microscopy, we further 131 demonstrate that PAAR is required for correct baseplate localization and thus for sheath 132 assembly. Taken together, our results confirm that the PAAR protein is an essential core 133 component of the T6SS. 134 PAAR caps the C-terminal needle domain of VgrG spike complex. 137 T6SS-1 encodes a unique predicted PAAR protein, downstream the tli1 gene coding for 138 immunity protein against the Tle1 toxin (20). Our previous cryo-EM structure of the EAEC 139 (VgrG)3-(Tle1)3 complex revealed that the toxins are loaded on the sides of the β-prism of the 140 VgrG spike, leaving the top of the β-prism free to accommodate the PAAR protein (21). To 141 test this hypothesis, we aimed to determine where precisely does the PAAR localize on this 142 structure. We heterologously co-produced PAARFLAG (PAARFL) and StrepVgrG (SVgrG), as 143 well as its truncated versions deleted for the TTR domain (SVgrG1-778 called SVgrGΔTTR) 144 or the whole β-prism (needle) part (SVgrG1-490 called SVgrGΔNeedle) and tested direct 145 interactions by streptactin pull-down assays (Fig. 1AB). As shown in Fig. 1B, PAARFLAG co- 146 precipitated with SVgrG and SVgrGΔTTR but not with VgrG deleted for the whole β-prism 147 (SVgrGΔNeedle). We conclude that PAAR directly interacts with the C-terminal domain of 148 VgrG without implication of the TTR domain. A complete spike complex loaded with toxin 149 PAARFL-SVgrG-Tle1H was purified using a double-affinity and size exclusion 150 chromatography (Fig. 1C) and analyzed using single particle cryo-EM (Fig. 1DE, Fig. S1A). 151 For comparison we have repeated the analysis of a previously reported SVgrG-Tle1H complex 152 without PAAR purified and analyzed in the same conditions. As previously reported, the 153 (VgrG)3-(Tle1)3 complexes dimerized through interactions at the top of the VgrG needle to 154 produce violin body-shaped 2D classes ((21), Fig. 1D, Fig. S1B). Such dimerization was 155 completely absent in the VgrG-Tle1-PAAR complex, suggesting that PAAR prevents the two 156 blunt ends of VgrG β-prisms from sticking together (Fig. 1D, Fig. S1A). A single conical 157 density on top of the trimeric VgrG needle prism was readily recognizable and likely 158 corresponds to the PAAR protein (Fig. 1D). Surprisingly, the 2D classification revealed 159 heterogeneity in particles yielding classes where 3, 2, 1 or none of the Tle1 toxins were 160 visible judging from the side and the top views (Fig. S1A). This is in contrast with the violin- 161 body-shaped 2D classes of the (VgrG)3-(Tle1)3 complex (without PAAR) where sharp 162 densities for all the toxins were typically observed (Fig. S1B) (21). This suggests that the 163 toxin positions are locked by the unnatural dimerization of the (VgrG)3-(Tle1)3 complexes 164 that occurs due to the absence of the PAAR protein. PAAR caps the C-terminal needle domain of VgrG spike complex. 137 This dimerization seems to lock the TTR 165 regions in fixed position, that are extended from the VgrG structure and hang on the flexible 166 linkers not visible in the structure (21). It is therefore likely that the toxins loaded on the 167 VgrG are indeed quite flexible, which explains the partial absence of the Tle1 densities in 168 VgrG-Tle1-PAAR complex where TTRs are not fixed. 169 Due to very small size and lack of symmetry the resolution was not sufficient to build an 170 i l i d l f h PAAR i i h b i d EM d i W 171 Due to very small size and lack of symmetry the resolution was not sufficient to build an 170 experimental atomic model of the PAAR protein in the obtained cryo-EM density maps. We 171 have therefore produced an AlphaFold2 co-folded model of the needle domain of VgrG 172 together with PAAR and aligned it to the experimental VgrG-Tle1 model (PDB:6SJL). The 173 resulting complete model could be readily docked into best resolved cryo-EM 3D volumes 174 representing (VgrG)3-(Tle1)2-PAAR (4.2 Å) or (VgrG)3-(Tle1)1-PAAR (at 3,7 Å) structures 175 (Fig. 1E; Fig S1C). The triangular shape of PAAR model perfectly fits into the extra conical 176 density that we assigned to PAAR (Fig. 1E). 177 representing (VgrG)3-(Tle1)2-PAAR (4.2 Å) or (VgrG)3-(Tle1)1-PAAR (at 3,7 Å) structures 175 (Fig. 1E; Fig S1C). The triangular shape of PAAR model perfectly fits into the extra conical 176 density that we assigned to PAAR (Fig. 1E). 177 representing (VgrG)3-(Tle1)2-PAAR (4.2 Å) or (VgrG)3-(Tle1)1-PAAR (at 3,7 Å) structures 175 (Fig. 1E; Fig S1C). The triangular shape of PAAR model perfectly fits into the extra conical 176 density that we assigned to PAAR (Fig. 1E). 177 PAAR binds a zinc atom via conserved cysteine and histidine residues. 179 PAAR binds a zinc atom via conserved cysteine and histidine residues. 179 The 88 amino-acid length protein PAAR (EC042_4537) from EAEC T6SS-1 belongs to class 180 1 PAAR family (28) carrying a PAAR_CT_2 domain (cd14744 of NCBI-CDD). The 181 PAAREAEC amino acid sequence was aligned with the sequences of structurally characterized 182 PAAR proteins from Vibrio cholerae and E. coli CFT073 (Fig. 2A) (28). These two proteins 183 were shown to bind a zinc atom that is coordinated by three histidines (His) and one cysteine 184 (Cys) localized at the extremity of the tip (28). Three motifs with hydrophobic residues that 185 could functionally resemble PAAR-motifs (Pro-Ala-Ala-Arg) could be identified using 186 sequence alignment (Fig. 2A). In the PAAREAEC protein, two His (His14 and His46) and two 187 Cys (Cys41 and Cys74) residues are conserved and localize at the sharp end of the 188 AlphaFold2 structural model of PAAR (Fig. 2B). We therefore hypothesized that these 189 residues could be involved in metal binding (Fig. 2AB) as previously reported for V. cholerae 190 and E. coli PAAR proteins (28). PAAREAEC protein was fused to a SUMO protein to increase 191 its solubility and a 6histidine tag for purification with affinity chromatography (Fig. 2C). 192 ICP-OES analysis of the purified protein showed that 74.2 % (+/- 13.7 SD) of PAAREAEC 193 contains zinc. (Fig. 2D). Only small amounts (< 0.75% of total protein) of iron, copper or 194 nickel were found in the samples (Fig. S2). PAAREAEC proteins with single and double 195 alanine substitutions in conserved cysteines C41 and C74 exhibited a significant decrease in 196 zinc content compared to the wild type (Fig. 2D). Only 34.7% (+/- 10,9 SD) of PAARC41A, 197 20.1% (+/- 12. 7 SD) of PAARC74A and 25.1 % (+/- 7.0) of PAARC41AC74A were found to 198 coordinate a zinc atom (Fig. 2D). To rule out the possibility that the overall fold of the small 199 The 88 amino-acid length protein PAAR (EC042_4537) from EAEC T6SS-1 belongs to class 180 1 PAAR family (28) carrying a PAAR_CT_2 domain (cd14744 of NCBI-CDD). The 181 PAAREAEC amino acid sequence was aligned with the sequences of structurally characterized 182 PAAR proteins from Vibrio cholerae and E. coli CFT073 (Fig. 2A) (28). PAAR binds a zinc atom via conserved cysteine and histidine residues. 179 Our model provides unambiguous case to study the role of PAAR, 209 since only one tip complex exists in EAEC T6SS-1 and the loading of toxins on the VgrG 210 spike does not require PAAR (Fig. 1D). To determine the importance of PAAR protein in the 211 function of T6SS of EAEC we have tested the capacity of a PAAR deletion mutant (ΔPAAR 212 Ω) to kill E. coli W3110 prey, using two different methods (34) (Fig. 3). First, the predator 213 strains and the prey cells were grown separately in T6SS-1 inducing medium (SIM) before 214 being mixed at a 1:4 ratio (for colorimetric method) or 4:1 ratio (for emergence time method) 215 and spotted on SIM-agar plates. In the case of the colorimetric method, after two hours of 216 contact, yellow CPRG substrate was deposited on the mixed-cell spots. CPRG substrate was 217 degraded into purple CPR product by ß-galactosidase released from the prey cells upon cell 218 lysis induced by the secreted phospholipase toxin Tle1 by the wild type (WT) strain. On the 219 contrary, competition with deletion strain of the whole T6SS-1 cluster (ΔT6SS) used for 220 control retains yellow color of the mixed spot reporting the absence of cell lysis. Interestingly, 221 the ΔPAARΩ mutant was not able to kill W3110 strain. However, its antibacterial activity 222 proteins was impaired, we have assayed the interaction of the mutants with VgrG. The 200 PAAREAEC proteins with single C41A and C74A, and to lesser extent double C41A/C74A, 201 substitutions retained their capacity to interact with VgrG suggesting that these mutations 202 mainly affect metal coordination (Fig. S3). Overall, these results indicate that PAAREAEC 203 protein binds a zinc atom via at least the two conserved cysteines C41 and C74. 204 proteins was impaired, we have assayed the interaction of the mutants with VgrG. The 200 PAAREAEC proteins with single C41A and C74A, and to lesser extent double C41A/C74A, 201 substitutions retained their capacity to interact with VgrG suggesting that these mutations 202 mainly affect metal coordination (Fig. S3). Overall, these results indicate that PAAREAEC 203 protein binds a zinc atom via at least the two conserved cysteines C41 and C74. 204 PAAR binds a zinc atom via conserved cysteine and histidine residues. 179 These two proteins 183 were shown to bind a zinc atom that is coordinated by three histidines (His) and one cysteine 184 (Cys) localized at the extremity of the tip (28). Three motifs with hydrophobic residues that 185 could functionally resemble PAAR-motifs (Pro-Ala-Ala-Arg) could be identified using 186 sequence alignment (Fig. 2A). In the PAAREAEC protein, two His (His14 and His46) and two 187 Cys (Cys41 and Cys74) residues are conserved and localize at the sharp end of the 188 AlphaFold2 structural model of PAAR (Fig. 2B). We therefore hypothesized that these 189 residues could be involved in metal binding (Fig. 2AB) as previously reported for V. cholerae 190 and E. coli PAAR proteins (28). PAAREAEC protein was fused to a SUMO protein to increase 191 its solubility and a 6histidine tag for purification with affinity chromatography (Fig. 2C). 192 ICP-OES analysis of the purified protein showed that 74.2 % (+/- 13.7 SD) of PAAREAEC 193 contains zinc. (Fig. 2D). Only small amounts (< 0.75% of total protein) of iron, copper or 194 nickel were found in the samples (Fig. S2). PAAREAEC proteins with single and double 195 alanine substitutions in conserved cysteines C41 and C74 exhibited a significant decrease in 196 zinc content compared to the wild type (Fig. 2D). Only 34.7% (+/- 10,9 SD) of PAARC41A, 197 20.1% (+/- 12. 7 SD) of PAARC74A and 25.1 % (+/- 7.0) of PAARC41AC74A were found to 198 coordinate a zinc atom (Fig. 2D). To rule out the possibility that the overall fold of the small 199 proteins was impaired, we have assayed the interaction of the mutants with VgrG. The 200 PAAREAEC proteins with single C41A and C74A, and to lesser extent double C41A/C74A, 201 substitutions retained their capacity to interact with VgrG suggesting that these mutations 202 mainly affect metal coordination (Fig. S3). Overall, these results indicate that PAAREAEC 203 protein binds a zinc atom via at least the two conserved cysteines C41 and C74. 204 205 PAAR is required for the antibacterial activity of T6SS. 206 In the systems with multiple T6SS tip complexes at least one PAAR protein was shown to be 207 required for functionality of the T6SS but some reports suggested that it is not always 208 necessary (28, 30-33). PAAR is required for the antibacterial activity of T6SS. 206 On the contrary, prey cells exposed to ΔT6SS-1 or ΔPAARΩ 231 mutants took less than five hours to reach the same A600, indicating that much more prey cells 232 remained viable in the mixed-cell spot. Both methods confirm that the PAAR protein is 233 required for the EAEC T6SS-1 antibacterial activity. We have also tested whether expression 234 of PAAR with the single amino acid substitutions in conserved metal-coordination residues 235 could complement lack of wild-type PAAR protein. Interestingly, except for histidine H14, 236 substitutions in all the other residues predicted to be involved in zinc binding, i.e., C41, C74 237 and H46 could not complement PAAR deletion. This further suggests the importance of zinc 238 binding at the tip of the PAAR for its function (Fig. 3). The functionality of H14 mutant may 239 suggest that it is not involved in zinc binding. However, it is not unusual that the deletion of a 240 single metal coordinating residue is not enough to affect metal binding. The Cys76 is well 241 located in the predicted structure to compensate the absence of His14 for metal binding (Fig 242 S4A). We thus constructed and analyzed C76A and His14AC76A mutants. Both H14A and 243 C76A single mutants, but not the double mutant H14AC76A, could complement PAAR 244 deletion in the killing assay (Fig S4B). Accordingly, the zinc content of the double mutant 245 H14AC76A, but not that of C76A or H14A, seems to be impaired compared to the wild type 246 (Fig S4C). All together, these data suggest that C41, C74, H46 and likely H14 (or C76) are 247 important for zinc binding and function of PAAREAEC. As a control, we have assayed alanine 248 substitutions of other cysteine residues of PAAREAEC protein, i.e., C70 and C72, that are 249 time method (34). After two hours of contact each cell spot was scraped from agar plates and 225 diluted into a liquid medium selective for prey cells. The re-growth of prey cells was 226 evaluated by measuring A600 for 15 hours using a TECAN. The emergence time, being the 227 time for the cultures to reach an A600 equal to 0.4, was plotted for each condition (Fig. 3B). PAAR is required for the antibacterial activity of T6SS. 206 In the systems with multiple T6SS tip complexes at least one PAAR protein was shown to be 207 required for functionality of the T6SS but some reports suggested that it is not always 208 necessary (28, 30-33). Our model provides unambiguous case to study the role of PAAR, 209 since only one tip complex exists in EAEC T6SS-1 and the loading of toxins on the VgrG 210 spike does not require PAAR (Fig. 1D). To determine the importance of PAAR protein in the 211 function of T6SS of EAEC we have tested the capacity of a PAAR deletion mutant (ΔPAAR 212 Ω) to kill E. coli W3110 prey, using two different methods (34) (Fig. 3). First, the predator 213 strains and the prey cells were grown separately in T6SS-1 inducing medium (SIM) before 214 being mixed at a 1:4 ratio (for colorimetric method) or 4:1 ratio (for emergence time method) 215 and spotted on SIM-agar plates. In the case of the colorimetric method, after two hours of 216 contact, yellow CPRG substrate was deposited on the mixed-cell spots. CPRG substrate was 217 degraded into purple CPR product by ß-galactosidase released from the prey cells upon cell 218 lysis induced by the secreted phospholipase toxin Tle1 by the wild type (WT) strain. On the 219 contrary, competition with deletion strain of the whole T6SS-1 cluster (ΔT6SS) used for 220 control retains yellow color of the mixed spot reporting the absence of cell lysis. Interestingly, 221 the ΔPAARΩ mutant was not able to kill W3110 strain. However, its antibacterial activity 222 could be restored by complementing the expression of PAAR protein from a plasmid 223 (PAAR+) (Fig. 3A). To perform quantitative analysis, we have further pursued the emergence 224 time method (34). After two hours of contact each cell spot was scraped from agar plates and 225 diluted into a liquid medium selective for prey cells. The re-growth of prey cells was 226 evaluated by measuring A600 for 15 hours using a TECAN. The emergence time, being the 227 time for the cultures to reach an A600 equal to 0.4, was plotted for each condition (Fig. 3B). 228 After being exposed to an EAEC WT strain or a complemented ΔPAARΩ mutant strain, prey 229 cells take seven hours to reach an A600 of 0.4 indicating very little prey cells have survived to 230 restore the growth in culture. PAAR is required for the antibacterial activity of T6SS. 206 253 positioned elsewhere in the structure and are thus not likely to contribute to metal binding 250 (Fig. S4AB). As expected, no effect on bacterial killing was observed using those mutants. 251 The production in ΔPAARΩ of the different versions of PAAR protein from pBAD plasmids 252 was confirmed by Western Blot (Fig. S4F). 253 positioned elsewhere in the structure and are thus not likely to contribute to metal binding 250 (Fig. S4AB). As expected, no effect on bacterial killing was observed using those mutants. 251 The production in ΔPAARΩ of the different versions of PAAR protein from pBAD plasmids 252 was confirmed by Western Blot (Fig. S4F). 253 PAAR is required for the antibacterial activity of T6SS. 206 228 After being exposed to an EAEC WT strain or a complemented ΔPAARΩ mutant strain, prey 229 cells take seven hours to reach an A600 of 0.4 indicating very little prey cells have survived to 230 restore the growth in culture. On the contrary, prey cells exposed to ΔT6SS-1 or ΔPAARΩ 231 mutants took less than five hours to reach the same A600, indicating that much more prey cells 232 remained viable in the mixed-cell spot. Both methods confirm that the PAAR protein is 233 required for the EAEC T6SS-1 antibacterial activity. We have also tested whether expression 234 of PAAR with the single amino acid substitutions in conserved metal-coordination residues 235 could complement lack of wild-type PAAR protein. Interestingly, except for histidine H14, 236 substitutions in all the other residues predicted to be involved in zinc binding, i.e., C41, C74 237 and H46 could not complement PAAR deletion. This further suggests the importance of zinc 238 binding at the tip of the PAAR for its function (Fig. 3). The functionality of H14 mutant may 239 suggest that it is not involved in zinc binding. However, it is not unusual that the deletion of a 240 single metal coordinating residue is not enough to affect metal binding. The Cys76 is well 241 located in the predicted structure to compensate the absence of His14 for metal binding (Fig 242 S4A). We thus constructed and analyzed C76A and His14AC76A mutants. Both H14A and 243 C76A single mutants, but not the double mutant H14AC76A, could complement PAAR 244 deletion in the killing assay (Fig S4B). Accordingly, the zinc content of the double mutant 245 H14AC76A, but not that of C76A or H14A, seems to be impaired compared to the wild type 246 (Fig S4C). All together, these data suggest that C41, C74, H46 and likely H14 (or C76) are 247 important for zinc binding and function of PAAREAEC. As a control, we have assayed alanine 248 substitutions of other cysteine residues of PAAREAEC protein, i.e., C70 and C72, that are 249 positioned elsewhere in the structure and are thus not likely to contribute to metal binding 250 (Fig. S4AB). As expected, no effect on bacterial killing was observed using those mutants. 251 The production in ΔPAARΩ of the different versions of PAAR protein from pBAD plasmids 252 was confirmed by Western Blot (Fig. S4F). PAAR is required for the polymerization of T6SS sheath. 255 We wanted to further understand whether the killing defect in the absence of PAAR is due to 256 its requirement for T6SS assembly or in the later stages such as target cell penetration. We 257 have therefore used fluorescence microscopy to follow the dynamics of TssB sheath protein 258 fused to the fluorescent protein sfGFP, that allows observation of sheath elongation and 259 contraction events. Time lapse fluorescence microscopy revealed that as compared to wild- 260 type strain that could assemble T6SS sheaths, the PAAR mutant (ΔPAARΩ) displayed a 261 diffuse fluorescence indicating that TssB-sfGFP proteins are produced but T6SS sheaths are 262 not assembled (Fig. 4A). However, the complementation of PAAR protein expression from a 263 plasmid (ΔPAARΩ + pBAD–PAAR) could restore the wild-type phenotype (Fig. 4A) which 264 confirms that PAAR protein is necessary for the assembly of T6SS sheaths in EAEC. In 265 addition, PAAR function is not affected by the presence of a C-terminal VSVG tag (Fig 3 and 266 Fig 4A). 267 To assess the role of conserved cysteines and histidines of PAAREAEC, that we have shown to 268 be involved in the binding of a zinc atom (C41/C74/H46) and required for T6SS mediated 269 killing (C41/C74/H46), we tried to complement the tssB-sfGFP ΔPAARΩ by plasmid 270 expression of PAARH14A, PAARC41A, PAARH46A or PAARC74A, as well as PAARC76A or 271 PAARH14AC76A. Strains producing PAARC41A, PAARH46A or PAARC74A showed diffuse 272 fluorescence confirming that these three residues are crucial for PAAR function in the T6SS 273 sheath assembly (Fig. 4BC). In contrast, wild type-like sheath assembly was observed by 274 complementing with PAARH14A, PAARC70A, PAARC72A, or PAARC76A which is consistent 275 with antibacterial competition assays indicating that these residues do not play an important 276 role in the function of PAAR protein (Fig. 4BC, Fig. S4E). Again, expression of the 277 PAARH14AC76A double mutant could not restore sheath assembly, suggesting that the presence 278 of at least H14 or C76 residue in PAAR is necessary for T6SS function. 279 280 PAAR is necessary for the correct localization of the T6SS baseplate, but not of the 281 membrane complex. 282 The membrane complex recruits the baseplate complex that is required for the coordinated 283 assembly of the tube and sheath components (8, 11, 13, 16). PAAR is required for the polymerization of T6SS sheath. 255 To determine at which step of the 284 T6SS biogenesis PAAR protein is required, we followed the localization of the membrane 285 complex component TssM and the baseplate component TssK by fluorescence microscopy. 286 We observed the wild type and PAAR mutant (ΔPAARΩKan) cells producing sfGFP-TssM or 287 TssK-sfGFP (Fig. 5). As described previously, in T6SS-active strains, the sfGFP-TssM and 288 TssK-sfGFP present foci at the membrane ((8, 11), Fig. 5). We noted that in the absence of 289 PAAR, TssM was correctly localized at the membrane, suggesting that PAAR is not required 290 for proper localization of sfGFP-TssM and thus for membrane complex formation (Fig. 5AB). 291 However, the localization of TssK-sfGFP became diffuse in the absence of PAAR, suggesting 292 that PAAR is required for proper formation of the baseplate and its recruitment to the 293 membrane complex (Fig. 5CD). Consequently, PAAR was also required for downstream 294 events – the TssB/C sheath assembly (Fig. 4A) and the T6SS-1 mediated killing (Fig. 3). 295 296 DISCUSSION 297 There is a long-standing debate about the necessity of the PAAR protein for the T6SS- 298 complementing with PAARH14A, PAARC70A, PAARC72A, or PAARC76A which is consistent 275 with antibacterial competition assays indicating that these residues do not play an important 276 role in the function of PAAR protein (Fig. 4BC, Fig. S4E). Again, expression of the 277 PAARH14AC76A double mutant could not restore sheath assembly, suggesting that the presence 278 of at least H14 or C76 residue in PAAR is necessary for T6SS function. 279 The membrane complex recruits the baseplate complex that is required for the coordinated 283 assembly of the tube and sheath components (8, 11, 13, 16). To determine at which step of the 284 T6SS biogenesis PAAR protein is required, we followed the localization of the membrane 285 complex component TssM and the baseplate component TssK by fluorescence microscopy. 286 We observed the wild type and PAAR mutant (ΔPAARΩKan) cells producing sfGFP-TssM or 287 TssK-sfGFP (Fig. 5). As described previously, in T6SS-active strains, the sfGFP-TssM and 288 TssK-sfGFP present foci at the membrane ((8, 11), Fig. 5). We noted that in the absence of 289 PAAR, TssM was correctly localized at the membrane, suggesting that PAAR is not required 290 for proper localization of sfGFP-TssM and thus for membrane complex formation (Fig. 5AB). PAAR is required for the polymerization of T6SS sheath. 255 291 However, the localization of TssK-sfGFP became diffuse in the absence of PAAR, suggesting 292 that PAAR is required for proper formation of the baseplate and its recruitment to the 293 membrane complex (Fig. 5CD). Consequently, PAAR was also required for downstream 294 events – the TssB/C sheath assembly (Fig. 4A) and the T6SS-1 mediated killing (Fig. 3). 295 contradictory results (28, 30-33). Most recently, it was concluded that PAAR is only 300 necessary when transporting toxic effectors (32). In this work we have employed a model 301 system that can tackle this question since (i) under well described conditions (ii) it has only 302 one T6SS firing complex (iii) which has a single PAAR protein that is (iv) uncoupled from 303 toxin loading. Using the well-studied EAEC model we have demonstrated that PAAREAEC 304 protein is required for antibacterial activity mediated by the T6SS-1. Our results indicate that 305 PAAREAEC is indispensable for T6SS-mediated killing by participating in the upstream event 306 – the recruitment of the baseplate that allows polymerization of contractile sheath of T6SS 307 (Fig. 5). 308 contradictory results (28, 30-33). Most recently, it was concluded that PAAR is only 300 necessary when transporting toxic effectors (32). In this work we have employed a model 301 system that can tackle this question since (i) under well described conditions (ii) it has only 302 one T6SS firing complex (iii) which has a single PAAR protein that is (iv) uncoupled from 303 toxin loading. Using the well-studied EAEC model we have demonstrated that PAAREAEC 304 protein is required for antibacterial activity mediated by the T6SS-1. Our results indicate that 305 PAAREAEC is indispensable for T6SS-mediated killing by participating in the upstream event 306 – the recruitment of the baseplate that allows polymerization of contractile sheath of T6SS 307 (Fig. 5). 308 Ten years ago, Shneider and colleagues have first reported structures of a small protein PAAR 309 that sharpens the T6SS spike (28). It was then proposed to stabilize the baseplate hub protein 310 VgrG, by nucleating the folding of its trimers, or to regulate the incorporation of the VgrG 311 into the T6SS machinery (28). However, we have found that the steady-state levels of 312 VgrGEAEC protein are not significantly different without the PAAREAEC, and that the 313 overproduction of VgrGEAEC in the absence of PAAREAEC does not restore the T6SS-1 314 assembly and activity (Fig. PAAR is required for the polymerization of T6SS sheath. 255 S5, Fig. S6). Similarly, the intracellular level of VgrG2 is not 315 impaired in the absence of PAAR proteins in Serratia marcescens (30). Nevertheless, we 316 have previously observed that purified VgrGEAEC without PAAREAEC tends to precipitate in 317 some buffers (21) and therefore we cannot disregard the idea that PAAR could facilitate the 318 folding of the VgrG trimer or its incorporation into the baseplate (28). It is important to 319 mention that previous PAAR structures were obtained in chimeric complexes that were 320 prepared using a soluble fragment of T4 gp5 β-helix and grafting the terminal β-helices of 321 various VgrG spikes. Our attempts to crystallize PAAR from EAEC, alone or in complex 322 with the gp5C part of VgrG were unsuccessful. Nevertheless, our protein interaction studies 323 supported by cryo-EM density maps docked with AlphaFold2 model presented in this study 324 Ten years ago, Shneider and colleagues have first reported structures of a small protein PAAR 309 that sharpens the T6SS spike (28). It was then proposed to stabilize the baseplate hub protein 310 VgrG, by nucleating the folding of its trimers, or to regulate the incorporation of the VgrG 311 into the T6SS machinery (28). However, we have found that the steady-state levels of 312 VgrGEAEC protein are not significantly different without the PAAREAEC, and that the 313 overproduction of VgrGEAEC in the absence of PAAREAEC does not restore the T6SS-1 314 assembly and activity (Fig. S5, Fig. S6). Similarly, the intracellular level of VgrG2 is not 315 impaired in the absence of PAAR proteins in Serratia marcescens (30). Nevertheless, we 316 have previously observed that purified VgrGEAEC without PAAREAEC tends to precipitate in 317 some buffers (21) and therefore we cannot disregard the idea that PAAR could facilitate the 318 folding of the VgrG trimer or its incorporation into the baseplate (28). It is important to 319 mention that previous PAAR structures were obtained in chimeric complexes that were 320 prepared using a soluble fragment of T4 gp5 β-helix and grafting the terminal β-helices of 321 various VgrG spikes. Our attempts to crystallize PAAR from EAEC, alone or in complex 322 with the gp5C part of VgrG were unsuccessful. PAAR is required for the polymerization of T6SS sheath. 255 Nevertheless, our protein interaction studies 323 supported by cryo-EM density maps docked with AlphaFold2 model presented in this study 324 provide ultimate proof of the localization of PAAR protein at the top of the β-prism of the 325 gp5-domain of VgrG (Fig. 1). The spike complex of the bacteriophage T4 adopts highly 326 similar structure where a small conical gp5.4 protein sits on top of the β-prim of the gp5C 327 (12). Strikingly, similar structures found in other phages such as P2 or Phi92 sharpen the apex 328 of their needle by tying the ends of the gp5 β-helix into the knot that overall resembles the 329 conical PAAR protein (Fig. 6) (35). Moreover, all these needle end structures are supported 330 by the coordinated metal at the far end of the sharp structures (Fig. 6). We unambiguously 331 showed that the purified PAAREAEC protein contains zinc. At least two conserved cysteines 332 and one histidine, and likely a second histidine are involved in the binding of this zinc atom. 333 Their essentiality in vivo (C41, C74 and H46, Fig. 3, Fig. 4) supports the crucial role of metal 334 coordinated tip structure in the T6SS function. While the AlphaFold2 model of PAAR shows 335 histidine residues oriented slightly outward, it is likely that they constitute the coordination 336 center for the zinc atom and are hence pulled inwards to yield compact and sharp tip (Fig. 2). 337 Similarly, 3 histidine and 1 cysteine residues were shown to be involved in zinc binding in the 338 homologous PAAR proteins from Vibrio or UPEC and in the gp5.4 protein of phage T4 (12, 339 28). In the case of Francisella PAAR-like T6SS tip protein IglG, 4 cysteines were shown to 340 be important for binding of iron or zinc (31). Interestingly, the apex domains of gp138 of 341 Phi92 phage, gpV of P2 phage, and gpV of the R2 pyocin all contain iron, hexacoordinated by 342 6 histidines symmetrically provided by 3 monomers (Fig. 6, PDB:4s3; (35, 36)). In the light 343 of these observations, our results further support the role of metal coordinating tip structures 344 in sharpening spikes of membrane attacking contractile systems. PAAR is required for the polymerization of T6SS sheath. 255 345 Whil h h i i h h ffi i i i f h b i d dil While the sharp tip might ensure the efficient piercing of the membrane it does not readily 346 explain the requirement of the PAAR for the assembly of T6SSEAEC. PAAR could be required 347 for the recruitment of the spike to the membrane complex. In this case, the sharp tip 348 maintained by the zinc atom may be important for this function. Interestingly, expression of 349 While the sharp tip might ensure the efficient piercing of the membrane it does not readily 346 explain the requirement of the PAAR for the assembly of T6SSEAEC. PAAR could be required 347 for the recruitment of the spike to the membrane complex. In this case, the sharp tip 348 maintained by the zinc atom may be important for this function. Interestingly, expression of 349 the VgrGEAEC-Tle1EAEC complex yielded virtually all spike complexes stuck head-to-head 350 through the hydrophobic blunt end of the VgrG β-prisms as observed by cryo-EM (Fig. 1D, 351 Fig. S1B). These interactions involve the hydrophobic patch otherwise covered by PAAR 352 ((21), Fig. 1, Fig. S1B). Indeed, the presence of PAAREAEC seems to prevent this aberrant 353 behavior of VgrGEAEC (Fig. 1DE, Fig. S1A). Strikingly, the same head-to-head interaction 354 occurs in the case of T4 phage gp5 needle when produced alone. Without the PAAR homolog 355 gp5.4, the β- prism of gp5 dimerizes through its C-terminal hydrophobic tips and these 356 dimers of trimers can form in two different orientations (37). We could also observe different 357 2D classes of twin VgrGEAEC-Tle1EAEC complexes likely representing continuous or 60° 358 rotated interactions of β-prisms (Fig. S1B). These observations further suggest that the 359 amyloid-like structure of gp5 domain must be concluded to achieve a closed sharp end. 360 Indeed, formation of a baseplate and its attachment to the membrane complex could be 361 corrupted in the case of self-dimerization of VgrGEAEC trimers. Moreover, this proposed role 362 of PAAR protein in T6SS assembly could explain previous results showing that gp27 and 363 gp5-OB domain of VgrG are sufficient for T6SS assembly and firing (20, 38). Indeed, 364 without its β-prism, the needle-less trimeric VgrG may not dimerize this way and thus PAAR 365 may not be required for T6SS assembly in this case. PAAR is required for the polymerization of T6SS sheath. 255 366 Of note, despite the addition of PAAR protein, no interpretable density was again observed 367 for the base of VgrG corresponding to the gp27-gp5 OB fold -like domains (21). Only one 368 cryo-EM 2D class of particles displayed a distinct triangular shape structure at the base of the 369 needle, suggesting that the flexibility of this domain is not stabilized by the presence of 370 PAAR (Fig. S1AB). On the contrary, in the presence of PAAR most of the cryo-EM 2D 371 classes were missing densities for one, two or three Tle1 toxins. Closer observations revealed 372 that the EM densities around the middle of the gp5 structure were frequently visible and 373 overall more intense. The missing toxin densities are therefore likely a result of the flexibility. 374 It is possible that the head-to-head dimer of VgrG-Tle1 fixes the Tle1 toxins in a more rigid 375 conformation than the VgrG-Tle1-PAAR complex. In particular, the TTR domain of VgrG 376 that is hanging on a flexible linker might be less constrained in the presence of PAAR than in 377 the head-to-head VgrG-Tle1 dimer. Gel filtrations consistently show uniform peak for VgrG- 378 Tle1-PAAR complex and Tle1 seems to be in a stoichiometric equilibrium with VgrG based 379 on band intensities in SDS-PAGE gels (Fig S7). This suggests that Tle1 toxins are efficiently 380 loaded on the spike but are likely more flexible than previously observed. This flexibility 381 might play an important role when encountering the target and liberating the toxin from spike. 382 To conclude, we suggest that while the overall large structure of the T6SS firing tube 383 is built on the hexameric symmetry, it has to be resolved in order to be sealed and sharpened. 384 First symmetry transition is enabled by the VgrG-trimer that starts with pseudo-hexameric 385 symmetry at its base and wraps it up to a trimeric β-prism. Further, PAAR protein initiates by 386 a pseudo-trimeric symmetry that connects to the trimeric β-prism. Towards the extremity 387 PAAR is sealed into a sharp end with the help of a metal coordinated by conserved cysteines 388 and histidines. The importance of such architecture is supported by the fact that the closure of 389 the puncturing tip with the help of metal coordinating center is structurally conserved across 390 contractile injection systems that carry gp5-like β-prism domains (12, 28, 31, 35, 36). PAAR is required for the polymerization of T6SS sheath. 255 In 391 some, but not all cases, the final conical tip is encoded as a separate protein. One has to take 392 into account, that in the case of T6SS, such separation provides an additional anchoring point 393 for toxins. Hence, the separate ORF coding for the tip of puncturing device might have been 394 evolutionary favored. Nevertheless, as demonstrated in this work, even without carrying toxic 395 domains, sharpening the T6SS tip by PAAR protein is essential for the assembly and activity 396 of the T6SS machinery in EAEC. 397 398 MATERIALS AND METHODS 399 Bacterial strains, growth conditions and chemicals. 400 Strains used in this study are listed in Table S1. Escherichia coli DH5ɑ, BL21(DE3) and 401 W3110 were used for cloning procedures, protein production and as a prey for antibacterial 402 competition assays, respectively. EAEC strain 17-2 and its isogenic derivatives were used for 403 functional studies. Cells were grown at 37°C with aeration in LB with antibiotics when 404 required (ampicillin (100 µg/ml), kanamycin (50 µg/ml) or chloramphenicol (30 µg/ml)) or in 405 T6SS-1-inducing medium (SIM; M9 minimal medium, 0.2% glycerol, 1 µg/ml vitamin B1, 406 100 µg/ml casamino acids, 10% LB, supplemented or not with 1.5% Bacto agar) (39). Gene 407 expression was induced by the addition of 0.02–0.2% of L-arabinose (Sigma-Aldrich) for 408 pBAD (40) and by 0.5 or 1 mM of isopropyl-thio-b-D-galactopyranoside (IPTG; Eurobio) for 409 pETDuet-1, pRSFDuet-1 and pACYCDuet-1 (Novagen) derivative vectors. 410 411 Cloning procedures, plasmid and strain constructions. 412 The plasmids and primers (obtained from Merck or IDT) used in this study are listed in Table 413 S1. Cloning was performed by standard restriction-ligation procedures – DNA fragments 414 coding for PAAR and VgrG were amplified from EAEC 17-2 chromosomal DNA using Q5 415 high-fidelity DNA polymerase (New England Biolabs), PCR fragments were purified on 416 NucleoSpin Gel and PCR Clean-up columns (Macherey-Nagel), digested as recommended by 417 the manufacturer (NEB), and purified before ligation. Recombinant plasmids were 418 transformed into E. coli DH5α cells and constructions were verified by colony-PCR and by 419 DNA sequencing (Eurofins) after extraction of the plasmids using Wizard Plus SV Minipreps 420 kit (Promega). 421 For pACYC-PAARFL construct, the sequence encoding PAAR was amplified by PCR using 422 5-NdeI-PAAR and 3-XhoI-PAARFL primers introducing NdeI and XhoI sites, respectively, 423 and cloned into the pACYCDuet-1 (Novagen) multiple cloning site 2. The 3- XhoI -PAARFL 424 Bacterial strains, growth conditions and chemicals. 400 Strains used in this study are listed in Table S1. Escherichia coli DH5ɑ, BL21(DE3) and 401 W3110 were used for cloning procedures, protein production and as a prey for antibacterial 402 competition assays, respectively. EAEC strain 17-2 and its isogenic derivatives were used for 403 functional studies. Cells were grown at 37°C with aeration in LB with antibiotics when 404 required (ampicillin (100 µg/ml), kanamycin (50 µg/ml) or chloramphenicol (30 µg/ml)) or in 405 T6SS-1-inducing medium (SIM; M9 minimal medium, 0.2% glycerol, 1 µg/ml vitamin B1, 406 100 µg/ml casamino acids, 10% LB, supplemented or not with 1.5% Bacto agar) (39). Gene 407 expression was induced by the addition of 0.02–0.2% of L-arabinose (Sigma-Aldrich) for 408 pBAD (40) and by 0.5 or 1 mM of isopropyl-thio-b-D-galactopyranoside (IPTG; Eurobio) for 409 pETDuet-1, pRSFDuet-1 and pACYCDuet-1 (Novagen) derivative vectors. 410 411 Cloning procedures, plasmid and strain constructions. 412 The plasmids and primers (obtained from Merck or IDT) used in this study are listed in Table 413 S1. Cloning was performed by standard restriction-ligation procedures – DNA fragments 414 coding for PAAR and VgrG were amplified from EAEC 17-2 chromosomal DNA using Q5 415 high-fidelity DNA polymerase (New England Biolabs), PCR fragments were purified on 416 NucleoSpin Gel and PCR Clean-up columns (Macherey-Nagel), digested as recommended by 417 the manufacturer (NEB), and purified before ligation. Recombinant plasmids were 418 transformed into E. coli DH5α cells and constructions were verified by colony-PCR and by 419 DNA sequencing (Eurofins) after extraction of the plasmids using Wizard Plus SV Minipreps 420 kit (Promega). 421 For pACYC-PAARFL construct, the sequence encoding PAAR was amplified by PCR using 422 5-NdeI-PAAR and 3-XhoI-PAARFL primers introducing NdeI and XhoI sites, respectively, 423 and cloned into the pACYCDuet-1 (Novagen) multiple cloning site 2. The 3- XhoI -PAARFL 424 For pACYC-PAARFL construct, the sequence encoding PAAR was amplified by PCR using 422 5-NdeI-PAAR and 3-XhoI-PAARFL primers introducing NdeI and XhoI sites, respectively, 423 and cloned into the pACYCDuet-1 (Novagen) multiple cloning site 2. The 3- XhoI -PAARFL 424 primer introduces a sequence encoding a FLAG-tag to allow in-frame fusion of PAAR with a 425 C-terminal FLAG extension. For pET-HSUMO-PAAR construct, the sequence encoding 426 PAAR was amplified by PCR using primers 5-BmtI-PAAR and 3-HindIII-PAAR introducing 427 BmtI and HindIII sites, respectively, and cloned into the pETDuet-HIS-SUMO multiple 428 cloning site. 429 primer introduces a sequence encoding a FLAG-tag to allow in-frame fusion of PAAR with a 425 C-terminal FLAG extension. For pET-HSUMO-PAAR construct, the sequence encoding 426 PAAR was amplified by PCR using primers 5-BmtI-PAAR and 3-HindIII-PAAR introducing 427 BmtI and HindIII sites, respectively, and cloned into the pETDuet-HIS-SUMO multiple 428 cloning site. 429 The pBAD-PAARVSVG was constructed by megapriming. The 3pBAD-4537-VSVG primer 430 introduces a sequence encoding a VSVG-tag to allow in-frame fusion of PAAR with a C- 431 terminal VSVG extension. Site directed mutagenesis of PAAR was performed on pACYC- 432 PAARFLAG , pETDuet His-SUMO-PAAR or pBAD33-PAARVSVG by QuickChange PCR- based 433 targeted mutagenesis using complementary pairs of oligonucleotides and the PfuTurbo high- 434 fidelity polymerase (Agilent Technologies). Cloning procedures, plasmid and strain constructions. 412 435 For strain construction, the PAAR gene (EC042_4537) was deleted into the EAEC 17-2 wild- 436 type strain, the tssB-sfGFP, the tssK-sfGFP and the sfGFP-tssM using a modified one-step 437 inactivation procedure (41) as previously described (42) using pKD4 plasmid and 438 oligonucleotide pairs DEL-4537-5-DW/DEL-4537-3-DW. Kanamycin resistant clones were 439 selected and verified by colony-PCR and sequencing of the region (Eurofins genomics). 440 441 For strain construction, the PAAR gene (EC042_4537) was deleted into the EAEC 17-2 wild- 436 type strain, the tssB-sfGFP, the tssK-sfGFP and the sfGFP-tssM using a modified one-step 437 inactivation procedure (41) as previously described (42) using pKD4 plasmid and 438 oligonucleotide pairs DEL-4537-5-DW/DEL-4537-3-DW. Kanamycin resistant clones were 439 selected and verified by colony-PCR and sequencing of the region (Eurofins genomics). 440 441 SDS PAGE and Western blot analyzes. 442 Sodium Dodecyl Sulfate-Poly Acrylamide Gel Electrophoresis (SDS-PAGE) was performed 443 using standard protocols. Proteins were stained using InstantBlue™ (Sigma-Aldrich) or 444 transferred onto 0.2 m nitrocellulose membrane (Amersham Protran). Immunoblots were 445 probed with anti-Strep-Tag Classic (clone Strep-tag II, Bio-Rad catalogue #MCA2489), anti- 446 FLAG (clone M2, Sigma-Aldrich catalogue #F3165), anti-His-Tag (clone 1B7G5, Proteintech 447 catalogue #66005-1-Ig), anti-VSVG (clone P5D4; Sigma-Aldrich) or anti-RecA (clone M2, 448 Sigma-Aldrich catalogue #F3165) primary monoclonal antibodies, and alkaline phosphatase- 449 conjugated goat anti-mouse secondary antibodies, and revealed in alkaline buffer (pH 9) using 450 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT) in the presence of 451 10 mM MgCl2. 452 conjugated goat anti-mouse secondary antibodies, and revealed in alkaline buffer (pH 9) using 450 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT) in the presence of 451 10 mM MgCl2. 452 Protein production and pull-down assays. 454 An overnight culture of E. coli BL21(DE3) co-transformed with pACYC-PAARFL (or 455 pACYC-PAARFL derivatives) and pET-SVgrG (or pET-SVgrG derivatives) was diluted 1/100 456 into 50 ml of LB supplemented with the required antibiotics, grown to A600 =0.8 and induced 457 with 1 mM IPTG for 18 h at 16°C. Cells were pelleted by centrifugation at 5000× g, 458 resuspended in buffer A (50 mM Tris–HCl pH8.5, 150 mM NaCl, 1 mM TCEP, cOmplete 459 EDTA-free protease inhibitor cocktail (Sigma)). After cell lysis by sonication, the cell extract 460 was cleared by centrifugation for 45 min at 15 000 × g and incubated for 1h at 4°C with 461 gentle mixing with 100 μl of Strep-Tactin Superflow resin (IBA Technology) previously 462 equilibrated in buffer A. The resin was washed 5 times with 300 μl of buffer A and the 463 SVgrG-PAARFL were eluted with 100 μl of buffer B (50 mM Tris–HCl pH8.5, 150 mM NaCl, 464 1 mM TCEP, 2.5 mM desthiobiotin). Ten microliters of the elution fractions were separated 465 by 12.5%-acrylamide SDS-PAGE, stained using InstantBlue™ (Sigma-Aldrich) or analyzed 466 by Western blot with Strep-Tag Classic or anti-FLAG antibodies. 467 For the production of Tle1-VgrG-PAAR complex, BL21(DE3) was co-transformed with 468 pRSF-Tle1H, pET-SVgrG and pACYC-PAARFL plasmids. SVgrG-Tle1H-PAARFL complex 469 was then produced and purified using the same double step affinity procedure as for the 470 purification of the SVgrG-Tle1H complex described in (21). 471 Cryo-EM sample preparation, data collection and modelling. 473 Cryo-EM sample preparation, data collection and modelling. 473 Quantifoil Cu300 mesh R2/2 grids were glow discharged at 0.2 mbar vacuum, 2mA current 474 for 45 seconds (in an ELMO glow discharge system (Cordouan Technologies). Four μl of 475 protein samples at concentration of 0.5 ml/ml were blotted and plunge-frozen in liquid ethane 476 using Vitrobot instrument (FEI) at 4°C and 100% humidity, using blot force 0 and blotting 477 time 3s. Grids were transferred to liquid nitrogen for storage. Observation and data were 478 collected with Talos Arctica (FEI) 200 kV microscope equipped with K2 camera (Gatan). 479 Data were collected at 45k magnification, pixel size of 0.93 Å, with 4.4 s exposition and total 480 dose of 50 e-/Å with the help of SerialEM version 3.8 software. 2180 movies were collected 481 for VgrG-Tle1 complex (without PAAR) dataset. 3237 movies were collected for VgrG-Tle1- 482 PAAR dataset. Data was processed with CryoSPARC v4.0.0. Particle picking was assisted by 483 4 iterations of Topaz (43) training for 20 epochs, each time resubmitting best quality particles 484 for new iteration of training. For VgrG-Tle1 (without PAAR) dataset: 256,139 particles were 485 extracted with 550-pixels box size. For VgrG-Tle1-PAAR dataset: 436,572 particles were 486 extracted with 400-pixels box size. Best defined 2D classes were used for generation 4 ab- 487 initio models and best models were refined using heterologous refinement. For high 488 resolution VgrG-Tle1-PAAR map, particles used for best class refinement were again used to 489 generate 4 ab initio models and best model was then refined using best defined 2D classes. 490 Final step of non-uniform refinement with 31,647 particles was used to gain resolution in best 491 regions. 492 VgrG-Tle1-PAAR model was generated as follows: gp5 domain of VgrG (3 copies) and 493 PAAR were co-folded using AlphaFold2 (44, 45). Using UCSF ChimeraX (46), the best 494 ranked AlphaFold2 model was aligned to VgrG-Tle1 model (PDB:6SJL, (21)) via the gp5 495 domains. This model was then fitted into cryo-EM density maps (46). 496 His-SUMO-PAAR purification and samples preparation for ICP-OES. 498 Overnight cultures of E. coli BL21(DE3) transformed with pETDuet-HSUMO-PAAR or its 499 mutated derivatives were diluted 1/100 into 2L of LB supplemented with the required 500 antibiotics. Cells were grown to an A600 =0.8 and induced with 1 mM IPTG for 18 h at 501 16°C. Cells were pelleted by centrifugation at 5000× g, resuspended in buffer C (20 mM 502 HEPES pH8, 250 mM NaCl, 1 mM TCEP, cOmplete EDTA-free protease inhibitor cocktail 503 (Sigma) 100 μg/ml DNase I, 100 μg/ml lysozyme, and 10 mM MgCl2). After cell lysis using 504 an Emulsiflex-C5 (Avestin), the cell extract was cleared by centrifugation for 40 min at 40 505 000 × g and incubated for 1h at 4°C with gentle mixing with 1 ml of Strep-Tactin Superflow 506 resin (IBA Technology) previously equilibrated in buffer C. The resin was washed 5 times 507 with 8 ml of buffer C and the H-SUMO-PAAR was eluted with 100 μl of buffer D (20 mM 508 HEPES pH8, 250 mM NaCl, 1 mM TCEP, 500 mM imidazole. Ten microliters of the elution 509 fractions were separated by 15%-acrylamide SDS-PAGE, stained using InstantBlue™ or 510 transferred onto nitrocellulose membrane and immunodetected with anti- His-Tag antibodies. 511 512 Fluorescence Microscopy. 527 Overnight cultures grown in LB medium with the appropriate antibiotics were diluted 1/100 528 into SIM to an A600 = 0.8, then 500µl of the culture was centrifuged and resuspended in fresh 529 SIM to a A600 = 10. Cells were spotted on a thin 2% agarose pad, prepared with SIM medium 530 in Frame-Seal™ slide chambers (Bio-Rad), and covered with a cover slip. After 20 min at 531 28°C, Hilo fluorescence microscopy was performed with a Nikon Eclipse Ti2 microscope 532 equipped with a 100x NA 1.45 Ph3 objective, an Orca-Fusion digital camera (Hamamatsu), a 533 perfect focus system, and an Ilas2 TIRF/FRAP module (Gataca Systems). Fluorescence 534 images were acquired with an exposure time of 100 ms for phase contrast, sfGFP-TssM, 535 TssK-sfGFP and 50 ms for TssB- sfGFP. The experiments were conducted in triplicate and a 536 representative result is shown. The microscopy images were analyzed using ImageJ 537 (http://imagej.nih.gov/ij/). 538 Metal quantification by Inductively Coupled Plasma analysis - Optical Emission 513 Spectrometry (ICP-OES). 514 50 to 300 µM of proteins in 500 µl of PBS were diluted in 500 µl of 69% nitric acid. Samples 515 were boiled for 40 minutes and 4 ml of 3% nitric acid was added to each sample. These 516 samples were analyzed for copper, nickel, zinc, and iron content with an ICAPTM 6000 series 517 optical emission spectrometer (Thermo Scientific). Serial dilutions of pure copper, nickel, 518 iron, and zinc standard solutions were used for calibration. Results are expressed in 519 percentage of total proteins in samples, each value representing the mean of three technical 520 replicates from different fractions of two to three different purification preparations. As a 521 control, 500µl of purification buffer was submitted to the same metal content analysis. For 522 statistical analysis, one-way ANOVA followed by Dunnett’s multiple comparisons test was 523 performed using GraphPad Prism version 9.3.1 for macOS (GraphPad Software, San Diego, 524 California USA). 525 performed using GraphPad Prism version 9.3.1 for macOS (GraphPad Software, San Diego, 524 California USA). 525 Antibacterial competition assay by the colorimetric method. 540 At 558 A600 = 0.8, the predator strain and the prey were mixed at 4:1 ratio and spotted on SIM-agar 559 plates supplemented 0,2% arabinose. After 4h of incubation at 37 °C, each spot was 560 resuspended in 1 ml of selective medium for prey and 100 µl of a 10-fold dilution were added 561 to 100 µl of selective medium in 96-wells microplate (Thermofisher, Nunc). Cultures were 562 grown for 15 hours at 37°C with agitation and A600 was measured each 5 minutes using a 563 microplate reader (TECAN). A time of emergence (Te) considered as time required for each 564 sample to reach A600 = 0.4 of was calculated. Each value represents the mean of three 565 technical replicates from at least three biological replicates and were compared between 566 strains by a One-way ANOVA followed by Dunnett’s multiple comparisons test performed 567 using GraphPad Prism (version 9.3.1 for macOS, GraphPad Software, San Diego, California 568 USA). Negative controls contained only the prey or the predator strain. EAEC 17-2 wild-type 569 strain was used as positive control for W3110 prey lysis. 570 Production in EAEC ΔPAARΩKan of the different derivatives of PAAR protein from pBAD 571 representative image is presented. For quantification of CPR, slices of agar-containing spots 549 were vortexed in 500 µL PBS and further centrifuged at 8,000× g for 1 min to pellet cells and 550 agar. 100µl of the supernatant were dropped into a 96-well plate. After addition of 10 μl of 2 551 mM CPRG into the supernatant, absorbance at λ = 572 was measured every 30 seconds for 1 552 hour with a TECAN. 553 Antibacterial competition assay by the colorimetric method. 540 Overnight cultures grown in LB medium with the appropriate antibiotics were diluted 1/100 541 into SIM medium to an A600 = 0.4. Then, 50 µM of IPTG was added in the culture of W3110 542 bacterial prey cells to induce the production of β-galactosidase. WT or mutated PAAR genes 543 expression was induced from pBAD33-PAARVSVG using 0,2% arabinose. At A600 = 0.8, the 544 predator and the prey cultures were mixed at 1:4 ratio and spotted on SIM-agar plates 545 supplemented with 50 µM of IPTG and 0,2 % arabinose. After 2 hours of incubation at 37 °C, 546 drops of 2 mM CPRG solution were deposited on the cell spots. Pictures were taken after the 547 emergence of the coloration. Experiment was performed at least three times and a 548 Overnight cultures grown in LB medium with the appropriate antibiotics were diluted 1/100 541 into SIM medium to an A600 = 0.4. Then, 50 µM of IPTG was added in the culture of W3110 542 bacterial prey cells to induce the production of β-galactosidase. WT or mutated PAAR genes 543 expression was induced from pBAD33-PAARVSVG using 0,2% arabinose. At A600 = 0.8, the 544 predator and the prey cultures were mixed at 1:4 ratio and spotted on SIM-agar plates 545 supplemented with 50 µM of IPTG and 0,2 % arabinose. After 2 hours of incubation at 37 °C, 546 drops of 2 mM CPRG solution were deposited on the cell spots. Pictures were taken after the 547 emergence of the coloration. Experiment was performed at least three times and a 548 representative image is presented. For quantification of CPR, slices of agar-containing spots 549 were vortexed in 500 µL PBS and further centrifuged at 8,000× g for 1 min to pellet cells and 550 agar. 100µl of the supernatant were dropped into a 96-well plate. After addition of 10 μl of 2 551 mM CPRG into the supernatant, absorbance at λ = 572 was measured every 30 seconds for 1 552 hour with a TECAN. 553 554 Antibacterial competition assay by the emergence time method. 555 Overnight cultures grown in LB medium with the appropriate antibiotics were diluted 1/100 556 into SIM medium to an A600 = 0.4. Arabinose 0,2% was added into predator strains cultures to 557 induce PAAR gene expression from pBAD33-PAARVSVG (or pBAD33-PAARVSVG mutants). Antibacterial competition assay by the emergence time method. 555 Overnight cultures grown in LB medium with the appropriate antibiotics were diluted 1/100 556 into SIM medium to an A600 = 0.4. Arabinose 0,2% was added into predator strains cultures to 557 induce PAAR gene expression from pBAD33-PAARVSVG (or pBAD33-PAARVSVG mutants). At 558 A600 = 0.8, the predator strain and the prey were mixed at 4:1 ratio and spotted on SIM-agar 559 plates supplemented 0,2% arabinose. After 4h of incubation at 37 °C, each spot was 560 resuspended in 1 ml of selective medium for prey and 100 µl of a 10-fold dilution were added 561 to 100 µl of selective medium in 96-wells microplate (Thermofisher, Nunc). Cultures were 562 grown for 15 hours at 37°C with agitation and A600 was measured each 5 minutes using a 563 microplate reader (TECAN). A time of emergence (Te) considered as time required for each 564 sample to reach A600 = 0.4 of was calculated. Each value represents the mean of three 565 technical replicates from at least three biological replicates and were compared between 566 strains by a One-way ANOVA followed by Dunnett’s multiple comparisons test performed 567 using GraphPad Prism (version 9.3.1 for macOS, GraphPad Software, San Diego, California 568 USA). Negative controls contained only the prey or the predator strain. EAEC 17-2 wild-type 569 strain was used as positive control for W3110 prey lysis. 570 Production in EAEC ΔPAARΩKan of the different derivatives of PAAR protein from pBAD 571 plasmids used for antibacterial competition and fluorescence microscopy observations were 572 controlled by Western Blotting using anti-VSVG antibodies. 573 Production in EAEC ΔPAARΩKan of the different derivatives of PAAR protein from pBAD 571 plasmids used for antibacterial competition and fluorescence microscopy observations were 572 controlled by Western Blotting using anti-VSVG antibodies. 573 574 Protein stability assay. 575 Overnight cultures of E. coli DH5ɑ transformed with pBAD18-PAARVSVG and pOK- 576 VgrGFLAG or empty pBAD18 and pOK-VgrGFLAG were diluted 1/100 into 50 ml of LB 577 medium supplemented with the required antibiotics, grown at 37°C to an A600 of 0.6 and 578 induced with 0,5 mM IPTG and 0,2% Arabinose. After 1h, spectinomycin (100 µg/ml) and 579 chloramphenicol (40 µg/ml) were added. Before and after the induction, at 1 h, 2 h and 3 h 580 after the inhibition of protein synthesis, 2 ml of cultures were collected, centrifuged for 5 min 581 at 6000 × g and supplemented with 60 or 100 µl of 4X SDS-PAGE loading dye with β- 582 mercaptoethanol. Proteins were separated by SDS-PAGE and transferred onto nitrocellulose 583 membrane for immunodetection with anti-VSVG, or anti-FLAG or anti-RecA monoclonal 584 antibodies, followed by recognition by secondary antibodies coupled to the Alkaline 585 Phosphatase. Intensity of the bands were measured using BioRad Imager. 586 Computer algorithms. 588 EAEC PAAR orthologs (EC042_4537; NCBI-protein ID: CBG37359) were retrieved using 589 EAEC PAAR orthologs (EC042_4537; NCBI-protein ID: CBG37359) were retrieved using 589 BlastP analysis against the KEGG genes database and aligned with MultiAlin tool (47, 48). 590 We thank Eric Cascales and the members of the Cascales research group for insightful 593 discussions, especially Thierry Doan and Yoann G. Santin for microscopy; Boris Taillefer for 594 CPRG killing and Maïalène Chabalier for cloning of pETDuet-FLAGVgrG. We thank Stéphane 595 Canaan for the use of BioRad Imager and software. We thank Moly Ba, Isabelle Bringer, 596 Annick Brun, Olivier Uderso and Mathilde Valade for technical assistance. This work was 597 We thank Eric Cascales and the members of the Cascales research group for insightful 593 discussions, especially Thierry Doan and Yoann G. Santin for microscopy; Boris Taillefer for 594 CPRG killing and Maïalène Chabalier for cloning of pETDuet-FLAGVgrG. We thank Stéphane 595 Canaan for the use of BioRad Imager and software. We thank Moly Ba, Isabelle Bringer, 596 Annick Brun, Olivier Uderso and Mathilde Valade for technical assistance. This work was 597 We thank Eric Cascales and the members of the Cascales research group for insightful 593 discussions, especially Thierry Doan and Yoann G. Santin for microscopy; Boris Taillefer for 594 CPRG killing and Maïalène Chabalier for cloning of pETDuet-FLAGVgrG. We thank Stéphane 595 Canaan for the use of BioRad Imager and software. We thank Moly Ba, Isabelle Bringer, 596 Annick Brun, Olivier Uderso and Mathilde Valade for technical assistance. This work was 597 supported by the Aix-Marseille Université (AMU), the Centre National de la Recherche 598 Scientifique (CNRS) and grants from the Agence Nationale de la Recherche (ANR-14-CE14- 599 0006 and ANR-18-CE15-0013 to LJ). 600 supported by the Aix-Marseille Université (AMU), the Centre National de la Recherche 598 Scientifique (CNRS) and grants from the Agence Nationale de la Recherche (ANR-14-CE14- 599 0006 and ANR-18-CE15-0013 to LJ). 600 SGB was supported by a doctoral fellowship from the French Ministère de l'Enseignement 601 Supérieur, de la Recherche et de l'Innovation. NF was supported by the ANR-14-CE14-0006 602 grant. MB was supported by the ANR-18-CE15-0013. D.J. was supported by a post-doctoral 603 fellowship from the Fondation pour la Recherche Médicale (SPF201809007142). 604 Author contributions 605 S.G.B.: conceptualization, formal analysis, investigation, visualization, writing - original 606 draft. N.F.: conceptualization, investigation, writing - review and editing. M.I.: formal 607 analysis, investigation, resources, writing - review and editing. M.B. and EGF: formal 608 analysis, investigation. R.F.: conceptualization, formal analysis, resources, supervision. D.J.: 609 conceptualization, formal analysis, investigation, supervision, visualization, writing - original 610 draft. L.J.: conceptualization, formal analysis, funding acquisition, investigation, resources, 611 supervision, validation, visualization, writing - original draft. 612 613 Conflict of Interests 614 The authors declare that they have no conflict of interest. 615 616 REFERENCES 617 618 S.G.B.: conceptualization, formal analysis, investigation, visualization, writing - original 606 draft. N.F.: conceptualization, investigation, writing - review and editing. M.I.: formal 607 analysis, investigation, resources, writing - review and editing. M.B. and EGF: formal 608 analysis, investigation. 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Deciphering key features in protein structures with the new 770 48. Robert X, Gouet P. 2014. Deciphering key features in protein structures with the new 770 END i t N l i A id R 42 W320 W324 htt //d i /10 1093/ / k 316 771 48. Robert X, Gouet P. 2014. Deciphering key features in protein structures with the new 770 ENDscript server. Nucleic Acids Res 42: W320 –W324. https://doi.org/10.1093/nar/gku316 771 772 Fig. 1. PAAR interacts with the VgrG needle domain and caps the VgrG-Tle1 complex. 783 (A) Schematic representation of the EAEC VgrG1 protein and the truncated variants used for 784 pull-down assays with PAAR. The different domains and their boundaries are indicated – 785 base domain gp27; OB fold domain; β helical domain gp5-C; H - helix; DUF2345 domain; 786 TTR - transthyretin like domain). (B) Pull-down assays. Lysates of BL21(DE3) cells co- 787 producing FLAG-tagged PAAR (PAARFL) and Strep-tag II-tagged VgrG (SVgrG) or 788 truncated versions of VgrG (SVgrG ΔTTR, SVgrG ΔNeedle) were loaded (L) on a Strep- 789 Tactin column. After washing steps, desthiobiotin-eluted (E) and flow through (FT) fractions 790 were analyzed by SDS-PAGE and Coomassie blue staining (upper panel), immunoblot using 791 anti-StrepII (middle panel) and anti-FLAG (lower panel). (C) VgrG-PAAR-Tle1 complex 792 purification. SDS-PAGE analysis and Coomassie blue staining (top panel), immunoblot 793 using anti-StrepII (upper middle panel), anti-His (lower middle panel) and anti-FLAG 794 (bottom panel) of the purification steps. Cell lysate (L) co-producing Strep-tag II-VgrG 795 (SVgrG), Tle1-6xHis (Tle1H) and PAAR-FLAG (PAARFL) was loaded onto a StrepTrapHP 796 column. After washing (W) the material was eluted with desthiobiotin directly into a HisTrap 797 column. The imidazole-eluted material (E) was then loaded onto a Superose 6 10/300 gel 798 filtration column (GF). 10 μl of each fraction was loaded onto the gel. The molecular weight 799 markers (in kDa) are indicated on the left and the positions of the different proteins are 800 indicated on the right. (D) Cryo-EM density maps of VgrG-Tle1 and VgrG-Tle1-PAAR 801 producing FLAG-tagged PAAR (PAARFL) and Strep-tag II-tagged VgrG (SVgrG) or 788 truncated versions of VgrG (SVgrG ΔTTR, SVgrG ΔNeedle) were loaded (L) on a Strep- 789 Tactin column. After washing steps, desthiobiotin-eluted (E) and flow through (FT) fractions 790 were analyzed by SDS-PAGE and Coomassie blue staining (upper panel), immunoblot using 791 anti-StrepII (middle panel) and anti-FLAG (lower panel). (C) VgrG-PAAR-Tle1 complex 792 purification. SDS-PAGE analysis and Coomassie blue staining (top panel), immunoblot 793 using anti-StrepII (upper middle panel), anti-His (lower middle panel) and anti-FLAG 794 (bottom panel) of the purification steps. Cell lysate (L) co-producing Strep-tag II-VgrG 795 (SVgrG), Tle1-6xHis (Tle1H) and PAAR-FLAG (PAARFL) was loaded onto a StrepTrapHP 796 column. After washing (W) the material was eluted with desthiobiotin directly into a HisTrap 797 column. The imidazole-eluted material (E) was then loaded onto a Superose 6 10/300 gel 798 filtration column (GF). Fig. 1. PAAR interacts with the VgrG needle domain and caps the VgrG-Tle1 complex. 783 Each value represents the mean of three 827 technical replicates of two to four different fractions of two (C41A, C74A) to three different 828 purifications preparations (WT, C41AC74A). 829 Fig. 1. PAAR interacts with the VgrG needle domain and caps the VgrG-Tle1 complex. 783 10 μl of each fraction was loaded onto the gel. The molecular weight 799 markers (in kDa) are indicated on the left and the positions of the different proteins are 800 indicated on the right. (D) Cryo-EM density maps of VgrG-Tle1 and VgrG-Tle1-PAAR 801 complex. Left: Cryo-EM density maps of VgrG needle bound to Tle1 (in green) 802 corresponding to a (VgrG)3-(Tle3)3 tip-to-tip dimer complex. Right: VgrG needle bound to 803 Tle1 in the presence of the PAAR protein (in yellow). In the presence of PAAR, most 804 particles correspond to VgrG needle bound to one or two Tle1 (represented here) and no 805 VgrG needle tip-to-tip dimerization. 2D classes are presented in Fig. S1. (E) VgrG-Tle1- 806 PAAR complex model. Alphafold2 co-folding model of the needle domain of VgrG (3 807 copies, green, yellow, red) with PAAR (blue) was aligned with the VgrG-Tle1 model 808 (PDB:6SJL) merging on the needle domain. This model was fitted in the cryo-EM density 809 maps (4.2 Å, two Tle1 visible). 810 Fig. 2. PAAR binds a zinc atom involving conserved His and Cys residues. 812 Fig. 2. PAAR binds a zinc atom involving conserved His and Cys residues. 812 (A) Alignment of amino acid sequences of PAAREAEC (EC042_4537), PAAR from E. coli 813 CFT073 (c1882) and Vibrio cholerae (VCA0105). PAAR motifs are shown in green boxes, 814 His and Cys residues involved in zinc binding characterized in (28) are indicated in black, 815 below the alignment. H14, C41, H46 and C74, the predicted residues involved in metal 816 binding in PAAREAEC are indicated in red above the alignment. (B) Alphafold2 model of 817 PAAR highlighting His14, Cys41, His46 and Cys74 predicted metal binding residues. PAAR 818 was co-modelled with VgrG needle part and only PAAR is shown here. (C) SDS-PAGE 819 followed by Coomassie blue staining (upper panel) and immunoblot using anti-His-Tag 820 (lower panel) of purified wild type His-SUMO-PAAR proteins and mutated versions (His-SUMO- 821 PAARC41A, His-SUMO-PAARC74A and His-SUMO-PAARC41AC74A). The molecular weight markers 822 (in kDa) are indicated on the left. (D) Zinc content analysis using ICP-OES of purified His- 823 SUMO-PAAR (WT) and cysteine mutants (His-SUMO-PAARC41A, C41A; His-SUMO-PAARC74A, 824 C74; His-SUMO-PAARC41AC74A, C41AC74A). Statistical significance was calculated using 825 Ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test using GraphPad 826 Prism (GraphPad Software). * p<0.0001. Fig. 3. PAAR is necessary for T6SS-1 mediated killing. 831 The T6SS-1 function of 843 the WT, ΔPAARΩ and ΔPAARΩ complemented with PAAR or PAAR-Cys and His-mutant 844 strains was tested by assessing their ability to kill W3110 E. coli K12 bacterial prey. Killing 845 efficiency was quantified by measuring the time (in hours) needed for prey cells exposed to 846 predator cells to regrow in selective media to the A600 = 0.4. Statistical significance was 847 calculated using Ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test 848 using GraphPad Prism. p<0.0001; * p=0.0009; ** p= 0.0092; p= 0.0401, ns=0.9711 849 (ΔT6SS vs. ΔPAARΩ ), ns=0.9967 (PAAR+ vs. H14A). 850 (B) Antibacterial competition assay by the Emergence time method. The T6SS-1 function of 843 the WT, ΔPAARΩ and ΔPAARΩ complemented with PAAR or PAAR-Cys and His-mutant 844 strains was tested by assessing their ability to kill W3110 E. coli K12 bacterial prey. Killing 845 efficiency was quantified by measuring the time (in hours) needed for prey cells exposed to 846 predator cells to regrow in selective media to the A600 = 0.4. Statistical significance was 847 calculated using Ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test 848 using GraphPad Prism. * p<0.0001; * p=0.0009; ** p= 0.0092; p= 0.0401, ns=0.9711 849 (ΔT6SS vs. ΔPAARΩ ), ns=0.9967 (PAAR+ vs. H14A). 850 Fig. 3. PAAR is necessary for T6SS-1 mediated killing. 831 (A) Antibacterial competition assay by the Colorimetric method. The T6SS-1 function of the 832 WT, ΔPAARΩ and ΔPAARΩ complemented with PAAR (PAAR+) or PAAR Cys and His 833 mutant strains (using pBAD33-PAARVSVG and corresponding PAAR Cys and His mutant 834 plasmids derivatives) was tested by assessing their ability to kill W3110 E. coli K12 bacterial 835 prey. Killing efficiency was monitored by observing degradation of yellow CPRG into purple 836 CPR by free ß-galactosidase released from lysed prey cells after being exposed to the predator 837 (A) Antibacterial competition assay by the Colorimetric method. The T6SS-1 function of the 832 WT, ΔPAARΩ and ΔPAARΩ complemented with PAAR (PAAR+) or PAAR Cys and His 833 mutant strains (using pBAD33-PAARVSVG and corresponding PAAR Cys and His mutant 834 plasmids derivatives) was tested by assessing their ability to kill W3110 E. coli K12 bacterial 835 prey. Killing efficiency was monitored by observing degradation of yellow CPRG into purple 836 CPR by free ß-galactosidase released from lysed prey cells after being exposed to the predator 837 cells. CPR absorbance (A572nm, upper graph) was measured from the spots (lower panel). The 838 means of three biological replicates is indicated. The error bars represent standard deviation. 839 Statistical significance was calculated using Ordinary one-way ANOVA followed by 840 Dunnett’s multiple comparisons test using GraphPad Prism (GraphPad Software). * 841 p<0.0001. 842 cells. CPR absorbance (A572nm, upper graph) was measured from the spots (lower panel). The 838 means of three biological replicates is indicated. The error bars represent standard deviation. 839 Statistical significance was calculated using Ordinary one-way ANOVA followed by 840 Dunnett’s multiple comparisons test using GraphPad Prism (GraphPad Software). 841 p<0.0001. 842 (B) Antibacterial competition assay by the Emergence time method. The T6SS-1 function of 843 the WT, ΔPAARΩ and ΔPAARΩ complemented with PAAR or PAAR-Cys and His-mutant 844 strains was tested by assessing their ability to kill W3110 E. coli K12 bacterial prey. Killing 845 efficiency was quantified by measuring the time (in hours) needed for prey cells exposed to 846 predator cells to regrow in selective media to the A600 = 0.4. Statistical significance was 847 calculated using Ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test 848 using GraphPad Prism. p<0.0001; * p=0.0009; ** p= 0.0092; *p= 0.0401, ns=0.9711 849 (ΔT6SS vs. ΔPAARΩ ), ns=0.9967 (PAAR+ vs. H14A). 850 (B) Antibacterial competition assay by the Emergence time method. Fig. 4. PAAR is necessary for T6SS assembly. 852 (C) Quantification of the number of extended sheaths per cell from the 865 strains tssB-sfGFP + pBAD (BsfGFP), tssB-sfGFP ΔPAARΩ + pBAD (BsfGFPΔPAAR), tssB- 866 sfGFP ΔPAARΩ + pBAD-PAARVSVG (PAAR+), and tssB-sfGFP ΔPAARΩ transformed with 867 pBAD33-PAARH14A-VSVG (H14A), pBAD33-PAARC41A-VSVG (C41A), pBAD33-PAARH46A-VSVG 868 (H46A) or pBAD33-PAARC74A-VSVG (C74A) analyzed in A and B. The total number of 869 analyzed cells (n) from 3 independent biological replicates is indicated. The error bars 870 represent standard deviation. 871 on the left (scale bar = 2 μm). Fluorescence images of TssB-sfGFP (green) are shown on the 863 right. Cells are outlined in white, white arrows indicate dynamic sheath assembly and/or 864 contraction events. (C) Quantification of the number of extended sheaths per cell from the 865 strains tssB-sfGFP + pBAD (BsfGFP), tssB-sfGFP ΔPAARΩ + pBAD (BsfGFPΔPAAR), tssB- 866 sfGFP ΔPAARΩ + pBAD-PAARVSVG (PAAR+), and tssB-sfGFP ΔPAARΩ transformed with 867 pBAD33-PAARH14A-VSVG (H14A), pBAD33-PAARC41A-VSVG (C41A), pBAD33-PAARH46A-VSVG 868 (H46A) or pBAD33-PAARC74A-VSVG (C74A) analyzed in A and B. The total number of 869 analyzed cells (n) from 3 independent biological replicates is indicated. The error bars 870 represent standard deviation. 871 Fig. 4. PAAR is necessary for T6SS assembly. 852 (A) Fluorescence microscopy of EAEC tssB-sfGFP strains transformed with the empty vector 853 pBAD33 (tssB-sfGFP + pBAD), EAEC tssB-sfGFP ΔPAARΩ Kan transformed with the empty 854 vector pBAD33 (tssB-sfGFP ΔPAARΩ + pBAD) and EAEC tssB-sfGFP ΔPAARΩ 855 complemented in trans with pBAD33-PAARVSVG plasmid (tssB-sfGFP ΔPAARΩ + pBAD- 856 PAAR). Phase contrast images are shown on the left (scale bar = 2 μm). Fluorescence 857 images of TssB-sfGFP (green) and membrane dye FM4-64 (red) are shown on the right. 858 Pictures taken every 30 sec are shown from left to right. White arrows indicate dynamic 859 sheath assembly and/or contraction events. (B) Fluorescence microscopy of EAEC tssB- 860 sfGFP ΔPAARΩKan strain transformed with pBAD33-PAARH14A-VSVG , pBAD33-PAARC41A- 861 VSVG, pBAD33-PAARH46A-VSVG or pBAD33-PAARC74A-VSVG . Phase contrast images are shown 862 (A) Fluorescence microscopy of EAEC tssB-sfGFP strains transformed with the empty vector 853 pBAD33 (tssB-sfGFP + pBAD), EAEC tssB-sfGFP ΔPAARΩ Kan transformed with the empty 854 vector pBAD33 (tssB-sfGFP ΔPAARΩ + pBAD) and EAEC tssB-sfGFP ΔPAARΩ 855 complemented in trans with pBAD33-PAARVSVG plasmid (tssB-sfGFP ΔPAARΩ + pBAD- 856 PAAR). Phase contrast images are shown on the left (scale bar = 2 μm). Fluorescence 857 images of TssB-sfGFP (green) and membrane dye FM4-64 (red) are shown on the right. 858 Pictures taken every 30 sec are shown from left to right. White arrows indicate dynamic 859 sheath assembly and/or contraction events. (B) Fluorescence microscopy of EAEC tssB- 860 sfGFP ΔPAARΩKan strain transformed with pBAD33-PAARH14A-VSVG , pBAD33-PAARC41A- 861 VSVG, pBAD33-PAARH46A-VSVG or pBAD33-PAARC74A-VSVG . Phase contrast images are shown 862 on the left (scale bar = 2 μm). Fluorescence images of TssB-sfGFP (green) are shown on the 863 right. Cells are outlined in white, white arrows indicate dynamic sheath assembly and/or 864 contraction events. (C) Quantification of the number of extended sheaths per cell from the 865 strains tssB-sfGFP + pBAD (BsfGFP), tssB-sfGFP ΔPAARΩ + pBAD (BsfGFPΔPAAR), tssB- 866 sfGFP ΔPAARΩ + pBAD-PAARVSVG (PAAR+), and tssB-sfGFP ΔPAARΩ transformed with 867 pBAD33-PAARH14A-VSVG (H14A), pBAD33-PAARC41A-VSVG (C41A), pBAD33-PAARH46A-VSVG 868 (H46A) or pBAD33-PAARC74A-VSVG (C74A) analyzed in A and B. The total number of 869 analyzed cells (n) from 3 independent biological replicates is indicated. The error bars 870 represent standard deviation. 871 on the left (scale bar = 2 μm). Fluorescence images of TssB-sfGFP (green) are shown on the 863 right. Cells are outlined in white, white arrows indicate dynamic sheath assembly and/or 864 contraction events. Fig. 5. PAAR is necessary for TssK but not TssM localization. 873 888 Ribbon diagrams of VgrG(gp5C)-PAAR from EAEC, gp5-gp5.4 from T4 phage (PDB:5iv 889 gpV from P2 phage (PDB:3aqj), gp138 from Phi92 phage (PDB:3pqi) and gpV from 890 pyocin (PDB:4s36). All β-prism spikes are capped and the tip is closed with coordina 891 metal. Structures are colored by chain. 892 893 894 Fig. 6. A conical tip structure consolidated by a metal atom is conserved in different 887 phages and bacterial injection systems. 888 Fig. 5. PAAR is necessary for TssK but not TssM localization. 873 (A) Fluorescence microscopy analysis of sfGFP-tssM and sfGFP-tssM ΔPAARΩKan. Phase 874 contrast images are shown on the left (scale bar = 2 μm) and fluorescence images of sfGFP 875 (green) are shown on the right. Cells are outlined in white, white arrows indicate fluorescent 876 foci localized at the membrane. (B) Quantification of the number of foci per cell from sfGFP- 877 tssM and sfGFP-tssM ΔPAARΩKan. The total number of analyzed cells (n) from 3 878 independent biological replicates is indicated. The error bars represent standard deviation. (C) 879 Fluorescence microscopy analysis of tssK-sfGFP and tssK-sfGFP ΔPAARΩKan. Phase 880 contrast images are shown on the left (scale bar = 2 μm) and fluorescence images of sfGFP 881 (green) are shown on the right. Cells are outlined in white, white arrows indicate fluorescent 882 foci localized at the membrane. (D) Quantification of the number of foci per cell from tssK- 883 sfGFP and tssK-sfGFP ΔPAARΩKan. The total number of analyzed cells (n) from 3 884 independent biological replicates is indicated. The error bars represent standard deviation. 885 886 (A) Fluorescence microscopy analysis of sfGFP-tssM and sfGFP-tssM ΔPAARΩKan. Phase 874 contrast images are shown on the left (scale bar = 2 μm) and fluorescence images of sfGFP 875 (green) are shown on the right. Cells are outlined in white, white arrows indicate fluorescent 876 foci localized at the membrane. (B) Quantification of the number of foci per cell from sfGFP- 877 tssM and sfGFP-tssM ΔPAARΩKan. The total number of analyzed cells (n) from 3 878 independent biological replicates is indicated. The error bars represent standard deviation. (C) 879 Fluorescence microscopy analysis of tssK-sfGFP and tssK-sfGFP ΔPAARΩKan. Phase 880 contrast images are shown on the left (scale bar = 2 μm) and fluorescence images of sfGFP 881 (green) are shown on the right. Cells are outlined in white, white arrows indicate fluorescent 882 foci localized at the membrane. (D) Quantification of the number of foci per cell from tssK- 883 sfGFP and tssK-sfGFP ΔPAARΩKan. The total number of analyzed cells (n) from 3 884 independent biological replicates is indicated. The error bars represent standard deviation. 885 Fig. 6. A conical tip structure consolidated by a metal atom is conserved in differ 887 phages and bacterial injection systems. Fig. 6. A conical tip structure consolidated by a metal atom is conserved in different 887 phages and bacterial injection systems. 888 Ribbon diagrams of VgrG(gp5C)-PAAR from EAEC, gp5-gp5.4 from T4 phage (PDB:5iv5), 889 gpV from P2 phage (PDB:3aqj), gp138 from Phi92 phage (PDB:3pqi) and gpV from R2 890 pyocin (PDB:4s36). All β-prism spikes are capped and the tip is closed with coordinated 891 metal. Structures are colored by chain. 892 894
https://openalex.org/W4283591814	https://www.research-collection.ethz.ch/bitstream/20.500.11850/557099/2/1167-12084-1-PB.pdf	English	null	Magical Realism for Water Governance Under Power Asymmetries in the Aracataca River Basin, Colombia	The international journal of the commons	2,022	cc-by	12,493	ETH Library ETH Library Author(s): Author(s): Fonseca-Cepeda, Valentina; Castillo-Brieva, Daniel; Baquero-Bernal, Luis; Rodríguez, Luz Angela; Steiner, Eliane; Garcia-Ulloa, John Permanent link: https://doi.org/10.3929/ethz-b-000557099 Rights / license: Creative Commons Attribution 4.0 International Rights / license: g Creative Commons Attribution 4.0 International KEYWORDS: Social-ecological systems; institutional arrangements; water governance; role- playing game; Aracataca river; Colombia ABSTRACT CORRESPONDING AUTHOR: Valentina Fonseca-Cepeda Pontificia Universidad Javeriana, CO The Ciénaga Grande de Santa Marta is a wetland complex characterized by its high productivity and different systems of biodiversity use associated with water. The Ciénaga receives fresh water from rivers descending from the Sierra Nevada de Santa Marta. Changes in the water dynamics of these rivers since the early twentieth century have been causing serious social and ecological changes in the region. The Aracataca river is one of these rivers that showed a drastic change in its water availability. In this research we study the relation between cooperation and hydrological dynamics that shapes the water governance system in this basin. The study combines quantitative data obtained from the hydrological description and qualitative information derived from interviews and a role-playing game workshop, which was analyzed from a social-ecological perspective. The analysis shows that the historical management of water, characterized by conflicts between individual and collective interests, power asymmetries, and the heterogeneity between actors, has established a problematic scenario. Our analysis at river basin scale showed difficulties in water governance regardless of water annual variability, thus requiring structural changes that allow the development of coordinated processes toward collective action. This research identifies elements that can enrich the management discourses of the Aracataca river basin and the Ciénaga as a whole, highlighting the importance of understanding environmental issues as problems of common pool resources. Pontificia Universidad Javeriana, CO v.fonseca@javeriana.edu.co Originally published in: Originally published in: International Journal of the Commons 16(1), https://doi.org/10.5334/ijc.1167 This page was generated automatically upon download from the ETH Zurich Research Collection. For more information, please consult the Terms of use. RESEARCH ARTICLE Magical Realism for Water Governance Under Power Asymmetries in the Aracataca River Basin, Colombia VALENTINA FONSECA-CEPEDA DANIEL CASTILLO-BRIEVA LUIS BAQUERO-BERNAL LUZ ANGELA RODRÍGUEZ ELIANE STEINER JOHN GARCIA-ULLOA Author affiliations can be found in the back matter of this article RESEARCH ARTICLE Magical Realism for Water Governance Under Power Asymmetries in the Aracataca River Basin, Colombia VALENTINA FONSECA-CEPEDA DANIEL CASTILLO-BRIEVA LUIS BAQUERO-BERNAL LUZ ANGELA RODRÍGUEZ ELIANE STEINER JOHN GARCIA-ULLOA Author affiliations can be found in the back matter of this article RESEARCH ARTICLE Magical Realism for Water Governance Under Power Asymmetries in the Aracataca River Basin, Colombia VALENTINA FONSECA-CEPEDA DANIEL CASTILLO-BRIEVA LUIS BAQUERO-BERNAL LUZ ANGELA RODRÍGUEZ ELIANE STEINER JOHN GARCIA-ULLOA Author affiliations can be found in the back matter of this article RESEARCH ARTICLE ABSTRACT INTRODUCTION to study the key challenges around water governance in the Aracataca River basin following a social-ecological systems’ (SES) perspective (McGinnis & Ostrom, 2014). Water management in the basin is described as a Common Pool Resource (CPR) problem (Ostrom, 1990) while the river basin constitutes the spatial and institutional domain of the study. We describe its hydrological characteristics using supply and demand parameters in dry, wet, and normal years and analyze the institutional arrangements and their relationship with water dynamics. We also study the relationships between the hierarchies of the governance systems to understand power relations and identify decision-making mechanisms with different levels of formality (Stoker, 1998). Wetland ecosystems play an essential role in maintaining ecological processes which yield benefits for different human groups that include the regulation of the chemical properties of water, the buffering of atmospheric phenomena (Mitsch & Gosselink, 2000), and the production of hydrobiological resources (Vilardy-Quiroga et al., 2011). The dynamics of global change and human pressure on the natural resources provided by wetlands have accelerated the degradation of these ecosystems, affecting their natural dynamics of variability and the provision of ecosystem services (Koch et al., 2009). These transformation processes are particularly relevant in a country such as Colombia, where wetland ecosystems represent an extensive portion of the continental territory (Vilardy-Quiroga & Cortés-Duque, 2014). The Ciénaga Grande de Santa Marta (CGSM) is the largest coastal wetland in the country, consisting of a complex of estuarine lagoons that is part of the deltaic plain of the Magdalena River in the Colombian Caribbean. This wetland system receives fresh water from the Magdalena River, the main river in the country, and rivers descending from the Sierra Nevada de Santa Marta (SNSM). It is characterized by its high biodiversity and various livelihoods closely linked to water, especially associated with fishing (Carvajalino- Fernández et al., 2017). It is worth noting here that in vertical water systems such as river basins and irrigation systems, tailenders hardly receive water in the necessary quantities, or they do it at a very high cost (Shivakoti & Ostrom, 2003). Spatial asymmetries among those who are near or distant from the resource (Ostrom & Gardner, 1993) are largely due to the conditions of subtraction of water as a CPR, as the use given by one person affects the possibility of other people to use the resource (Tang, 1992). TO CITE THIS ARTICLE: Fonseca-Cepeda, V., Castillo- Brieva, D., Baquero-Bernal, L., Rodríguez, L. A., Steiner, E., & Garcia-Ulloa, J. (2022). Magical Realism for Water Governance Under Power Asymmetries in the Aracataca River Basin, Colombia. International Journal of the Commons, 16(1), pp. 155–172. DOI: https://doi. org/10.5334/ijc.1167 Fonseca-Cepeda et al. International Journal of the Commons DOI: 10.5334/ijc.1167 156 INTRODUCTION This occurs in the Aracataca River basin, where the problem of collective action in the irrigation system of palm oil producers (headenders), among others, limits the access to water of the fishing community (tailenders). Thus, we focus the analysis on the lower basin because the collective action problems become most evident there given the conditions of vulnerability and reduced capacity of fishermen to transform the system. The CGSM main transformations began to occur at the beginning of the 20th century (Vilardy-Quiroga, 2009). These have consisted of hypersalinization of soils, mangroves dieback, modifications in the structure of fish communities, water pollution, and changes in sediment circulation (Botero & Mancera-Pineda, 1996). The degraded environmental conditions of the CGSM are a consequence of economic development policies with poor understanding of the system, demographic changes, and political instability associated with the armed conflict (Torres et al., 2016; Vilardy-Quiroga et al., 2011). STUDY AREA The Aracataca River basin, with an area of 166.258 ha (Figure 1), is part of the alluvial plain formed by rivers that descend from the western slope of the Sierra Nevada de Santa Marta (SNSM) and flow into the CGSM (Bernal, 1996), in the Department of Magdalena.1 The influence of the trade winds of the Northern Hemisphere creates arid and semiarid conditions, which are strongly affected by the El Niño Southern Oscillation (Vilardy-Quiroga & González- Novoa, 2011). The rainfall seasonality in the lagoon delta complex is characterized by a rainy season that extends from April to November, with one peak in May or June, a local minimum in July and the most intense peak in October, and by a dry season that runs from December to March. The spatially well-differentiated rainfall regime in the lagoon delta complex determines a water deficit gradient that grows from -211.6 mm yr-1 in the southern to -1146.8 mm yr-1 in northern areas (Blanco et al., 2006). The Aracataca river is one of the freshwater inlets One of the CGSM’s tributary rivers is the Aracataca River. Agricultural production is the main driver of change in the river basin, especially palm oil. Land occupation has followed a dual scheme of peasant and enclave economies, with intensive use of water for irrigation (Cabeza, 2014). The historical context of water management has led to inequalities in water distribution, conflicts over its use, and the reduction of the flow at the mouth of the river which has affected the fishing population in the lower basin. The purpose of the paper is to study the relation between cooperation and hydrological dynamics that shapes the water governance system in the Aracataca river basin. We follow a mixed-methods approach to integrate hydrological and institutional variables with the purpose The Aracataca river is one of the freshwater inlets (17.4 m3s-1) that influence the seasonal variation in the water level and contribute to oxygenation, circulation of Fonseca-Cepeda et al. International Journal of the Commons DOI: 10.5334/ijc.1167 157 Figure 1 Aracataca River Basin. The lower basin corresponds to the coastal plain (0–100 m.a.s.l.). Figure 1 Aracataca River Basin. The lower basin corresponds to the coastal plain (0–100 m.a.s.l.). Figure 1 Aracataca River Basin. The lower basin corresponds to the coastal plain (0–100 m.a.s.l.). Colombian government’s interest in positioning itself in the international market (Cabeza, 2014; Herrera & Romero, 1979). STUDY AREA The government developed irrigation systems with canals, dams, and reservoirs (Cabeza, 2014), leading to conflicts due to the unequal distribution of the resource, initially among farmers, settlers, and banana companies (LeGrand, 1983) and, more recently, among palm oil producers, banana producers and small-scale artisanal fishermen (Salzwedel et al., 2016). sediments, temperature variation and salinity levels of the southeastern part of the estuarine complex. These characteristics have significant effects on the composition and distribution of biological communities (Bernal & Betancur, 1996; Invemar, 2018). The flow of the river varies based on this seasonality (Hoyos et al., 2019). The occupation history of the region and the development models implemented have ignored this ecological complexity and the hydrological connection between the Aracataca river, the CGSM, and the sea, as well as its relationship with livelihoods and socioeconomic dynamics (Vilardy-Quiroga, 2009). This has caused changes in the hydrology of the river and therefore in the ecology of the CGSM (Botero & Mancera-Pineda, 1996; Botero & Salzwedel, 1999; Röderstein et al., 2014). This has been particularly evident in the lower basin, where agricultural practices have defined the strategies for water harvesting causing the most critical appropriation and provision problems as CPR and on the ecology of the CGSM. Actors within the Aracataca River who make decisions regarding water are diverse. In the upper part of the basin, Arhuaco indigenous communities predominate with a population of 22,134 people (DANE, 2005). In the middle part of the basin, coffee producers are the main economic actors, and in the lower basin, palm oil producers are organized in an association of irrigation district users, with 380 associates in 2019. Although banana producers are also in the basin, they occupy a very small portion in the watershed area and in the irrigation district users. At this point is located the municipal seat of Aracataca, with 30.149 people (DANE, 2005). At the mouth of the river on CGSM land is the Trojas de Cataca, a floating community of artisanal fishermen of 154 people (Aguilera, 2011), which Since the 18th century, the alluvial plain of the Aracataca River has had a strong agricultural tradition (Herrera & Romero, 1979). The introduction of commercial banana cultivation in 1887 (LeGrand, 1983) was motivated by the Fonseca-Cepeda et al. HYDROLOGICAL ANALYSIS For our hydrological analysis, we used the water scarcity index (1). This index represents the relationship between water use by different users and the surface water supply available for the same spatial and temporal resolutions (García et al., 2010). The index is reported as a percentage, indicating the relationship between total water demand and net water supply (Domínguez et al., 2010) and the degree of pressure on water supply and water supply vulnerability (IDEAM, 2015). Net water supply and total demand were calculated from the parameters of the following equation, where Ie is the scarcity index, Dt is the volume of total water demand, and On is the net water supply in the basin: 100% Dt Ie On = (1) (1) Different classes of water resource pressure are distinguished: high, when the demand reaches 41% or more of the water provided by the supply source; medium, when the demand is between 21 and 40%; moderate, when the demand is between 11 and 20%, and low, when the demand is below 10% (Rivera et al., 2004). STUDY AREA International Journal of the Commons DOI: 10.5334/ijc.1167 158 asymmetries and challenges faced by tailenders (Lam, 1998; Ostrom & Gardner, 1993; Shivakoti & Ostrom, 2003; Tang, 1992) and on prior knowledge about conflicts over water in this specific area, we decided to focus our analysis on the problem between palm oil producers and fishermen in the lower basin. Since the wellbeing of fishermen in the Trojas de Cataca community depends on upstream water management, our methodological tools focused on describing the dynamics of water use among palm oil producers. represent 20% of the original population that decided to return after a massacre executed by paramilitary groups in 2000 (CNMH, 2014). The situation of this community is critical, as 98% of the population consumes water with no treatment. Several cases of acute respiratory infections and acute diarrheal diseases have been reported, caused by the consumption of non-potable water, poor sewerage, and aqueduct service, as well as water pollution through agrochemical waste from palm crops (CNMH, 2014). Oil palm plantations in the region appeared around 1950, inheriting historical dynamics and conflicts over water (Rangel et al., 2009). Oil palm (Elaeis guineensis) has been successful in the area due to the high solar radiation and nutrient conditions of the soil. However, this crop requires high levels of rainfall. A secure irrigation is therefore essential for its productivity (Cabeza, 2014). The irrigation demand from this crop in the Colombian Caribbean is high compared to the national demand. This is due to the low water supply in the dry months (Mejía, 2000) and the poor efficiency of gravity or flood irrigation systems (Álvarez et al., 2007). The regional environmental authority formally regulates the use of water in the Aracataca basin by allocating water permits. Nevertheless, compliance with water regulation is low and illegal use of water is common. On the other hand, the office of National Natural Parks of Colombia is responsible for ecosystem conservation in the lower basin. METHODS We did a hydrological analysis of the Aracataca river basin and conducted fieldwork in Aracataca and Santa Marta during 2019. The purpose of the hydrological analysis was to describe the dynamics of the water in the basin while fieldwork consisted of semi-structured interviews and a role-playing game workshop (Steiner, 2020). The interviews were carried out to collect general information on water management in the region and issues within the watershed perceived by each type of actor. The role-playing game workshop allowed the analysis and synthesis of a socioecological problem while exploring the reactions of the participants to different configurations of the system. This mixed-methods approach had the purpose to articulate institutional and hydrological variables, in order to explore relationships between water catchment strategies and hydric dynamics of the basin. The information collected was integrated based on the SES analytical approach. Annex 1 presents the tools used to document the SES variables. We used a percentile-based approach to define dry and wet conditions from average monthly precipitation data of 39 pluviometric stations in the region of interest. The pluviometric data is available on the IDEAM’s (Institute of Hydrology, Meteorology, and Environmental Studies) Hydrometeorological Monitoring Network website (IDEAM, 2019). This approach, as explained by Steinemann et al. (2015), allows statistically consistent scenario creation processes capable of representing a wide range of precipitation conditions. We fitted precipitation data to parametric statistical distribution models of Pearson family type and then used the percentile 10 and 90 to respectively define dry and wet conditions. To define normal conditions, using the same precipitation dataset, we calculate the mean value of the fitted statistical distribution. A more detailed description of the followed fitting method can be found in Domínguez et al. (2008). The basin was delimited as a hydrological domain and as the unit for the analysis of the institutional arrangements. Based on a literature review on the topics of power The calculated average monthly precipitation in dry, normal, and wet conditions and average monthly Fonseca-Cepeda et al. International Journal of the Commons DOI: 10.5334/ijc.1167 159 of water related issues in the basin. Therefore, we preferred to implement few in-depth interviews with key actors that would provide detailed information, rather than having a large sample with redundant information. Interviews also were used for the adaptation of the role-playing game (RPG) called Upstream (Steiner, 2020). ROLE-PLAYING GAME WORKSHOP The RPG was implemented to approach the CPR problem from a socio-ecological perspective as this methodology allows identifying aspects of the problem that are not evident during an interview. Specifically, the role-playing game had more importance in the description of the governance variables, interactions, and outcomes of the SES, than in the characterization of the other subsystems (Annexes 1 and 3). For this purpose, we adapted an already existing RPG to represent the Aracataca river basin and its dynamics. The original RPG called Upstream was designed to represent water management in the Cravo Sur River basin, located in the Colombian Llanos. We used information obtained from our hydrological analysis, a literature review, and the semi-structured interviews to adapt and calibrate the RPG to the Aracataca River basin (for details see Steiner, 2020). Y X ETa = − (2) (2) We estimated the water demand (Mm3) by adding the urban and rural domestic demand (Ministerio de Ambiente Vivienda We estimated the water demand (Mm3) by adding the urban and rural domestic demand (Ministerio de Ambiente Vivienda y Desarrollo Territorial, 2003; Ministerio de Vivienda Ciudad y Territorio, 2010), the livestock demands for pig, cattle, and poultry consumption (DANE, 2016; Resolución 00011912 de 2019), agricultural demand (Allen et al., 1998; IDEAM, 2015), and industrial demand (Atencia, 2014; DAABON, 2019; Resolución 2263 de 2011; Resolución 4147 de 2018). The agricultural water demand was calculated using the cultivated area per crop in the water basin, a calculation that was made based on the reported areas per municipality by DANE (2016) and the relative area occupied by each municipality in the water basin. Permanent crops were assumed to have a constant crop coefficient (Kc) equal to its final growth stage, meanwhile transitory crops were assigned with a mean Kc to sum up the Kc differences in the crop stages. Field capacity was assumed as 50 mm because of the well-drained soils of the water basin (CORPAMAG, 2013). Industrial demand was represented by the water consumption of 6 oil mills present in the water basin. When the water demand of a specific oil mill was unknown, an average data completion was applied. Because of the data availability, we calculated water demand on an annual scale for 2019, the same year in which the institutional analysis was performed. ROLE-PLAYING GAME WORKSHOP The game modeled the action arena that involves the management of water for irrigation in the lower basin, proposing different configurations of water availability and possibilities for dialog, from which the responses of the actors were evaluated (Allington et al., 2018). Annex 3 illustrates the variables, and the observed elements present in the RPG. The RPG consists of a game board, which represents the river basin (Figure 2). Blue fields in the middle of the board represent the Aracataca River and small water tokens simulate the flow of water. At the end of the river, a green vessel represents the CGSM. The game setup includes the CGSM ecosystem and its need for freshwater inflow to uphold its ecological functions. Players are located along the board. Their goal is to capture water tokens, which they need to make their oil palm and banana farms work. To represent the upstream-downstream subtraction problem, players located in the upper part of the gameboard were allowed to capture water before those located in the lower part. The number of available water tokens varies in each round, representing the seasonal variation in the water supply (i.e., rainy and dry tested interspersed). Water storage infrastructure is not available by default in the game so, if it was identified by the players as a factor that needed to be solved, it had to be designed by them as a water management strategy. We used ArcGIS, ESRI (10.5) and Python (2.7 & 3.8) software (ESRI Inc., 2016; Python Software Foundation, 2020) to conduct our calculations and for spatial representation. Annex 2 shows the hydrological parameters used. METHODS temperature data from 41 climatological stations of IDEAM’s network were used as input variables to calculate the annual surface water supply, following the water balance equation (2), where Y is the runoff, X is the precipitation and ETa is the actual evapotranspiration. The actual evapotranspiration was calculated using the Thornthwaite-Mather’s method (García & Montoya, 1972). For both the precipitation and temperature data, the process of data selection and completion followed the methods presented in CAR (2015). SEMI-STRUCTURED INTERVIEWS We conducted 11 semi-structured interviews (Ander-Egg, 1995; Bonilla-Castro & Rodriguez, 1995) with managers of irrigation districts, leaders of the Arhuaco council, representatives of the oil palm guild, experts in palm certification, managers of coffee associations, municipal officials, palm oil producers, and representatives of the regional environmental authority. The purpose of the interviews was to capture different actors’ understanding The workshop was attended by nine participants, including palm oil producers, oil palm and banana technicians, one representative of a palm oil extractor and Fonseca-Cepeda et al. International Journal of the Commons DOI: 10.5334/ijc.1167 160 permits and conditions at his own discretion. Players were given five minutes to negotiate their permits. In the second situation, players had to discuss how to build and implement a watershed management plan. Five minutes were given for this too. And last, an intense drought was presented, where water tokens decreased. These three conditions represented changes in the rules of the game that forced players to design strategies to harvest water efficiently, to decide whether to leave available water to the tailenders, and to build agreements for water distribution. Table 1 shows the possibilities of action and decision by role under two seasons and three situations. representatives of an environmental NGO. All participants voluntarily agreed to be part of the activity. Eight of the participants adopted the role of producers, and one adopted the role of representative of the irrigation district who was responsible for distributing water among producers. Five rounds were played, in a span of three hours. In our workshop, we presented three situations: 1) negotiation of water harvesting permits, 2) watershed management plan, and 3) intense drought. In the first situation, producers must negotiate their water harvesting permits with the irrigation district representative. The person with this role could determine the limits of the Figure 2 RPG workshop in Aracataca. WATER AVAILABILITY SCENARIOS SITUATIONS ROLE GOAL DECISION OPTIONS Rainy Dry Negotiation of water permits Producer Catch water tokens to keep farms producing How much water to take How much water to negotiate Watershed management plan To leave water for tailenders or not To build and fulfill agreements or not Intense drought Representative of water district Distributing water among producers Permit limits assigned per player Distribute water equitably or not Table 1 Player roles, goals, and decision options along five rounds. 161 Fonseca-Cepeda et al. International Journal of the Commons DOI: 10.5334/ijc.1167 RESULTS SUPPLY, DEMAND, AND SCARCITY INDEX: RESOURCE UNITS AND RESOURCE SYSTEM (RU, RS) As to water demand on an annual scale, the total demand is equivalent to 103.14 Mm3. The scarcity index for the Aracataca River basin is moderate for normal years (11%), high for dry years (64%) and low in wet years (5%) (Domínguez et al., 2008; IDEAM, 2015) (Table 2). As seen in the map (Figure 3), the highest water supply values (more than 900 mm) are found above 100 m.a.s.l. in normal (A) and wet (B) scenarios, while the lowest water supply production is recorded below that altitude in both conditions, exactly where the oil palm landscape is located. This finding indicates that in the upper basin, precipitation is greater than the actual evapotranspiration, and there is always surface water in this part of the basin, while in the lower basin, the precipitation does not exceed the ETa at the annual scale, and thus, the supply is provided only by the water that runs off the SNSM. In wet conditions (B), water supply values are the highest, reaching more than 1800 mm in the upper basin and never acquiring a value of 0 mm in the lower basin. In dry conditions (C), however, water supply in the whole basin area is below 900 mm, mostly with a value of 0 mm. This indicates that in dry years, precipitation does not exceed actual evapotranspiration in any part of the basin, and the supply values do not exceed 400 mm in the upper basin. CHARACTERIZATION OF THE SOCIOECOLOGICAL SYSTEM AND INSTITUTIONAL ARRANGEMENTS We characterized the basin as a social-ecological system following Ostrom’s SESs framework using the results from the semi-structured interviews. Then, for the governance subsystems and interactions, we focused on the lower basin, given its relevance in the appropriation-provision problem among palm oil producers and fishermen. Social, economic, and political context (S) Water management is framed within a historical and political context that defines the forms of water use. First, governments at the departmental and municipal levels have promoted the development of the agricultural and industrial sectors without implementing specific strategies that regulate agricultural expansion as a function of water supply. In addition, economic strategies do not align with Figure 3 Spatially distributed annual water supply for normal (A), wet (B) and dry (C) conditions. Figure 3 Spatially distributed annual water supply for normal (A), wet (B) and dry (C) conditions. Fonseca-Cepeda et al. International Journal of the Commons DOI: 10.5334/ijc.1167 162 Average supply (Mm3) Normal 992.18 Dry 161.38 Wet 2047.66 Demand (Mm3) Domestic 2.26 Industrial 0.46 Livestock 0.58 Agricultural 99.84 TOTAL 103.14 Scarcity index Normal 11% Dry 64% Wet 5% Table 2 Summary of the hydrological analysis. Average supply (Mm3) Normal 992.18 Dry 161.38 Wet 2047.66 Demand (Mm3) Domestic 2.26 Industrial 0.46 Livestock 0.58 Agricultural 99.84 TOTAL 103.14 Scarcity index Normal 11% Dry 64% Wet 5% Table 2 Summary of the hydrological analysis. “The water problem has meant food security problems for the floating population as, due to lack of fresh water, fish have left, and people have to go further to fish”. Municipal representative, August 28, 2019. “The water problem has meant food security problems for the floating population as, due to lack of fresh water, fish have left, and people have to go further to fish”. Municipal representative, August 28, 2019. Thus, the extraction of water by the oil palm guild reduces water availability for the population of Trojas de Cataca, carrying with it changes in fishing resources. Actors (A) Actors involved in the Aracataca River basin are very diverse. Water access differences arise from infrastructures for water access, location and/or asymmetries in decision- making power. First, while palm oil producers have a consolidated irrigation infrastructure, fishermen do not have an aqueduct or sewage system. On the other hand, those actors located upper in the system access water first and in greater quantities than do those located in the lower part of the basin, which have the most negative impacts on fishermen, the tailenders in this water vertical system. Table 2 Summary of the hydrological analysis. planning instruments and protection figures that regulate the use of land, water, and biodiversity, such as the declaration of the CGSM as an Exclusive Reserve Zone for the artisanal extraction of fishery resources, or the watershed management plan (Decreto 1640 de 2012; Decreto 2811 de 1974). Additionally, the historical armed conflict in the region has been related to the establishment of illicit crops and drug trafficking routes in the SNSM foothills and the incursion of paramilitarism in the CGSM, which has led to distrust among actors and erosion of the social capital. Nevertheless, the most critical difference between actors is the asymmetry in decision-making power with large palm oil producers having much more power than small producers and fishermen of Trojas de Cataca. In the case of small palm oil producers, for instance, this is mainly because the board of directors of the water user association usually make decisions that benefit large producers. Here it is worth noting that the members of this board are appointed based on the favors related to the distribution of water and not on voting, thereby reducing the participation of some producers. Similarly, fishermen are disadvantaged compared to palm oil producers in the collective processes of construction and transformation of institutional arrangements because as there are not effective spaces for dialog and negotiation. Ensuring spaces for dialog and participation in decision- making regarding water management are important challenges in the basin. Different local initiatives have emerged since the 1980s to establish communication bridges based on the management of natural resources, for example, ProSierra, Fondo del Agua or ProCiénaga. However, the lack of continuity and guarantees of participating in those spaces prevent the consolidation of collective processes that consolidate a joint strategy for water governance. Governance system (GS) The regional environmental authority grants catchment permits to users’ associations, such as irrigation districts (Ley 41 de 1993). These permits determine the volume of water granted and a fee that must be paid for water used. The districts must distribute the granted amount of water to the affiliated producers. The amount of water each producer receives depends on the cultivated area. In the lower basin, for oil palm crops, the irrigation district is managed by a water user association. Irrigation districts are public entities administered under a private system of user associations (Decreto 1881 de 1994), in this case, palm oil producers. Distribution is conducted in shifts, where producers who have paid their fees are prioritized (Resolución 498 de 1997). The interviewees identified a clear relationship between water extraction of the Aracataca River and the dynamics of the CGSM. This relationship is revealed in the reduction in freshwater volume that reaches the wetland complex, mainly attributed to the infrastructure of the irrigation district. The lack of fresh water affects fish populations and the resource availability for floating communities. “Water does not reach the Ciénaga, and for that reason, you can no longer see the fish migration. Only on weekends does the floodgate [of the irrigation district] open, and that is when a little water is released”. Palm plantation technician in Aracataca, September 18, 2019. Fonseca-Cepeda et al. International Journal of the Commons DOI: 10.5334/ijc.1167 163 Water is managed under a primarily public scheme, which eventually acquires private characteristics for both individuals and groups. That is, water is a CPR with conditions of high rivalry and low exclusion, whose formal access is not open but regulated by the state as a public entity that temporarily grants private use rights to individuals or user associations. Any water uses in excess or not granted by the environmental authority is considered illicit. the riverbed and guaranteeing the collective rights of the population of Trojas de Cataca. The popular movement is considered an expression of constructing and modifying institutional arrangements, aimed at creating spaces for dialog, transforming conflicts, encouraging participation of different actors in decision-making, and developing monitoring and sanctioning strategies. “If it weren’t for this type of measure, nothing would happen. None of the measures of the regional environmental authority work. What works is now the follow-up that will be done for the popular movement from the national oversight entities. Governance system (GS) There has never been a judicial action of this type, and we may end up serving as a reference for the other watersheds that also deliver water to Ciénaga and find themselves in similar situations. There has never been, at least in the Department of Magdalena, a measure of protection of collective rights with respect to ecosystems in the framework of a popular movement”. Municipal representative, August 28, 2019. However, this regulation for the appropriation of water designed at the constitutional level is misaligned with what occurs at the collective and operational levels where informal and illegal institutional arrangements dominate water access. Such arrangements are characterized by extraction of greater water volumes than needed for cultivated areas, no-payment of tariffs, construction of infrastructure independent of the irrigation district and lack of monitoring and sanction. Furthermore, money transactions and favors in exchange for water outside the concessions granted are common. Thus, formal and informal governance systems overlap as illegal practices have been normalized. On one hand, formal institutions specify strategies for the appropriation and provision of water, such as rules that sanction individualistic behavior (Ley 1333 de 2009), that explicitly regulate the time frame for collection (Decreto 1541 de 1978) as well as the amount of water allowed (Acuerdo 193 de 2009; Ley 41 de 1993; Resolución 498 de 1997). On the other hand, in the field illegal practices have consolidated as the institutional arrangements for water management. This has resulted in the inequitable distribution of water, conflicts between small and large producers, erosion of trust and a shortage of water in Trojas de Cataca due to reduced flow at the mouth of the Aracataca river. However, it does not address the structural problem of the contradictory formal and informal governance systems. Since this movement is so new and due to the robustness of the dynamics of distrust and illegality, expectations about tangible outcomes of the popular movement and the potential of transforming the situation of action in the basin are unknown. Interactions (I) Interactions are manifested as emerging conflicts between users. In addition to the conflict among palm users and fishers (described above), there are also conflicts among palm producers associated with the distribution of water between large (more than 100 hectares) and small (less than 20 hectares) producers, which are more pronounced during the dry seasons of the year, when there is less rainfall, and dependence on irrigation becomes critical. Although, based on the water user association of the irrigation district, the criterion for water distribution is providing service to those who pay for it, small producers still feel that water is reserved for large producers, regardless of who pays or does not. One emerging response to this problem has been a pro- collective rights movement (span. acción popular) in the community of Trojas de Cataca. In Colombia, a popular movement is a legal mechanism that is exercised to prevent damage or stop violations of collective rights. These rights include the enjoyment of a healthy environment, ecological balance, the management and rational use of natural resources, the protection of areas of ecological importance, and access to a service infrastructure that guarantees public health (Ley 472 de 1998). The main purpose of the popular movement of Trojas de Cataca is to update the regulation of water use to guarantee a healthy environment for the floating population and to recover freshwater input to the CGSM. It is an initiative that appeals to the collective level but has effects at both the operational and constitutional levels because it involves actors and proposes actions at all three levels. “In this part of the basin, we always have water, but you know how it works; the big fish eats the small fish, and the big producers always have water […]. Large producers always have priority; they never run out of water”. Small palm producer, September 17, 2019. This movement, initiated in 2018, requests public and private entities at the local, regional, and national levels to take certain actions aimed at stopping the threats to Fonseca-Cepeda et al. International Journal of the Commons DOI: 10.5334/ijc.1167 164 Outcomes (O) sanctioned, while those in the lower basin stated that they ran out of water during the drought rounds. The most critical outcomes of the system are inequitable distribution of water, illegality, lack of cooperation, and reduced flow at the mouth of the river, with effects on the water and ecological dynamics of the CGSM. These results are mediated by the absence of spaces of dialog between actors, deficiencies in monitoring and sanctions, the exclusion of certain actors in the modification of institutional arrangements during the processes of decision-making, and contradictory overlapping governance systems. It is interesting to note that even when players saw that emerging institutional arrangements did not work, no new agreements or sanction strategies were discussed. This could have been related to the fact that only some players participated in the dialogue spaces and proposed strategies, while others remained passive and condescending with those who showed leadership. It is also interesting that this resembles the real-basin situation where breaking the formal rules is common and no new collective agreements were created to regulate water access. These limitations reduce the possibility that the different actors share their knowledge about the resource system, the problems perceived in the basin, the points of view about existing conflicts. This hinders the learning process and the needed collective action to modify the structures of the governance system that have resulted in undesirable outcomes (Figure 4). “Being many people and all agree for the common good, that does not happen in real life. It does not work because there is no consciousness of water, we believe that it is an eternal resource and that we will always have it. There is also no honesty. There is also no interest in collectivity”. Palm plantation technician in Aracataca, September 17, 2019. “Being many people and all agree for the common good, that does not happen in real life. It does not work because there is no consciousness of water, we believe that it is an eternal resource and that we will always have it. There is also no honesty. There is also no interest in collectivity”. Palm plantation technician in Aracataca, September 17, 2019. HETEROGENEITY AND POWER ASYMMETRIES Results found for the three scenarios suggest that, regardless of the variation in water supply, the main outcomes remain: inequality in water distribution, decrease in flow in Trojas de Cataca and conflicts among users. This suggests that the water issues in the basin are more complex than the numerical relationship between demand and supply. Thus, the governance system acquires relevance because the decisions made by water users and other actors are fundamental (Linton & Budds, 2014). The dynamics of the appropriation and provision of water are critical in the socioecological configurations of the system associated with the distribution of water, and in particular, its flow in the lower basin and the surroundings of the CGSM. Heterogeneity in CPR systems may take different forms and either facilitate or hinder cooperation (Baland & Platteau, 1996; Naidu, 2005; Usón et al., 2017), creating challenges for water governance. Although there is no consensus on whether how some dimensions of inequality promote collective action toward the conservation of CPRs (Baland & Platteau, 1999), in the Aracataca River heterogeneity is hindering cooperation. Although diversity manifests itself in many ways and some specific configurations in certain contexts can shape conditions for collective action (Poteete et al., 2009), it raises the cost of negotiation in the construction of institutional arrangements and in defining desired collective outcomes (Bardhan & Dayton-Johnson, 2001). Groups with different socioeconomic and cultural attributes tend to lack trust and lack shared interpretations of reality, which contributes to processes of agreement mediated by conflict and a lack of collective action (Araral, 2009; Dayton-Johnson, 2000; Fujiie et al., 2005; Varughese & Ostrom, 2001). Also, those who have more interest in transforming the CPR problem, may have fewer opportunities to influence decision-making processes, given less infrastructure access and lower negotiation power, as in the case of fishers in Trojas de Cataca. To ensure the necessary flow to maintain the ecological functions of the CGSM and access to water in Trojas de Cataca, it is key to solve the collective action problem and power asymmetries in the lower basin. In addressing this problem, the emphasis lies on institutional arrangements at the operational and collective choice levels among palm oil producers. From this starting point, problems of governance systems can be addressed at higher scales, such as cross-scale cooperation for the implementation of the watershed management plan. RESULTS OF THE ROLE-PLAYING GAME WORKSHOP The outcomes of the RPG workshop showed specific responses of the actors in the lower basin to the changing water availability (Annex 4). Regarding monitoring and sanction, although players were aware of the actions of others and were able to identify who breached the agreements in rounds 3 and 4, the participants never designed a sanction that would promote compliance with the norms. During deliberation and decision-making moments, players socialized their individual results from each round. Public accusations were also made about the breach of agreements and demands were made for their compliance, again, without In rounds 3 and 4, institutional arrangements for water appropriation emerged regarding water distribution among participants, first dictated by the representative of the irrigation district and then by consensus. In round 4, a norm to guarantee water for the CGSM also emerged. Nevertheless, these three norms did not work as intended because illegal water harvesting still occurred and was not Fi 4 I t ti d t f th SES Figure 4 Interactions and outcomes of the SES. Fonseca-Cepeda et al. International Journal of the Commons DOI: 10.5334/ijc.1167 165 The level of water scarcity perceived by water users is largely associated with conflicts and a lack of coordination (Meinzen-Dick, 2007; Schlüter & Pahl-Wostl, 2007). Whether the abundance of resources (Gurung et al., 2006), moderate levels of scarcity (Torres et al., 2016), or high scarcity of resources leads to cooperation has been discussed (Bardhan, 2000; Ostrom, 2000). For the Aracataca River basin, despite the three annual water supply scenarios, the inequitable distribution of water remains, and the asymmetries of power related to decision-making are constant, so collective action has not been the main outcome. establishing sanctions. Agreements were established to solve water allocation and to guarantee the provision of the resource in the dry rounds, but they did not reach any collective strategy to guide water management in the basin. Concrete conflicts did not emerge, other than the claim of downstream players about the scarcity of water at their water uptake point. HETEROGENEITY AND POWER ASYMMETRIES Addressing the problem of collective action involves the design of congruent appropriation and provision rules, as well as its coherence with local conditions of the CPR, such as spatial and temporal heterogeneity of water supply, and with the heterogeneity of livelihood strategies among users (Cox et al., 2010). Also, it implies the renewal of a governance configuration that prevents the participation of less powerful actors (e.g., small palm oil producers and fishermen) in modifying operational and collective institutional arrangements (Ostrom, 1990). Material and bargaining power asymmetries must be addressed towards a multilateral decision-making model (Cascão, 2009). A renovation of the monitoring system is also relevant since the regional environmental authority has demonstrated not to be entirely efficient. Strengthening positive feedback between trust, reciprocity, and reputation among actors, is also essential to achieve and maintain collective action (Ostrom, 1998). Differences between decision makers affect the joint understanding of a situation with respect to collective management and participation in solutions to the problems posed for CPRs, affecting which institutional arrangements the users choose, how they are maintained and whether users decide to follow the agreements or not (Bardhan & Dayton-Johnson, 2001). At the individual level, uncertainty about the preferences of others and the degree of dependence between actors can affect the incentives to contribute, and at the collective level, it can affect communication, monitoring and the reinforcement of institutional arrangements (Schlager & Blomquist, 1996). In contrast, common characteristics suggest common interests, which can increase the predictability of the interactions and actions of others, increasing trust among actors (Poteete & Ostrom, 2004). Fonseca-Cepeda et al. International Journal of the Commons DOI: 10.5334/ijc.1167 166 outcomes (Schlager & Ostrom, 1992). Actors build power dynamics through actions at the operational level. Power involves the perceived legitimacy of formal institutional arrangements and the ability to define the rules in use (Clement, 2013). Thus, some actors have more power than others in decision-making about the distribution of benefits, either because of their economic advantages and physical infrastructure or because of their negotiation skills (Zeitoun & Jägerkog, 2011). Although power asymmetries do not necessarily determine the outcome of decision-making processes, the general benefits of an action situation will be available to agents with more power (Qin et al., 2019). In the context of the Aracataca River, we identified different dimensions of heterogeneity. HETEROGENEITY AND POWER ASYMMETRIES One of them involves the verticality implied by water flow, where the differences in where the actors are located, i.e. the upper or lower basin, defines their role in the appropriation and provision of water. These differences have effects on the incentives perceived by each actor, reciprocity, trust, reputation, and collective action (Ostrom & Gardner, 1993). Verticality in water flow supposes an asymmetric relationship between actors in terms of access to water in time and quantity (Cárdenas et al., 2015). The physical and social distance between actors limits communication and any interactions that might lead to building trust and transforming conflicts (Swallow et al., 2006), representing an important challenge in governance in the basin. Specifically, in the Aracataca River’s lower basin, palm oil producers can access water first and can develop water maintenance and distribution systems, such as irrigation districts managed by water user associations. Fishermen in Trojas de Cataca access the water later and receive the externalities caused upstream, affecting their well-being (Cárdenas et al., 2015). In the lower basin, the asymmetries of power consist of not only infrastructural disadvantages, i.e., in contrast to palm oil producers, the fishing community has no infrastructure to collect and distribute water, but also the loss of autonomy and marginalization of this community when influencing the decision-making agenda. Their limitations of individual and collective power translate into their inability to transform rigid institutional structures (Cascão, 2009). Although spatial and technological differences asymmetries can encourage non cooperative behaviors and weaken norms, rules, and strategies for collective action (Bardhan & Dayton-Johnson, 2001), not all dimensions of diversity have deleterious effects on cooperation. It is important to ask which dimensions of diversity should be transformed and which should be sought, conserved, or reinforced to enrich the governance of water as a CPR. These attributes and desirable functions should be identified by the stakeholders themselves (Lebel et al., 2006). Given the power asymmetries and the various dimensions of heterogeneity that we identified in the Aracataca River’s lower basin, it is possible to affirm that, just as water problems are not merely technocratic and related to infrastructure, the social dimension of water management not only consists of access and distribution dilemmas. Water management directly involves political and power issues, which are manifested both formally and informally (Sultana, 2018; Zwarteveen & Boelens, 2014). HETEROGENEITY AND POWER ASYMMETRIES The discussion on institutional arrangements and water governance must explicitly address issues of equity, participation and justice for human well-being and the maintenance of ecological processes (Perreault, 2014). The analysis of the case study considering these elements is particularly relevant insofar as the need to transform the current structure of water governance has been identified (Neal et al., 2014). Although the differences pose challenges for self- organized water management, innovation in institutional arrangements can contribute to navigating local conditions of heterogeneity (Varughese & Ostrom, 2001). Attributes of the group of actors may be less important in the success of collective water management, while the ability to adapt and learn, to redesign institutional arrangements, to establish dialog and negotiation processes, and to ensure autonomy in the redistribution of costs and benefits become more relevant. Even heterogeneous social groups may be able to devise institutional arrangements that allow them to identify shared issues for collective action (Poteete & Ostrom, 2004; Varughese & Ostrom, 2001). Equity is related to how benefits and burdens associated with water use are distributed (Perreault, 2014), as well as rights to participate in decision making processes (Zwarteveen & Boelens, 2014). Participation is relevant since there are actors excluded from the possibility of getting involved in the development of autonomous water management mechanisms, i.e., their own infrastructure and their own rules in use. Fishermen and small producers are not participants in decision-making to distribute benefits. As long as all the actors can participate in the design of strategies for the distribution of water, fair situations will be created in the management of the resource (Neal et al., 2014). Another observed dimension of heterogeneity involves the asymmetries of power in decision-making regarding water management and participation in the construction and transformation of institutional arrangements. This asymmetry of power, particularly evident in the lower basin among the most disadvantaged, i.e., fishermen of Trojas de Cataca and the small palm oil producers, affects participants’ incentives, actions and, therefore, the system Justice has to do with how disadvantaged actors experience and point out unfair situations regarding Fonseca-Cepeda et al. International Journal of the Commons DOI: 10.5334/ijc.1167 167 methodology allows the experimentation of different institutional configurations, functioning as a platform for the design and testing of strategies, norms, and rules of provision and appropriation. 1 According to the Political Constitution of Colombia of 1991, departments are geographical and political-administrative units with autonomy for economic and social development within its territory. They mediate between the Nation and the Municipalities. CONCLUSIONS The three annual conditions demonstrated the variation of supply in the basin, with low, moderate, and high indices, in which supply is always exceeded by demand (the index is never equal to 0). Despite the variety of scenarios, collective action has not turned out to be the main outcome of the SES. This indicates that the configuration of the governance system outweighs the availability of the CPR in the construction of collective action. For future research, the calculation of the supply on a monthly scale, as well as the interactions between users, could provide greater resolution to the analysis. HETEROGENEITY AND POWER ASYMMETRIES However, the construction and maintenance of institutional arrangements implies long processes of consultation, building trust to attend the sessions, continuity of spaces for dialogue, the integration of different decision makers and, above all, being immersed in a long-term process at the basin scale that implies specific commitments of users, associations, and government entities. The scope of a RPG workshop is limited when addressing power asymmetries and the strengthening of trust, reciprocity, and reputation feedback. Therefore, it is only a tool that can accompany deeper strategies to manage changes in the political dimension of water management. water management (Boelens, 2009). This arises when a group feels that others are not contributing their share to the maintenance of water and the infrastructure for its allocation, or are taking more than their fair share from the CPR (Patrick, 2014). In the lower basin, given the power asymmetry, injustice is manifested in the absence of recognition of the right to access water for all actors, and of the claims and needs of the most disadvantaged. A fair system would consist of guaranteeing the means to address the social dilemma implied by the distribution of water and the right to make decisions, facing different scenarios of water availability in the basin, as we have shown. The perception of fairness is also a powerful predictor of trust (Neal et al., 2014), related to cooperation (Ostrom, 1998). Thus, water governance must include equity, participation, and justice as institutional configurations to promote collective action. It is important that all actors across scales are included, so that those with less power are included in the processes of water allocation and decision- making to solve the social dilemma (Sultana, 2018). CONSIDERATIONS REGARDING THE METHOD Our mixed-method approach made it possible to characterize the Aracataca River as a SES based on interactions and outcomes, emphasizing the institutional arrangements within the lower basin. While the hydrological analysis allows us to explore the conditions of the water system, the interviews were useful to identify the actors and conflicts within the basin. The RPG, on the other hand, helped us to get information on the dynamics and patterns that emerge when different resource system and governance configurations are recombined (Bousquet et al., 2002). Because RPG offer new options for obtaining information through the observation of body language, attitudes, and direct actions of the players during a session (Barnaud et al., 2007), it was possible to identify institutional arrangements and interactions among users that would not have been made explicit through other methodologies (Voinov et al., 2018). The structure of the governance system in the Aracataca River basin is largely defined by conflict, illegality and the lack of effective monitoring and sanction mechanisms, conditioning an institutional configuration that contributes to the water problem in the study area. This assumes that individuals have little incentive to cooperate and perceive little legitimacy from control entities and formal institutional arrangements affecting disproportionally the fishermen, who are not just the actors in the tail of the system but also those with lower opportunities to modify its structural conditions. During the debriefing (Lederman, 1992) it was possible to identify specific issues within the basin that were most important to actors, for example, the need for technical information related to the supply of water, the illegality in water collection, the lack of a collective strategies, the inequality in water distribution, the ineffectiveness of monitoring and sanctioning processes, and the difficulty of reaching a consensus. Thus, the RPG workshop offered learning opportunities for both researchers and players (Camargo et al., 2007; Rodela et al., 2019). The RPG workshop is a tool that can facilitate long- term process for the design institutional arrangements in the lower basin, but it requires being part of a process of structural change in power asymmetries. ACKNOWLEDGEMENT Álvarez, M., Posada, J. F., Mosquera, M., García, T., & Ardila, G. (2007). Eﬁciencia en el uso del agua en una plantación de palma de aceite. Importancia económica de su optimización. Revista Palmas, 28(2), 11–22. We thank the Usoaracataca irrigation district for providing the spaces for the role-playing game, the actors interviewed for their willingness and all the people who participated in the pilot studies for their contributions to the calibration of the methodology. We also thank the OPAL project for financial support for the development of this work, and Pontificia Universidad Javeriana for funding the publication of the article. We want to pay tribute to our colleague Efraín Domínguez-Calle, who passed away during the writing process of this manuscript. His legacy is vital for hydrological studies in Colombia. Ander-Egg, E. (1995). Técnicas de investigación social (24th ed.). Editorial LUMEN. Araral, E. (2009). What Explains Collective Action in the Commons? Theory and Evidence from the Philippines. World Development, 37(3), 687–697. DOI: https://doi.org/10.1016/j. worlddev.2008.08.002 Atencia, L. (2014). Estudio de impacto ambiental en planta extractora Aceites S.A. Baland, J. M., & Platteau, J. P. (1996). Halting degradation of natural resources: is there a role for rural communities? In International Affairs, 72(4). DOI: https://doi. The authors have no competing interests to declare. Baland, J. M., & Platteau, J. P. (1999). The ambiguous impact of inequality on local resource management. World Development, 27(5), 773–788. DOI: https://doi.org/10.1016/ S0305-750X(99)00026-1 ADDITIONAL FILES Acuerdo 193 de 2009. Por el cual se definen los lineamientos para establecer las tarifas aplicables a los usuarios de los distritos de Adecuación de Tierras ejecutados por el Incoder. Instituto Colombiano de Desarrollo Rural – Incoder. The additional files for this article can be found as follows: • Annex 1. Research methods and SES variables (adapted from Meinzen-Dick, 2007). DOI: https://doi.org/10.5334/ ijc.1167.s1 Aguilera, M. (2011). Habitantes del agua: el complejo lagunar de la Ciénaga Grande de Santa Marta. Documentos de Trabajo Sobre Economía Regional, 144, 1–46. DOI: https://doi. org/10.32468/dtseru.144 • Annex 2. Parameters of the hydrological analysis. DOI: https://doi.org/10.5334/ijc.1167.s2 • Annex 3. Variables for the collection and analysis of information derived from the role-playing game (Source: adapted from Ostrom, Gardner, & Walker, 1994; Ostrom, 1990; Agrawal, 2002). DOI: https://doi. org/10.5334/ijc.1167.s3 Allen, R. G., Pereira, L. S., Raes, D., & Smith, M. (1998). FAO Irrigation and Drainage Paper No. 56 – Crop Evapotranspiration (guidelines for computing crop water requirements) (p. 281). FAO. Allington, G. R. H., Fernandez-Gimenez, M. E., Chen, J., & Brown, D. G. (2018). Combining participatory scenario planning and systems modeling to identify drivers of future sustainability on the Mongolian Plateau. Ecology and Society, 23(2), 9. DOI: https://doi.org/10.5751/ES-10034-230 209 • Annex 4. Outcomes of the RPG workshop. DOI: https:// doi.org/10.5334/ijc.1167.s4 NOTE The game evidence potentialities and challenges to address the problem of collective action among palm oil producers and its relationship with the well-being of fishermen in Trojas de Cataca. On the one hand, the 168 Fonseca-Cepeda et al. International Journal of the Commons DOI: 10.5334/ijc.1167 COMPETING INTERESTS The authors have no competing interests to declare. AUTHOR AFFILIATIONS Bardhan, P. (2000). Irrigation and cooperation: An empirical analysis of 48 irrigation communities in South India. Economic Development and Cultural Change, 48(4), 847–865. DOI: https://doi.org/10.1086/452480 Valentina Fonseca-Cepeda orcid.org/0000-0001-7614-0569 Pontificia Universidad Javeriana, CO Daniel Castillo-Brieva orcid.org/0000-0001-9251-6124 Pontificia Universidad Javeriana, CO Luis Baquero-Bernal orcid.org/0000-0003-1062-9235 Pontificia Universidad Javeriana, CO Luz Angela Rodríguez orcid.org/0000-0002-6583-6197 Pontificia Universidad Javeriana, CO Eliane Steiner orcid.org/0000-0002-2845-8289 Swiss Federal Institute of Technology (ETH), CH John Garcia-Ulloa Swiss Federal Institute of Technology (ETH), CH Valentina Fonseca-Cepeda orcid.org/0000-0001-7614-0569 Pontificia Universidad Javeriana, CO Daniel Castillo-Brieva orcid.org/0000-0001-9251-6124 Pontificia Universidad Javeriana, CO Luis Baquero-Bernal orcid.org/0000-0003-1062-9235 Pontificia Universidad Javeriana, CO Luz Angela Rodríguez orcid.org/0000-0002-6583-6197 Pontificia Universidad Javeriana, CO Eliane Steiner orcid.org/0000-0002-2845-8289 Swiss Federal Institute of Technology (ETH), CH John Garcia-Ulloa Swiss Federal Institute of Technology (ETH), CH Bardhan, P., & Dayton-Johnson, J. 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International Journal of the Commons, 16(1), pp. 155–172. DOI: https://doi.org/10.5334/ijc.1167 Submitted: 17 November 2021 Accepted: 01 May 2022 Published: 27 June 2022 COPYRIGHT: © 2022 The Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. See http://creativecommons.org/licenses/by/4.0/. International Journal of the Commons is a peer-reviewed open access journal published by Ubiquity Press.
https://openalex.org/W2047877941	https://link.springer.com/content/pdf/10.1007/s11104-012-1245-0.pdf	English	null	Contribution of 15N-labelled leaf litter to N turnover, nitrous oxide emissions and N sequestration in a beech forest during eleven years	Plant and soil	2,012	cc-by	8,701	Plant Soil (2013) 362:67–77 DOI 10.1007/s11104-012-1245-0 Plant Soil (2013) 362:67–77 DOI 10.1007/s11104-012-1245-0 REGULAR ARTICLE Abstract production (0.02 %). Between 0.3 % (eleventh year) and 0.6 % (second year) of total annual N2O emissions were attributed to beech leaf litter of a single year. Conclusions Most of the annual N2O emissions (1.33–1.54 kg N ha−1 yr−1) were probably derived from older soil N pools. Aims Decomposition of leaf litterfall plays a major role for nitrogen (N) dynamics in soils. However, little is known as to which extent beech leaf litter contrib- utes to N turnover and nitrous oxide (N2O) emissions within one decade after litterfall. g y Conclusions Most of the annual N2O emissions (1.33–1.54 kg N ha−1 yr−1) were probably derived from older soil N pools. Methods In 1997, we exchanged recently fallen leaf litter by 15N-labelled litter in a beech stand (Fagus sylvatica) at the Solling, Germany. Measurements were conducted 2–3 and 10–11 years after litter exchange. Results Two years after litter exchange, 92 % of added 15N was recovered in the surface 10 cm of the soil. The labelled N was primarily found in the upper part of the F layer of the moder type humus. Eleven years after litter exchange, 73 % of the added 15N was lost and the remaining 27 % was mainly recovered in the lower part of the F layer indicating N sequestration. The remaining leaf litter N was subject to measurable N mineralisation (2–3 % of litter N) and N2O Methods In 1997, we exchanged recently fallen leaf litter by 15N-labelled litter in a beech stand (Fagus sylvatica) at the Solling, Germany. Measurements were conducted 2–3 and 10–11 years after litter exchange. Keywords Nitrous oxide . Beech leaf litter. 15N isotope technique . N mineralisation . Litter N dynamics Keywords Nitrous oxide . Beech leaf litter. 15N isotope technique . N mineralisation . Litter N dynamics Results Two years after litter exchange, 92 % of added 15N was recovered in the surface 10 cm of the soil. The labelled N was primarily found in the upper part of the F layer of the moder type humus. Eleven years after litter exchange, 73 % of the added 15N was lost and the remaining 27 % was mainly recovered in the lower part of the F layer indicating N sequestration. The remaining leaf litter N was subject to measurable N mineralisation (2–3 % of litter N) and N2O Contribution of 15N-labelled leaf litter to N turnover, nitrous oxide emissions and N sequestration in a beech forest during eleven years Nadine Eickenscheidt & Rainer Brumme Received: 24 January 2012 /Accepted: 30 March 2012 /Published online: 8 May 2012 # The Author(s) 2012. This article is published with open access at Springerlink.com production (0.02 %). Between 0.3 % (eleventh year) and 0.6 % (second year) of total annual N2O emissions were attributed to beech leaf litter of a single year. Introduction Weathering of geological parent material contributes very marginally to mineral nitrogen (N) content of soils (Berg and Matzner 1997; Parton et al. 2007). Thus, decomposition of litter and mineralisation inhere partic- ular importance for N availability in forest ecosystems (Parton et al. 2007). Decomposition has been described as a three-stage process: (i) the early stage, (ii) the late stage, and (iii) the humus-near stage, latter is character- ised by a decomposition rate close to zero (Berg and McClaugherty 2003; Berg and Matzner 1997). Responsible Editor: Per Ambus. Responsible Editor: Per Ambus. N. Eickenscheidt: R. Brumme Soil Science of Tropical and Subtropical Ecosystems, Buesgen Institute, University of Goettingen, Buesgenweg 2, 37077 Goettingen, Germany During the early stage, initial litter N concentrations primarily drive net N release from litter (Parton et al. 2007). A rapid mass loss of native litter N but a simultaneous net increase in N and lignin contents of the decaying litter during the early stage was generally reported (Berg and McClaugherty 2003; Laganière et al. 2010; Melillo et al. 1982; Zeller et al. 2000). Fahey N. Eickenscheidt (*) Institute of Forest Ecology and Forest Inventory, Johann Heinrich von Thuenen Institute, Alfred-Moeller-Strasse 1, 16225 Eberswalde, Germany e-mail: nadine.eickenscheidt@vti.bund.de Plant Soil (2013) 362:67–77 68 oxide is a potent greenhouse gas and contributes to the catalytic decomposition of stratospheric ozone (Crutzen 1979; IPCC 2007). High emissions of N2O have been reported from European beech forest soils, which have been exposed to chronic high N depositions (Brumme and Beese 1992; Papen and Butterbach-Bahl 1999; Zechmeister-Boltenstern et al. 2002). However, one of our previous studies revealed that despite the high N2O emissions of 2.6 kg N ha−1 yr−1 from an acid beech forest soil in Germany, only 6 % of the total emissions were derived from N depositions of the recent year whereas the remaining part was derived from the soil N pool (Eickenscheidt et al. 2011). Therefore the ques- tion arises as to what extent the turnover of leaf litter N contributes to N2O emissions. et al. (2011) observed a continuous decrease in native litter 15N of 20–30 % in the first year after an addition of 15N-labelled litter to a sugar maple stand. The loss, however, was balanced by a large N incorporation from external sources into the litter that resulted in an increase in N contents of 26–32 %. Introduction External N incorporation balanced N loss over a 2-years period in three European beech forest soils and net release of N was measured in the third year (Zeller et al. 2000; Zeller et al. 2001). Translocation of N from the soil to the decaying litter by fungi accounted for 25–35 % of the exogenous N, whereas atmospheric N deposition accounted for 30– 50 % in a French beech forest (Zeller et al. 2000). ( ) In the late stage of decomposition, condensation of degradation products combined with import of nutrients produces recalcitrant humic compounds, which accumulate (Berg and McClaugherty 2003). Berg and Matzner (1997) reported a rate-retarding effect of high initial litter N and soil N concentrations during the late stage of decomposition, which was ascribed to (i) the suppressing effect of N on the formation of ligninase in white-rot fungi, and (ii) condensation reactions in which lignin incorporates N, resulting in stable chemical structures. In forest ecosystems exposed to chronic high N depositions, leaching of organic N from the decaying litter but simultaneously reduced fungal N transport, due to sufficient supply of external N by depositions, may further promote N sequestration in soil organic matter (SOM; Fahey et al. 2011). In highly N enriched acid forest soils, absence of earthworms and bioturbation, which are key factors for decomposition, additionally reduce N loss (e.g. Schaefer et al. 2009). An increase in organic layer and soil N has indeed been reported from forests exposed to chronic high N and acid depositions (e.g. Meiwes et al. 2009). Turnover of litter N is mostly studied through mass loss observations (e.g. Berg and McClaugherty 2003) and only few studies have used stable isotope tracers (e.g. Fahey et al. 2011; Zeller and Dambrine 2011), where the emphasis has been on the early-stage of decomposition. Long-term 15N tracer studies are re- quired to improve our present knowledge about the processes of litter N dynamics and potential long-term N sequestration in the soil. The objective of our study was to assess the contri- bution of beech leaf litter N to N mineralisation, N2O emissions, and N sequestration in the soil 2–3 years and 10–11 years after litterfall application. We con- ducted a 15N isotope litter exchange experiment in a beech stand exposed to chronic high N depositions at the Solling, Germany. Study area Beech leaf litter is generally rich in N, water soluble compounds, tannin, and lignin (e.g. Berg and McClaugherty 2003; Lorenz et al. 2004; Melillo et al. 1982) and European beech forests have often been exposed to chronic high N and acid depositions. Thus, rapid decomposition in the early stage but slow decom- position in the late stage and temporary N sequestration may be expected (Berg and McClaugherty 2003). The study was conducted in a 160-yr-old beech stand (Fagus sylvatica) at the Solling in Central Germany (51° 46′ N, 9° 34′ E, 500 ma.s.l., mean temperature: 6.97 °C, annual precipitation: 1193 mm, stand depo- sition: 20 kg N ha−1 yr−1). The soil type is a Dystric Cambisol (pHH2O≤3.6; silty loam), which derived from Triassic sandstone covered by loess. The surface organic layer is a typical moder consisting of an L (litter) layer, F (fermentation) layer, and H (humus) layer with a thickness of 1–2 cm, 4–5 cm, and 2 cm, respectively. The organic layer in total stores 2790 gC m−2 and 129 gN m−2 (Table 1). Leaf litter production Mineral N, which is released from the decaying litter, may be utilised by micro-organisms for nitrifi- cation and denitrification processes during which ni- trous oxide (N2O) may be produced either as a by- product or intermediate (Davidson et al. 2000). Nitrous Plant Soil (2013) 362:67–77 69 2008 using the closed chamber method (chamber vol.: 5.4 L). Three N2O gas samples were collected in evacuated glass flasks (100 mL) at three regular time intervals following chamber closure (36 min at most) using a portable gas sampler equipped with a pressure sensor (Loftfield et al. 1997). Analysis was done with a gas chromatograph equipped with an EC detector (GC 14A, Shimadzu, Duisburg, Germany). Samples for 15N-N2O measurements were removed at 0.5 and 36 min (1999–2000) and at 0.5 and 20 min (2007–08) and were stored in evacuated 100 mL glass bottles. An isotope ratio mass spectrometer coupled with a precon- centration unit was used for 15N analysis (PreCon-GC- IRMS, Thermo Finnigan MAT, Bremen, Germany). Isotope abundances were expressed as absolute propor- tion [atom%]. We used the Keeling plot approach to calculate the 15N-N2O abundance of the N2O emitted from the soil (Tilsner et al. 2003), which was described in detail in Eickenscheidt et al. (2011). Experimental design In December 1997, six chamber bases (area: 0.0559 m2) were installed. The bases were inserted into the organic layer. We removed the freshly fallen beech leaf litter of the recent year and replaced it by 17 g 15N-labelled beech leaf litter (0.71±0.01 %N; 3.57±0.07 atom% 15N), which corresponded to the average annual leaf litter production. The N content of the replaced litter was lower than of the nativ leaf litter, which was 1.2 %N in 1997, since the 15N- labelled litter was derived from beech trees that were grown in a greenhouse and irrigated by a 15N solution. Three of the chamber bases were only used for soil sampling in 1999 and the other three bases were used for N2O and 15N-N2O gas flux measurements in 1999 and 2008 as well as for soil sampling in 2008. Three additional chamber bases, which contained the native unlabelled litter fall (no exchange), were installed as control chambers for 15N-N2O flux measurements. Control soil samples for natural 15N abundances were taken randomly at the study site where no litter ex- change was conducted. Study area amounted to 277±25 gm−2 yr−1 (standard deviation sd) from 1991 to 2003 with an average N content of 1.24±0.16 % (sd), which corresponded to 3.4±0.4 gN m−2 yr−1 (sd; Khanna et al. 2009). The N content of live foliage was 2.46±0.11 % (sd) from 1995 to 2002 and thus, withdrawal of N of live foliage during leaf senescence amounted to 51 % (Khanna et al. 2009). A detailed site description can be found in Brumme and Khanna (2009) and in Eickenscheidt et al. (2011). Soil sampling and in situ incubation After gas flux measurements, soil sampling and an in- situ incubation of the soil was conducted within the chamber bases to determine net rates of ammonifica- tion, nitrification, and 15Nexcess ammonification in summer 2008. For each chamber, two soil samples of the surface 10 cm of the soil were taken with a stain- less steel core (diameter: 5.5 cm). The sample included the surface organic layer and the surface mineral soil (Table 1). Soil from one core was removed and divid- ed into 2 cm sections, of which soil extracts for mineral N (Nmin0NH4 +-N+NO3 −-N) and 15Nmin analyses were prepared within few hours (t0 sample). The other soil core (t1 sample) was put into a PE bag and was returned into the hole for undisturbed in situ incubation, which lasted 16 days. During the incubation, the mean temper- ature in 10 cm soil depth, which was logged every 2 h, was 15.1±1.2 °C (standard deviation). After the Calculation of the 15N recovery and of the mean residence time Calculation of the 15N recovery and of the mean residence time Calculation of the 15N recovery and of the mean residence time The recovery of the 15N label of the exchanged leaf litter in N2O, Nt, and net ammonification were calculated using 15Nexcess values. For calculations of 15Nexcess val- ues, natural 15N abundances of unlabelled samples (N2O: Fig. 4; Nt: Table 1; NH4 +: 0.3641 atom% (organic layer), 0.3666 atom% (0–5 cm mineral soil)) were subtracted from total 15N abundances obtained from chambers with 15N-labelled leaf litter exchange. On average 69.8 mg 15Nexcess m−2 were applied by the leaf litter exchange in 1997 (the natural abundance of the native leaf litter was 0.365 atom% 15N in 1997). For recovery assessments the ratio of 15Nexcess masses of N2O, Nt, and net ammonifi- cation, respectively, to masses of applied 15Nexcess by litter exchange were calculated. b) 11 years after litter exchange b) 11 years after litter exchange b) 11 years after litter exchange 0 5 10 15 20 25 30 35 40 45 50 55 60 65 0–2 2–4 4–6 6–8 8–10 Soil depth [cm] a c b bc ac 0 5 10 15 20 25 30 35 40 45 50 55 60 65 0–2 2–4 4–6 6–8 8–10 Soil depth [cm] a c b bc ac 0 3 15 7 The mean residence time (MRT) of N (and C) in the surface organic layer (0–8 cm) was assessed using the measured mass of N (C) in the surface organic layer divided by the mass of N (C) of the annual aboveground total litterfall (6.8 gN m−2 yr−1 and 258 gC m−2 yr−1; Khanna et al. 2009). N2O and 15N-N2O flux measurements Analyses of 15N abundance (in Nt and NH4 +) were conducted with a Delta C plus IRMS (Finnigan MAT, Bremen, Germany). N2O and 15N-N2O flux measurements We measured N2O and 15N-N2O fluxes from January 1999 to December 2000 and from May 2007 to July Table 1 Characteristics of the surface organic layer and upper 2 cm of the Ah horizon of the beech forest at Solling, Germany Horizon Depth [cm] C [%] Nt [%] C [g m−2] Nt [g m−2] C/N ratio 15N-Nt [atom%] L 0–2 46.9±0.2 1.67±0.04 379±25 13.9±1.0 27.2 0.3658±0.0000 F 2–6 45.3±0.6 2.01±0.04 848±79 38.4±4.2 22.1 0.3656±0.0000 H 6–8 25.5±4.1 1.24±0.19 1563±195 77.1±10.6 20.3 0.3660±0.0000 Ah 8–10 9.1±1.7 0.47±0.08 1229±138 63.7±5.9 19.3 0.3669±0.0001 Plant Soil (2013) 362:67–77 70 incubation, soil cores were subject to the same treatment as the t0 samples. Mineral N extracts were obtained after shaking 30 g fresh mineral soil with 100 mL 0.5 M K2SO4 solution or 15 g of the organic layer with 50 mL K2SO4 solution. We analysed Nmin by using continuous flow injection colorimetry (Cenco/Skalar Instruments, Breda, The Netherlands). A modified diffusion method (Corre et al. 2003; Stark and Hart 1996) was applied for the determination of 15N in the extracted NH4 + and nitrate (NO3 −). However, net 15Nexcess nitrification rates were not available due to failure of the isotope ratio mass spectrometer. In addition, total carbon (C) and nitrogen (Nt) contents (CNS Elemental Analyzer, Heraeus Ele- mentar Vario EL, Hanau, Germany) and 15N abundance in Nt were determined in 1999 and 2008. Analyses of 15N abundance (in Nt and NH4 +) were conducted with a Delta C plus IRMS (Finnigan MAT, Bremen, Germany). incubation, soil cores were subject to the same treatment as the t0 samples. Mineral N extracts were obtained after shaking 30 g fresh mineral soil with 100 mL 0.5 M K2SO4 solution or 15 g of the organic layer with 50 mL K2SO4 solution. We analysed Nmin by using continuous flow injection colorimetry (Cenco/Skalar Instruments, Breda, The Netherlands). A modified diffusion method (Corre et al. 2003; Stark and Hart 1996) was applied for the determination of 15N in the extracted NH4 + and nitrate (NO3 −). However, net 15Nexcess nitrification rates were not available due to failure of the isotope ratio mass spectrometer. In addition, total carbon (C) and nitrogen (Nt) contents (CNS Elemental Analyzer, Heraeus Ele- mentar Vario EL, Hanau, Germany) and 15N abundance in Nt were determined in 1999 and 2008. Recovery and movement of 15N in the soil Recovery and movement of 15N in the soil Two years after 15N-labelled beech litter addition, 92 % of the 15N was recovered in the surface 10 cm of the soil, most of which (80 % of the total recovery) was found in the upper part of the F layer of the surface organic layer at 2–4 cm soil depth (Fig. 1a). Eleven years after the addition, 27 % of the 15N label was recovered with the highest recovery (15 % of the total recovery) in 4–6 cm soil depth, the lower part of the F layer (Fig. 1b). The changes of 15N-Nt-excess contents in the soil profile from 1999 to 2008, with a a) 2 years after litter exchange Nt [mg 15Nexcess m−2] Rec. [%] a) 2 years after litter exchange 0 5 10 15 20 25 30 35 40 45 50 55 60 65 0–2 2–4 4–6 6–8 8–10 Soil depth [cm] a c b bc ac 0 3 15 7 1 b) 11 years after litter exchange 0 5 10 15 20 25 30 35 40 45 50 55 60 65 0–2 2–4 4–6 6–8 8–10 Soil depth [cm] Nt [mg 15Nexcess m−2] Rec. [%] a ac a b c 5 3 4 80 1 a) 2 years after litter exchange Fig. 1 Distribution of soil 15N in the surface 10 cm of the soil b) 11 years after litter exchange 0 5 10 15 20 25 30 35 40 45 50 55 60 65 0–2 2–4 4–6 6–8 8–10 Soil depth [cm] Nt [mg 15Nexcess m−2] a ac a b c a) 2 years after litter exchange Statistical analyses Fig. 1 Distribution of soil 15N in the surface 10 cm of the soil 2 years (a) and 11 years (b) after 15N-labelled litter exchange in the beech stand. Among soil depths, means (± SE; n03) fol- lowed by the same letter were not significantly different (ANOVA, Tukey HSD-test at P≤0.05). On the right hand side, the recovery (Rec.) of the 15N-labelled litter in soil Nt is given for each layer Fig. 1 Distribution of soil 15N in the surface 10 cm of the soil 2 years (a) and 11 years (b) after 15N-labelled litter exchange in the beech stand. Among soil depths, means (± SE; n03) fol- lowed by the same letter were not significantly different (ANOVA, Tukey HSD-test at P≤0.05). On the right hand side, the recovery (Rec.) of the 15N-labelled litter in soil Nt is given for each layer Statistical analyses were conducted using R 2.12.0 (R Development Core Team 2010). We applied linear mixed effects models on time series data (Eickenscheidt et al. 2011). Results were given as mean±1 standard error and effects were considered significant where P≤0.05. 71 Plant Soil (2013) 362:67–77 -5 0 5 10 15 20 25 0–2 2–4 4–6 6–8 8–10 Soil depth [cm] [mg N m−2 d−1] Ammonification Nitrification a a ab b a a) -1 0 1 2 3 4 5 0–2 2–4 4–6 6–8 8–10 Soil depth [cm] 15N ammonification [µg 15Nexcess m−2 d−1] Rec. [% d−1] 0.0004 0.0005 0.0029 0.0047 0.0000 b) a ab ab b a Fig. 2 Net ammonification and nitrification rates (a) and net 15N ammonification rates (b) of the surface 10 cm of the soil in the beech stand. On the right hand side of Fig. 2b, the daily recovery (Rec.) of the 15N-labelled litter in net ammonification is given for each soil layer. The undisturbed in situ incubation lasted 16 days and was conducted within the chamber bases of the litter exchange experiment in summer 2008. Among soil depths, means (±SE; n03) followed by the same letter were not significantly different (ANOVA, Tukey HSD-test at P≤0.05). Statistical analyses Letters in figure a) belong to net ammonification rates and no significant differences occurred among net nitrification rates in diff il d h N 15N i ifi i -5 0 5 10 15 20 25 0–2 2–4 4–6 6–8 8–10 Soil depth [cm] [mg N m−2 d−1] Ammonification Nitrification a a ab b a a) a) decrease in 0–2 cm (P00.038) and 2–4 cm soil depths (P00.006) and an increase in 4–6 cm soil depth (P0 0.001), were significant. The ratio of 15N-Nt-excess to native Nt (14N+natural 15N) within the surface organic layer (0–8 cm) decreased from 0.03 % after 2 years to 0.01 % after 11 years. The mean residence time (MRT) of N derived from the annual aboveground litterfall in the surface organic layer averaged to approx. 19 years and was higher than the MRT of C (10.5 years) in the surface organic layer. Mineralisation of N and 15N Net ammonification and mineralisation, measured at about 15 °C in the field, was low in 0–2 cm soil depth (L layer), highest in 2–4 cm soil depth (upper F layer; Fig. 2a), and decreased gradually below this depth. Net NH4 + immobilisation occurred in 8–10 cm soil depth (mineral soil). Net nitrification increased with soil depth within the organic layer but differences were not statis- tically significant (Fig. 2a). In total, net mineralisation amounted to 58 mg N m−2 d−1 within the surface 10 cm of the soil, half of which was nitrified. -1 0 1 2 3 4 5 0–2 2–4 4–6 6–8 8–10 Soil depth [cm] 15N ammonification [µg 15Nexcess m−2 d−1] b) a ab ab b a b) Net 15Nexcess ammonification in the surface 10 cm of the soil in general followed the trend of total net ammonification and in total amounted to 6±1 μg 15N m−2 d−1 (Fig. 2b). Half of the total 15N ammoni- fication could be attributed to the 2–4 cm soil depth. During the incubation period, 0.0086 % of the 15N label was net ammonified in the surface 10 cm of the soil per day (Fig. 2b). Assuming a similar contribution of the 15N label to net nitrification than to net ammo- nification and assuming annual net N mineralisation rates between 0.7 and 1.0 gN m−2 yr−1 (Brumme et al. 2009), 2–3 % of the litter N was net mineralised 11 years after the litter exchange. Fig. 2 Net ammonification and nitrification rates (a) and net 15N ammonification rates (b) of the surface 10 cm of the soil in the beech stand. On the right hand side of Fig. 2b, the daily recovery (Rec.) of the 15N-labelled litter in net ammonification is given for each soil layer. The undisturbed in situ incubation lasted 16 days and was conducted within the chamber bases of the litter exchange experiment in summer 2008. Among soil depths, means (±SE; n03) followed by the same letter were not significantly different (ANOVA, Tukey HSD-test at P≤0.05). Letters in figure a) belong to net ammonification rates and no significant differences occurred among net nitrification rates in different soil depths. Net 15N nitrification rates were not available Fig. 2 Net ammonification and nitrification rates (a) and net 15 Fig. Mineralisation of N and 15N No significant differences occurred between 15N and native N regarding the different soil depths (Student’st-test at P≤ 0.05) -0.05 0 0.05 0.1 0.15 0–2 2–4 4–6 6–8 8–10 Soil depth [cm] Net ammonification / Ntot [%] 15N label nativ N ac a a a bc ab b b a a emissions during the winter months (Fig. 4). The cumulative N2O emission amounted to 1.33± 0.26 kg N ha−1 yr−1 in 1999, 1.54±0.88 kg N ha−1 yr−1 in 2000, and 1.35±0.57 kg N ha−1 yr−1 in 2007–08. In 1999–2000 15N-N2Oemitted abundances were sig- nificantly higher in the litter exchange chambers than in the control chambers (Table 2, Fig. 4; P00.036). Cumulative 15N-N2Oexcess emissions were 0.29 g 15N ha−1 yr−1 in 2000 which was 0.04 % of the added 15N in leaf litter. Eleven years after the litter applica- tion, the recovery of 15N in emitted N2O decreased to 0.13 g 15N ha−1 yr−1 in 2007–08, which was 0.02 % of the added 15N in leaf litter. However, the abundances of 15N in N2O emitted from the soil in 2007–08 did not significantly differ between the litter exchange chambers and the control chambers (Table 2, Fig. 4). Fig. 3 Ratio of daily net ammonification to Nt given for 15Nexcess derived from labelled beech litter and for native N (14N+natural 15N) for the five depth intervals. Among soil depths, means (±SE; n03) followed by the same letter were not significantly different regarding 15N or native N, respectively (ANOVA, Tukey HSD-test at P≤0.05). No significant differences occurred between 15N and native N regarding the different soil depths (Student’st-test at P≤ 0.05) Assuming that native N contained in the exchanged leaf litter participated equally in N2O emissions as the 15N label, then 0.3 % (2007–08) and 0.6 % (1999–00) of total annual N2O emissions were derived from total N of a single litterfall year. Assuming that native N contained in the exchanged leaf litter participated equally in N2O emissions as the 15N label, then 0.3 % (2007–08) and 0.6 % (1999–00) of total annual N2O emissions were derived from total N of a single litterfall year. Mineralisation of N and 15N 2 Net ammonification and nitrification rates (a) and net 15N ammonification rates (b) of the surface 10 cm of the soil in the beech stand. On the right hand side of Fig. 2b, the daily recovery (Rec.) of the 15N-labelled litter in net ammonification is given for each soil layer. The undisturbed in situ incubation lasted 16 days and was conducted within the chamber bases of the litter exchange experiment in summer 2008. Among soil depths, means (±SE; n03) followed by the same letter were not significantly different (ANOVA, Tukey HSD-test at P≤0.05). Letters in figure a) belong to net ammonification rates and no significant differences occurred among net nitrification rates in different soil depths. Net 15N nitrification rates were not available About 0.6 % of total net N ammonification could be ascribed to the exchanged leaf litter, assuming that the native N contained in the 15N-labelled litter partic- ipated equally in net ammonification as the 15N label. Data for net 15Nexcess nitrification were not available due to technical problems. Also, no significant differences between these propor- tions occurred when the different depth intervals of the soil were considered separately (Fig. 3). On average, the daily net 15Nexcess ammonification of the surface 10 cm of the soil accounted for 0.030± 0.012 % of the 15N-Nt-excess. The proportion was two times higher than the proportion of net native N am- monification per day of the native Nt regarding the same depth interval, which was 0.015±0.005 %. How- ever, the differences were not statistically significant. N2O and 15N-N2O fluxes N2O and 15N-N2O fluxes Fluxes of N2O displayed pronounced seasonality with high emissions during the summer months and low Plant Soil (2013) 362:67–77 72 -0.05 0 0.05 0.1 0.15 0–2 2–4 4–6 6–8 8–10 Soil depth [cm] Net ammonification / Ntot [%] 15N label nativ N ac a a a bc ab b b a a Fig. 3 Ratio of daily net ammonification to Nt given for 15Nexcess derived from labelled beech litter and for native N (14N+natural 15N) for the five depth intervals. Among soil depths, means (±SE; n03) followed by the same letter were not significantly different regarding 15N or native N, respectively (ANOVA, Tukey HSD-test at P≤0.05). Relocation and sequestration of litter N in the soil ) Only limited amount of the applied 15N had been relocated to the lower part of the F layer (Fig. 1), despite the huge amount of 2.8 kg C m−2 and 0.07 kg N m−2 which had been deposited through total aboveground litterfall since the beginning of the label- ling experiment (Khanna et al. 2009). The surprisingly constant accumulation of 15N in the H layer over the 11 years of observation revealed the lack of bioturba- tion, though leaching of soluble organic and inorganic compounds between the F and H layer would have occurred. Labelled 15N accumulated in the H layer was probably derived from initially high leaching of soluble organic N during the early-stage decomposi- tion (first 2 years) as was also observed by Zeller et al. (2000). However, the downward movement of 15N into the lower F layer might primarily be caused by litter deposition and decomposition. Limited reloca- tion of litter 15N from the organic horizons to the mineral soil within 4–5 years after litter application was also reported from three European beech forest soils which accumulated 0.7–1.3 kg C on the mineral soil (Zeller and Dambrine 2011; Zeller et al. 2001). The slow incorporation rate of litter N into the mineral-bound soil fraction was attributed to the ab- sence of earthworms. Bird and Torn (2006) recovered very little 13C and 15N from pine needle litter below a Recovery of 92 % of the 15N label 2 years after the 15N-labelled beech litter application was in line with other studies and pointed to low N losses during the first years of decomposition. Zeller et al. (2000) found between 80 % and 85 % of 15N-labelled beech leaf litter 3 years after the litter application in the surface 30 cm of an acid soil that had a poorly developed moder type humus. Fahey et al. (2011) reported a recovery of over 90 % for 15N derived from sugar maple leaf litter application in the surface organic layer and surface 10 cm of the soil 1 year after litter application. However 11 years after litter application, 73 % of the initially added 15N had been mineralised, taken up by trees, leached, or emitted as gaseous N compounds in our study. Mineralisation of N and 15N 4 Mean (± SE; n03) N2O fluxes in the litter exchange experiment of the Solling beech stand from January 1999 to December 2000 (a) and from May 2007 to July 2008 (b) and mean (±SE; n03) 15N abundances of N2O emitted from the soil from the chambers where litter was exchanged and from the control chambers for the measurements in 1999–2000 (c) and in 2007–08 (d) 73 Plant Soil (2013) 362:67–77 Table 2 Abundances of 15N in N2O emitted from the soil of the beech stand during the measurements in 1999–2000 and 2007–08 15N-N2Oemitted [atom%] Control Litter exchange 1999–2000 0.3574 (0.0006)a 0.3782 (0.0090)b 2007–2008 0.3582 (0.0012)ab 0.3636 (0.0024)ab Among treatments, means (± SE; n03) followed by the same letter were not significantly different (ANOVA, Tukey HSD-test at P≤0.05) Table 2 Abundances of 15N in N2O emitted from the soil of the beech stand during the measurements in 1999–2000 and 2007–08 the 15N of exchanged litter 11 years after litter addi- tion. The annual amount of litter-N sequestration thus equals to about 0.9 gN m−2 yr−1 and significantly contributes to the mean total N sequestration of 2.1 g N m−2 yr−1 in the surface organic layer observed over a period of 35 years at the studied site (Meiwes et al. 2009). The site is characterized by high load of acid and nitrogen deposition (Meesenburg et al. 2009), which has led to an N-enriched but very acid soil with a low decomposition of the litter material and the absence of deep borrowing earthworms (Meiwes et al. 2009; Schaefer et al. 2009). Although the C/N ratio of the surface organic layer was constant over the last 35 years (Meiwes et al. 2009) the soil still is accumu- lating N as indicated by the low leaching losses of 0.17 gN m−2 yr−1 and input–output balances of 2.1 gN m−2 yr−1 (Brumme and Khanna 2009). The most rea- sonable explanation is the stabilisation process which was induced by N depositions either directly by mi- crobial N immobilisation or indirectly by plant uptake and the production of N-enriched litter including a big increase in fructification products (Khanna et al. 2009; Brumme and Khanna 2009). Mineralisation of N and 15N -20 0 20 40 60 80 100 120 N2O flux [µg N m −2 h −1] -20 0 20 40 60 80 100 120 0.32 0.34 0.36 0.38 0.40 0.42 Jan Mar May Jul Sep Nov Jan Mar May Jul Sep Nov 15N-N2Oemitted [atom% 15N] litter exchange control 0.32 0.34 0.36 0.38 0.40 0.42 May Jul Sep Nov Jan Mar May Jul 2007 2008 c) d) 1999 2000 a) b) Fig. 4 Mean (± SE; n03) N2O fluxes in the litter exchange experiment of the Solling beech stand from January 1999 to December 2000 (a) and from May 2007 to July 2008 (b) and mean (±SE; n03) 15N abundances of N2O emitted from the soil from the chambers where litter was exchanged and from the control chambers for the measurements in 1999–2000 (c) and in 2007–08 (d) -20 0 20 40 60 80 100 120 0.32 0.34 0.36 0.38 0.40 0.42 May Jul Sep Nov Jan Mar May Jul 2007 2008 d) b) from the chambers where litter was exchanged and from the control chambers for the measurements in 1999–2000 (c) and in 2007–08 (d) -20 0 20 40 60 80 100 120 0.32 0.34 0.36 0.38 0.40 0.42 May Jul Sep Nov Jan Mar May Jul 2007 2008 d) b) from the chambers where litter was exchanged and from the control chambers for the measurements in 1999–2000 (c) and in 2007–08 (d) -20 0 20 40 60 80 100 120 N2O flux [µg N m −2 h −1] a) -20 0 20 40 60 80 100 120 d) b) b) a) d) c) 0.32 0.34 0.36 0.38 0.40 0.42 May Jul Sep Nov Jan Mar May Jul 2007 2008 d) 0.32 0.34 0.36 0.38 0.40 0.42 Jan Mar May Jul Sep Nov Jan Mar May Jul Sep Nov 15N-N2Oemitted [atom% 15N] litter exchange control c) 1999 2000 Jan Mar May Jul Sep Nov Jan Mar May Jul Sep Nov 1999 2000 Fig. Mineralisation of N and 15N In 2007–08, the daily 15N-N2Oexcess emissions amounted to 0.0002 % of 15N-Nt-excess of the surface 10 cm of the soil, which was the same as the ratio of native N2O emissions to native Nt of the surface 10 cm of the soil assuming N2O only derived from the top 10 cm of the soil. Discussion Relocation and sequestration of litter N in the soil Relocation and sequestration of litter N in the soil The mineral soil was covered by only 0.36 kg C indicating a rapid incorporation of litter into the mineral soil. Beside downward movement there is evidence that fungal hyphae may be responsible for an upward movement of N and C (Zeller et al. 2000). We observed that a significant proportion of applied 15N was still present in the L layer 2 years after litter exchange (Fig. 1a). This may be due to fungal hy- phae transporting N from decaying 15N-labelled litter to fresh litter. Fungal transport of N amounted to 25– 35 % of exogenous N that was incorporated into decaying litter in an acid beech forest soil with low throughfall N depositions of 6–8 kg ha−1 yr−1 where- as 30–50 % of the incorporated N might have come from deposited N (Zeller et al. 2000). In the sugar maple stand investigated by Fahey et al. (2011), 18 % of external incorporated N in fresh litter was derived from the litter of the previous year. Bird and Torn (2006) suggested that as a result of fungal N translocation, organic horizons may provide a sink for leachable litter N. However, 11 years after litter exchange this process was of less importance in the present study as was indicated by a non-significant 15N enrichment in the L layer (Fig. 1b). About 0.6 % of total net N ammonification could be ascribed to the exchanged leaf litter and approxi- mately 2–3 % of the litter N was net mineralised 11 years after litterfall, indicating that the (labelled) leaf litter N still participated in measureable amounts in N turnover. Compared to the native N twice as much labelled N was mineralised from the labelled N pool in the lower F layer and the H layer (Fig. 3). This demonstrated that the N from the comparably new 15N-labelled beech litter was not preferentially stabi- lised compared to “older” native N, which was decom- posed more slowly. Results from three European beech forests, where mineralisation of litter 15N was 2–4-fold higher than mineralisation of native soil N 4– 5 years after litter application, were in line with our findings (Zeller and Dambrine 2011). The 15N re- leased from the decaying litter was mainly recovered in the coarse particulate organic matter fraction (>200 μm; Zeller and Dambrine 2011). Bird et al. Relocation and sequestration of litter N in the soil Uptake of litter N by trees probably represented the main sink for litter N, since total annual N uptake by trees amount to approximate- ly 11.0 gN m−2 compared to N leaching of 0.17 g and gaseous N emissions of 0.19 g N2O-N (Brumme and Khanna 2009) and of <0.05 g NOx-N (unpublished data) at the Solling beech stand. The low turnover rate of the N pool of the surface organic layer expressed by a high MRT of 19 years, in contrast to a MRT of 10.5 years for C, indicated high recovery of 27 % of Plant Soil (2013) 362:67–77 74 labelled beech litter by Zeller et al. (2000) showed that the shift from prevailing immobilisation to net mineralisation of N occurred after the first 2 years of decomposition when the C/N ratio had lowered to 25. In the present study ammonification and miner- alisation continuously decreased with increasing soil depth below 2–4 cm depth (Fig. 2). Due to small changes in the C/N ratio other mechanisms might be responsible for this decrease. Respiration measure- ments conducted with surface organic matter of an adjacent beech stand showed a strong decrease in the release of CO2 from 59 μg C ha−1 g−1 in the L layer to 12 and 3.9 μg C ha−1 g−1 in the F and H layer, respectively (Chodak et al. 2003). This clearly indicated a considerably decreased of mineralisation in the lower part of the surface organic layer prob- ably caused by chemical stabilisation processes since physical stabilisation processes should be of minor importance in organic horizons. At the same beech stand, 15N pool dilution studies conducted by Corre et al. (2003) revealed a gross mineralisation rate that was approx. four times higher than our net mineralisation rate indicating a high microbial recycling of N. Similar results were reported from other forest soils but their relevance for the N sequestration is still not clear (Venterea et al. 2004). 8 cm thick O-horizon in which the litter was placed 2 years after the beginning of the experiment. In contrast, Fahey et al. (2011) measured a comparable fast relocation of litter 15N in the soil of a sugar maple forest with low earthworm activities. In their study, already 1 year after litter application the greatest pro- portion of recovered 15N was found in 0–5 cm mineral soil depth. Relocation and sequestration of litter N in the soil (2008) also reported that 58 % of the 15N of needle litter was recovered in the light fraction, which repre- sents the SOM fraction with the highest turnover, in a Conclusions Our study demonstrated that one quarter of the 15N label of initially added beech leaf litter was still pres- ent in the surface 10 cm of the soil 11 years after litter addition. The high recovery of added 15N indicated a tight microbial N cycle. Movement of beech leaf litter N through the surface organic layer was very slow. Loss of litter N from the chamber bases was mainly attributed to tree uptake whereas losses by gaseous N2O and NOx emissions and by N leaching probably accounted for less than 1 % of the litter N during the 10 years of observation. The 15N-labelled beech leaf litter still participated measurably in N turnover and N2O production and obviously was not completely stabilised 11 years after litter exchange. The contribu- tion of beech leaf litter of a single year to total annual N2O emissions ranged between 0.3 % (eleventh year) and 0.6 % (second year). We suggest that the high N2O emissions observed in the investigated beech stand resulted from accumulated N in older humus or soil N pools, since neither leaf litterfall of the last 2 to 11 years nor N depositions of the recent year repre- sented a significant source for N2O. The 15N experiment indicated that the 15N-labelled beech litter N measurably contributed to total N2O fluxes. The contribution decreased from the 2–3 years measurement to the 10–11 years measurement after litter exchange due to the decrease in 15N-labelled beech litter mass. Eleven years after the litter ex- change, about 1 % of the net mineralised litter N was emitted as N2O. Probably less than 1 % of the litter N was lost as N2O during 2 and 11 years after litter exchange. The proportions of N2O-N of total N were the same for labelled 15N and native N in the surface 10 cm of the soil, despite the two times higher relative ammonifica- tion of labelled 15N than that of native N 11 years after the exchange. This result may be due to a significant contribution of native N2O produced below 10 cm depth. However, we have no information about the proportion of 15Nexcess nitrification of 15N-Nt-excess but which probably could further clarify the observation. Acknowledgment We thank the German Research Foundation for financial support (BR 1524/6). N2O emissions and contribution of leaf litter N The observation that neither leaf litter N during the 2–11 years period nor inorganic N depositions of the recent year (contribution of 6 % to total N2O emis- sions; Eickenscheidt et al. 2011) were primarily re- sponsible for high N2O emissions measured at the Solling beech stand, still raised the question of the origin of these emissions. The other sources of N2O emissions may include the first year litterfall N with possibly a rapid initial release of 15N as also was reported by Zeller et al. (2000). However, we suppose that the older N pool of accumulated older litter ma- terial and accumulated deposited N may represents the major source for N2O emissions from this soil. Emissions measured during our experiment were sim- ilar to the mean value of a 10-year measuring period at the Solling beech stand, which amounted to 1.9 kg N ha−1 yr−1 (Brumme and Borken 2009). The beech stand belonged to the seasonal emission type where due to low oxygen availability in the soil, caused by high microbial and root respiration and low gas diffu- sivity in the surface organic layer, denitrification was promoted during the summer months (Brumme et al. 1999; Wolf and Brumme 2002). Low gas diffusivity was the result of laminar undecomposed beech leaves in the L layer of the surface organic layer combined with high precipitation rate (Ball et al. 1997; Brumme and Borken 2009). Leaching of NO3 −to the transitional zone between the H layer and the surface mineral soil presumably provided the substrate for denitrification. Brumme (1995) reported that high N2O emissions oc- curred at temperatures of >10 °C and water tensions <200 hPa. Hence, years with high summer precipitation, as was the case for 2007 but not for 2008, exhibit high N2O emissions (compare annual fluxes of Fig. 4). Mineralisation of leaf litter N The summed net N mineralisation of the surface 10 cm of the soil amounted to 58 mg N m−2 d−1 (Fig. 2a), similar as was reported for this site in previous studies (Brumme et al. 2009). The surface organic layer of the investigated stand was the primary horizon for N mineralisation and contributed to 77 % to total net N mineralisation of the surface 20 cm of the soil (Brumme et al. 2009). The present study showed an increased in the net ammonification and mineralisation rates within the soil profile from the L layer (0–2 cm) to the upper F layer (2–4 cm; Fig. 2), which was the most important layer for N turnover. The change in the N turnover could be attributed to the decrease in the C/N ratio from 28 to 22 (Table 1). Litter-bag studies with 15N 75 Plant Soil (2013) 362:67–77 Scots pine forest during the initial 1.5 years of their study. eleventh litterfall year only contributed approx. 5 % to total annual N2O emissions using an average emis- sion of 0.46 % (harmonic mean) for a single litterfall year. References Springer, Berlin, pp 353–367 Lorenz K, Preston CM, Krumrei S, Feger K-H (2004) Decom- position of needle/leaf litter from Scots pine, black cherry, common oak and European beech at a conurbation forest site. Eur J Forest Res 123:177–188 Brumme R, Khanna PK (2009) Functioning and management of European beech ecosystems. Springer, Berlin Heidelberg, 490 pp Meesenburg H, Eichhorn J, Meiwes KJ (2009) Chapter 15: Atmospheric deposition and canopy interactions. In: Brumme R, Khanna PK (eds) Functioning and manage- ment of European beech ecosystems. Springer, Berlin, pp 265–302 Brumme R, Borken W, Finke S (1999) Hierarchical control on nitrous oxide emission in forest ecosystems. 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https://openalex.org/W2227036535	https://europepmc.org/articles/pmc2968197?pdf=render	English	null	Dimethyl 2,2′-(<i>p</i>-phenylenedioxy)diacetate	Acta crystallographica. Section E	2,009	cc-by	2,163	organic compounds Acta Crystallographica Section E Structure Reports Online ISSN 1600-5368 Acta Crystallographica Section E Structure Reports Online ISSN 1600-5368 = 0.11 mm1 T = 293 (2) K 0.30 0.20 0.10 mm 0.30 0.20 0.10 mm Data collection Enraf–Nonius CAD-4 diffractometer Absorption correction: scan (North et al., 1968) Tmin = 0.954, Tmax = 0.977 1123 measured reﬂections Reﬁnement R[F 2 > 2(F 2)] = 0.058 wR(F 2) = 0.173 S = 1.00 1123 reﬂections Data collection Enraf–Nonius CAD-4 diffractometer Absorption correction: scan (North et al., 1968) Tmin = 0.954, Tmax = 0.977 1123 measured reﬂections Related literature For details of the synthesis procedure and the applications of benzothiazoles, see: Chakraborti et al. (2004); Seijas et al. (2007); Wang et al. (2009). For details of the synthesis proce- dure and the applications of aryloxyacetic acids, see: Nagy et al. (1997); Wei et al. (2005). For the use of phase-transfer catalysis in organic synthesis, see: Perreux et al. (2001). For bond-length data, see: Allen et al. (1987). The authors thank the Center of Testing and Analysis, Nanjing University, for support. Supplementary data and ﬁgures for this paper are available from the IUCr electronic archives (Reference: DN2426). Table 1 Received 11 January 2009; accepted 23 January 2009 Cg1 is the centroid of the C1–C3/C1A–C3A ring. Cg1 is the centroid of the C1–C3/C1A–C3A ring. Cg1 is the centroid of the C1–C3/C1A–C3A ring. D—H A D—H H A D A D—H A C6—H6C Cg1i 0.97 2.98 3.674 (2) 130 Symmetry code: (i) x þ 3 2; y 1 2; z þ 1 2. Key indicators: single-crystal X-ray study; T = 293 K; mean (C–C) = 0.004 A˚; R factor = 0.058; wR factor = 0.173; data-to-parameter ratio = 13.7. The title compound, C12H14O6, was prepared by the Williamson reaction of 1,4-dihydroxybenzene and methyl chloroacetate with phase-transfer catalysis. The compound lies on an inversion center. The structure is stabilized by weak C— H interactions. Data collection: CAD-4 Software (Enraf–Nonius, 1989); cell reﬁnement: CAD-4 Software; data reduction: XCAD4 (Harms & Wocadlo, 1995); program(s) used to solve structure: SHELXS97 (Sheldrick, 2008); program(s) used to reﬁne structure: SHELXL97 (Sheldrick, 2008); molecular graphics: SHELXTL (Sheldrick, 2008); software used to prepare material for publication: SHELXTL. Acta Cryst. (2009). E65, o403 Supplementary data and ﬁgures for this paper are available from the IUCr electronic archives (Reference: DN2426). Ling-hua Zhuanga and Guo-wei Wangb* aDepartment of Applied Chemistry, College of Science, Nanjing University of Technology, Nanjing 210009, People’s Republic of China, and bDepartment of Light Chemical Engineering, College of Science, Nanjing University of Technology, Nanjing 210009, People’s Republic of China Correspondence e-mail: kingwell2004@sina.com.cn Table 1 Hydrogen-bond geometry (A˚ , ). References Allen, F. H., Kennard, O., Watson, D. G., Brammer, L., Orpen, A. G. & Taylor, R. (1987). J. Chem. Soc. Perkin Trans. 2, pp. S1–19. Chakraborti, A. K., Selvam, C., Kaur, G. & Bhagat, S. (2004). Synlett, pp. 851– 855. Enraf–Nonius (1989). CAD-4 Software. Enraf–Nonius, Delft, The Nether- lands. Harms, K. & Wocadlo, S. (1995). XCAD4. University of Marburg, Germany. N C Fili S V S d E & S d V (1997) S h C 27 Harms, K. & Wocadlo, S. (1995). XCAD4. University of Marburg, Germany. Nagy, C., Filip, S. V., Surducan, E. & Surducan, V. (1997). Synth. Commun. 27, 3729–3736. Nagy, C., Filip, S. V., Surducan, E. & Surducan, V. (1997). Synth. Commun. 27, 3729–3736. North, A. C. T., Phillips, D. C. & Mathews, F. S. (1968). Acta Cryst. A24, 351– 359. Experimental Experimental Crystal data C12H14O6 Mr = 254.23 Monoclinic, P21=n a = 7.4190 (15) A˚ b = 7.0990 (14) A˚ c = 11.785 (2) A˚ = 95.49 (3) V = 617.8 (2) A˚ 3 Z = 2 Mo K radiation p Crystal data C12H14O6 Mr = 254.23 Monoclinic, P21=n a = 7.4190 (15) A˚ b = 7.0990 (14) A˚ Perreux, L. & Loupy, A. (2001). Tetrahedron, 57, 7957–7966. Seijas, J. A., Vazquez, T. M. P., Carballido, R. M. R., Crecente, C. J. & Romar, L. L. (2007). Synlett, pp. 313–317. Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122. ( ) y Wang, G., Wu, L., Zhuang, L. & Wang, J. (2009). Acta Cryst. E65, o158. Wang, G., Wu, L., Zhuang, L. & Wang, J. (2009). Ac Wei, T. B., Liu, H., Li, M. L. & Zhang, Y. M. (2005). Synth. Commun. 35, 1759– 1764. o403 Zhuang and Wang o403 Zhuang and Wang doi:10.1107/S1600536809002980 Experimental 5.5 g (0.05 mole) hydroquinone was dissolved in 50 ml acetone, 6.9 g (0.05 mole) potassium carbonate, potassium iodide 0.8 g and tetrabutyl ammonium bromide 1.0 g were added. Then 8.8 ml L (0.10 mole) of methyl chloroacetate was dropped into the mixture. The mixture was boiled for 5 h with intensive stirring, cooled to room temperature, and filtered. The organic solution was evaporated under vacuum to dryness and the dry residue was recrystallized from methanol to obtain title compound. Crystals of (I) suitable for X-ray diffraction were obtained by slow evaporation of ethyl acetate. 1H NMR (CDCl3, δ, p.p.m.) 6.85 (m, 4H), 4.58 (s, 4H), 3.79 (s,6H). Comment The derivatives of aryloxyacetic acids and their derivatives constitute a class of compounds for both biological activity and plant growth regulators (Nagy et al., 1997; Wei et al.,2005). The phase-transfer catalysis, with the advantages of simple experimental operations, mild reaction conditions, and inexpensive and environmentally benign reagents, has established its significance in organic synthesis as one of the most useful methods for the acceleration of heterogeneous reactions (Perreux & Loupy, 2001). Benzothiazole are remarkable heterocyclic ring systems. They have been found to exhibit a wide spectrum of biological activities. Many kinds of 2-substituted benzothiazoles are utilized as vulcanization accelators in the manufacture of rubber,as fluorescent brightening agents in textile dyeing,and in the leather industry (Chakraborti et al.,2004; Seijas et al.,2007; Wang et al.,2009). There are numerous synthetic methods to produce 2-arylbenzothiazoles. The most important ones include the reaction of o-aminothiophenols with benzoic acids or their derivatives (Chakraborti et al.,2004; Seijas et al.,2007; Wang et al.,2009). We are focusing on the synthesis of new products of bisbenzothiazole. We here report the crystal structure of the title compound (I). The compound (I) lies on an inversion center(Fig.1). All bond lengths are within normal ranges (Allen et al., 1987). There are no typical hydrogen bonds, while weak intermolecular C—H···π interactions involving benzene ring (C1/C3/C2/ C1a/C3a/C2a) (Table 1) may help in stabilizing the structure. L. Zhuang and G. Wang L. Zhuang and G. Wang L. Zhuang and G. Wang supplementary materials supplementary materials supplementary materials Acta Cryst. (2009). E65, o403 [ doi:10.1107/S1600536809002980 ] Refinement All H atoms were positioned geometrically and treated as riding on their parent C atoms with C—H = 0.93 Å (aromat- ic), 0.97Å (methylene) and 0.96Å (methyl) with Uiso(H) = xUeq(C), where x= 1.5 for methyl H and 1.2 for aromatic and methylene H atoms. sup-1 supplementary materials Figures Fig. 1. A view of the molecular structure of (I) showing the atom-numbering scheme. Ellips- oids are drawn at the 30% probability level. H atoms are represented as small spheres of arbit- rary radii. [symmetry code: (A) 1/2-x, 1/2+y, 1/2-z]. g Fig. 1. A view of the molecular structure of (I) showing the atom-numbering scheme. Ellips- oids are drawn at the 30% probability level. H atoms are represented as small spheres of arbit- rary radii. [symmetry code: (A) 1/2-x, 1/2+y, 1/2-z]. Special details Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes) are estimated using the full covariance mat- rix. The cell e.s.d.'s are taken into account individually in the estimation of e.s.d.'s in distances, angles and torsion angles; correlations between e.s.d.'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s. planes. Refinement. Refinement of F2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F2, convention- al R-factors R are based on F, with F set to zero for negative F2. The threshold expression of F2 > σ(F2) is used only for calculating R- factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F2 are statistically about twice as large as those based on F, and R- factors based on ALL data will be even larger. supplementary materials 82 parameters Δρmin = −0.24 e Å−3 Primary atom site location: structure-invariant direct methods Extinction correction: none Δρmin = −0.24 e Å−3 Δρmin = −0.24 e Å−3 Primary atom site location: structure-invariant direct methods Extinction correction: none Primary atom site location: structure-invariant direct methods Extinction correction: none Extinction correction: none Dimethyl 2,2'-(p-phenylenedioxy)diacetate Dimethyl 2,2'-(p-phenylenedioxy)diacetate Crystal data C12H14O6 F000 = 268 Mr = 254.23 Dx = 1.367 Mg m−3 Monoclinic, P21/n Mo Kα radiation λ = 0.71073 Å Hall symbol: -P 2yn Cell parameters from 27 reflections a = 7.4190 (15) Å θ = 1–25º b = 7.0990 (14) Å µ = 0.11 mm−1 c = 11.785 (2) Å T = 293 (2) K β = 95.49 (3)º Block, yellow V = 617.8 (2) Å3 0.30 × 0.20 × 0.10 mm Z = 2 Data collection Enraf–Nonius CAD-4 diffractometer Rint = 0.0000 Radiation source: fine-focus sealed tube θmax = 25.3º Monochromator: graphite θmin = 3.1º T = 293(2) K h = −8→8 ω/2θ scans k = 0→8 Absorption correction: ψ scan (North et al., 1968) l = 0→14 Tmin = 0.954, Tmax = 0.977 3 standard reflecti 1123 measured reflections every 200 reflecti 1123 independent reflections intensity decay: 9 Enraf–Nonius CAD-4 diffractometer Rint = 0.0000 Radiation source: fine-focus sealed tube θmax = 25.3º Monochromator: graphite θmin = 3.1º T = 293(2) K h = −8→8 ω/2θ scans k = 0→8 Absorption correction: ψ scan (North et al., 1968) l = 0→14 3 standard reflections every 200 reflections intensity decay: 9% Secondary atom site location: difference Fourier map Hydrogen site location: inferred from neighbouring sites H-atom parameters constrained w = 1/[σ2(Fo 2) + (0.1P)2 + 0.23P] where P = (Fo 2 + 2Fc 2)/3 (Δ/σ)max = 0.001 Δρmax = 0.26 e Å−3 w = 1/[σ2(Fo 2) + (0.1P)2 + 0.23P] where P = (Fo 2 + 2Fc 2)/3 (Δ/σ)max = 0.001 Δρmax = 0.26 e Å−3 sup-2 supplementary materials Special details Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) x y z Uiso/Ueq O1 1.1983 (3) 0.2968 (2) −0.07350 (16) 0.0520 (6) O2 1.3969 (3) 0.5931 (3) −0.13385 (19) 0.0636 (7) O3 1.6159 (2) 0.3997 (3) −0.17937 (17) 0.0531 (6) C1 0.8429 (3) 0.0447 (3) 0.0519 (2) 0.0431 (7) H1A 0.7382 0.0734 0.0859 0.052 C2 0.9507 (3) 0.1863 (4) 0.0139 (2) 0.0444 (7) H2A 0.9186 0.3114 0.0235 0.053* C3 1.1039 (3) 0.1452 (4) −0.0376 (2) 0.0425 (7) C4 1.3668 (3) 0.2569 (4) −0.1199 (2) 0.0460 (7) H4A 1.3449 0.1844 −0.1896 0.055* H4B 1.4456 0.1845 −0.0658 0.055* C5 1.4535 (3) 0.4417 (4) −0.1439 (2) 0.0441 (7) C6 1.7243 (4) 0.5536 (4) −0.2101 (3) 0.0617 (8) H6A 1.8350 0.5067 −0.2354 0.093* H6B 1.7513 0.6340 −0.1452 0.093* H6C 1.6595 0.6240 −0.2705 0.093* Atomic displacement parameters (Å2) U11 U22 U33 U12 U13 U23 O1 0.0553 (11) 0.0450 (11) 0.0577 (12) 0.0023 (9) 0.0163 (9) 0.0020 (9) O2 0.0667 (14) 0.0491 (12) 0.0760 (16) 0.0059 (10) 0.0130 (11) −0.0036 (11) O3 0.0517 (11) 0.0536 (12) 0.0555 (12) −0.0040 (9) 0.0125 (9) −0.0034 (9) C1 0.0441 (14) 0.0464 (14) 0.0381 (14) 0.0046 (11) −0.0002 (10) −0.0050 (11) C2 0.0433 (14) 0.0448 (14) 0.0444 (16) 0.0021 (11) 0.0006 (11) −0.0049 (12) C3 0.0451 (14) 0.0459 (14) 0.0356 (14) 0.0001 (11) −0.0011 (11) 0.0007 (11) C4 0.0395 (13) 0.0500 (15) 0.0473 (15) −0.0007 (11) −0.0018 (11) 0.0007 (12) C5 0.0519 (15) 0.0476 (15) 0.0314 (13) 0.0028 (12) −0.0031 (11) −0.0023 (11) C6 0.0703 (19) 0.0603 (18) 0.0553 (18) −0.0192 (15) 0.0100 (14) 0.0034 (15) Atomic displacement parameters (Å2) sup-3 supplementary materials supplementary materials Geometric parameters (Å, °) O1—C3 1.372 (3) C2—C3 1.370 (4) O1—C4 1.440 (3) C2—H2A 0.9300 O2—C5 1.164 (3) C4—C5 1.499 (4) O3—C5 1.347 (3) C4—H4A 0.9700 O3—C6 1.424 (3) C4—H4B 0.9700 C1—C2 1.385 (4) C6—H6A 0.9600 C1—C3i 1.419 (3) C6—H6B 0.9600 C1—H1A 0.9300 C6—H6C 0.9600 C3—O1—C4 116.7 (2) C5—C4—H4A 110.2 C5—O3—C6 116.9 (2) O1—C4—H4B 110.2 C2—C1—C3i 118.4 (2) C5—C4—H4B 110.2 C2—C1—H1A 120.8 H4A—C4—H4B 108.5 C3i—C1—H1A 120.8 O2—C5—O3 125.3 (3) C3—C2—C1 121.2 (2) O2—C5—C4 128.6 (3) C3—C2—H2A 119.4 O3—C5—C4 106.1 (2) C1—C2—H2A 119.4 O3—C6—H6A 109.5 C2—C3—O1 116.0 (2) O3—C6—H6B 109.5 C2—C3—C1i 120.4 (2) H6A—C6—H6B 109.5 O1—C3—C1i 123.5 (2) O3—C6—H6C 109.5 O1—C4—C5 107.6 (2) H6A—C6—H6C 109.5 O1—C4—H4A 110.2 H6B—C6—H6C 109.5 C3i—C1—C2—C3 0.9 (4) C3—O1—C4—C5 −175.2 (2) C1—C2—C3—O1 178.9 (2) C6—O3—C5—O2 −1.9 (4) C1—C2—C3—C1i −0.9 (4) C6—O3—C5—C4 178.7 (2) C4—O1—C3—C2 175.4 (2) O1—C4—C5—O2 −3.6 (4) C4—O1—C3—C1i −4.8 (4) O1—C4—C5—O3 175.74 (19) Symmetry codes: (i) −x+2, −y, −z. Hydrogen-bond geometry (Å, °) D—H···A D—H H···A D···A D—H···A C6—H6C···Cg1ii 0.97 2.98 3.674 (2) 130 Symmetry codes: (ii) −x+3/2, y−1/2, −z+1/2. sup-4 supplementary materials Fig. 1 Fig. 1 sup-5
https://openalex.org/W2803076177	https://www.scielo.br/j/rbef/a/Rny9KVk9WBZ8nhzWFVSmBRF/?lang=pt&format=pdf	Portuguese	null	Espalhamento Bhabha: Um Estudo Detalhado	Revista Brasileira de Ensino de Física	2,018	cc-by	6,518	Recebido em 18 de Dezembro, 2017. Revisado em 26 de Fevereiro, 2018. Aceito em 14 de Março, 201 Neste artigo, vamos estudar alguns conceitos fundamentais em física de partículas através do estudo detalhado de um processo especíﬁco da Eletrodinâmica Quântica (QED): o espalhamento Bhabha em ordem dominante (Leading Order - LO). Este ocorre na interação entre um elétron e sua antipartícula, o pósitron, sendo um dos processos básicos da QED. Nossa escolha em trabalhar este processo deve-se a riqueza de detalhes proporcionada pelas duas possibilidades (canais) de interação, que servem para ilustrar o cálculo da interferência entre as possibilidades. Além disso, esse processo é utilizado para determinar a luminosidade de um determinado colisor, o que garante maior precisão nas medidas de outras grandezas relevantes para a análise das interações entre partículas. Finalmente, comparamos a predição da QED com os resultados do experimento DESY-PETRA-TASSO. Palavras-chave: física de partículas, processos elementares, eletrodinâmica quântica. In this article, we will look at some fundamental concepts in Particle Physics through the detailed study of a speciﬁc process of Quantum Electrodynamics (QED): the Bhabha scattering. It occurs in the interaction between an electron and its antiparticle, the positron, one of the basic QED processes. Our choice for work this process is due to the wealth of details provided by the two possibilities (channels) of interaction which serve to illustrate the calculation of interference between possibilities. Furthermore, this process is used to determine the luminosity of a particular collider, which ensures greater accuracy in measurements of other relevant quantities for the analysis of interactions between particles. Finally, comparing the QED prediction with the experimental results of the DESY-PETRA-TASSO experiment. Keywords: particle physics, elementary processes, quantum electrodynamics. Endereço de correspondência: fabio.kopp@ufrgs.br. Revista Brasileira de Ensino de Física, vol. 40, nº 3, e3318 (2018) www.scielo.br/rbef DOI: http://dx.doi.org/10.1590/1806-9126-RBEF-2017-0395 Revista Brasileira de Ensino de Física, vol. 40, nº 3, e3318 (2018) www.scielo.br/rbef DOI: http://dx.doi.org/10.1590/1806-9126-RBEF-2017-0395 Artigos Gerais cb Artigos Gerais cb Licença Creative Commons cb Copyright by Sociedade Brasileira de Física. Printed in Brazil. Espalhamento Bhabha: Um Estudo Detalhado Bhabha Scattering: A Detailed Study Fábio Köpp Nóbrega1, Luiz Fernando Mackedanz2 Fábio Köpp Nóbrega*1, Luiz Fernando Mackedanz2 1Universidade Federal do Rio Grande do Sul, Instituto de Física, Porto Alegre, RS, Brasil 2Universidade Federal do Rio Grande, Instituto de Matemática, Estatística e Física, Programa de Pós-Graduação em Educação em Ciências, Rio Grande, RS, Brasil Recebido em 18 de Dezembro, 2017. Revisado em 26 de Fevereiro, 2018. Aceito em 14 de Março, 2018. 1. Introdução que tenham interesse em aprofundar conhecimentos na física de partículas, além de buscar dar uma base para a Iniciação Cientíﬁca ao longo da graduação. A divulgação cientíﬁca de física de partículas, apesar das notícias apresentadas pela mídia nacional e internacio- nal da descoberta do Bóson de Higgs pelo CERN, ainda não possui muitos livros em português. O que diﬁculta um pouco o interesse de alunos de graduação por tal tema. No entanto, há dois livros de divulgação cientíﬁca, em português, de pesquisadores nesta área: ”O Discreto Charme das Partículas Elementares”da Professora Ma- ria Cristina Batoni Abdalla, destinado a pessoas leigas em física. E ”O Cerne da Matéria”do Professor Rogério Rosenfeld, o qual relata toda a trajetória dos físicos e experimentos que culminaram na descoberta do Bóson de Higgs noticiada no dia 4 de Julho de 2012 pelo CERN. O livro deste professor é recomendado tanto para físicos de outras áreas quanto propriamente para pesquisadores desta área. Assim, a principal motivação deste artigo é fornecer um material em português bem como os có- digos e scripts necessários para reproduzir os valores e gráﬁcos aqui apresentados. Por isso, direcionamos este texto para estudantes de graduação, nos primeiros anos, O estudo de processos básicos do Modelo Padrão (Stan- dard Model) ocorre, normalmente, nos últimos anos do bacharelado em física ou em disciplinas especíﬁcas de pós-graduação. Neste sentido, buscamos proporcionar ao aluno uma visão geral do processo Bhabha, bem como uma breve revisão sobre a teoria da Eletrodinâmica Quântica (QED: Quantum electrodynamics). O processo Bhabha, assim como outros processos da QED, possuem um ”sinal”limpo quando comparado a processos que en- volvam a colisão de núcleons (prótons e nêutrons). Em processos deste tipo, envolvendo energias maiores do que a massa destas partículas, a interação se dá a nível dos constituintes (quarks e glúons), produzindo uma miríade de novas partículas. A descrição da dinâmica destas in- terações é feita pela Cromodinâmica Quântica (QCD: Quantum Chromodynamics), associada a um novo nú- mero quântico: a carga de cor que cada quark pode portar. Esta pode ser comparada, de forma ingênua, com a carga elétrica, pois também possui dois tipos de carga: cor e anticor. Por outro lado, segundo a QCD, todos os estados Espalhamento Bhabha: Um Estudo Detalhado e3318-2 ligados detectáveis devem ter um estado ﬁnal branco - o chamado singleto de cor. 2”As equações de movimento são invariantes sob uma transforma- ção de Gauge local”. 1. Introdução propagador do elétron e do fóton devido ao escopo deste trabalho, para maiores detalhes vide [11–14]. Já na seção 3, fazemos uma revisão sobre algumas grandezas básicas utilizadas modernamente na Física de Partículas, bem como a atual forma de cálculo, através dos diagramas de Feynman. Na seção 4, apresentamos os cálculos do processo de forma detalhada, principalmente o termo de interferência, normalmente não incluído nos livros texto da área. Finalmente, elencamos algumas conclu- sões relevantes e aplicações do processo dentro das atuais colaborações nos aceleradores de partículas, como o ILC. Um exemplo deste tipo de interação é realizado no Grande Colisor de Hádrons (LHC: Large Hadron Colli- der). As partículas produzidas nesse tipo de colisor pos- suem diferentes propriedades, tais como spin, massa, e carga elétrica. No momento da análise da colisão, diferen- ciar uma partícula X de todas as outras requer detectores especíﬁcos e simulações entre a estrutura do detector e a física subjacente. Ainda, devemos lembrar que processos envolvendo partículas e antipartículas também surgem nesse tipo de colisão, porém, desta vez, regidos pela teoria Eletrofraca: uniﬁcação da teoria eletromagnética (QED) com a teoria para as interações fracas, responsáveis pelo decaimento nuclear beta e também pelo decaimento do píon e do múon, entre outras. Desta forma, dizemos que processos analisados pela teoria eletrofraca resultam num sinal experimental com menos ruído. O Colisor Linear Internacional1 (ILC: International Linear Collider) coli- dirá elétrons e pósitrons e utilizará o processo Bhabha para determinar a sua luminosidade [1]. Além disso, pro- curará determinar as propriedades do Bóson de Higgs, Dimensões extras e a partícula supersimétrica mais leve. 1https://www.linearcollider.org/ILC 2. Introdução à Eletrodinâmica Quântica A eletrodinâmica quântica (QED, do inglês Quantum Electrodynamics) é a teoria que uniﬁca os fenômenos ele- tromagnéticos, como ondas de rádio e radiações, com a física quântica, levando a aplicações práticas na eletrô- nica, computação e descreve as interações entre quarks e léptons. Ainda, devemos salientar que a QED é quase toda a base da química que conhecemos. Além disso, é uma teoria Abeliana, ou seja, os campos de gauge comu- tam entre si ([Aµ, Aν] = 0) pertencendo ao grupo U(1), sendo estes os campos eletromagnéticos. Por Abeliano, dizemos que os mediadores de campo (os fótons), não possuem carga e, portanto, não podem interagir entre si. Nos diagramas de Feynman temos vértices, ponto de ”contato”, entre partículas/anti-particulas com o pro- pagador do bóson que media a interação. Este vértice surge ao aplicarmos o princípio de Gauge2 nos campos fermiônico. Para termos uma ideia de como o obtemos, partimos do Lagrangeano para partícula livre de spin 1/2 (Dirac) dado por A utilidade mais próxima de nós do estudo desse tipo de processo pode ser visto em [2] o qual trata da antimatéria na medicina moderna. Por ﬁm, devemos lembrar que há estudos igualmente importantes nesta área como estados ligados, decaimentos, oscilações de neutrinos e medidas de momentos dipolares magnéticos e elétricos. Neste sentido, propomos trabalhar a transposição ex- terna do conteúdo, aquela onde o pesquisador traz os conhecimentos da área para um nível acessível ao estu- dante interessado nesta área da Física, inserindo novas visões para temas já apresentados nos primeiros anos da graduação, ou novos conceitos apresentados pela primeira vez [3]. Nosso foco, neste artigo, é discutir fundamentos da Física de Partículas Elementares, através dos pro- cessos da Eletrodinâmica Quântica (QED) [4–9], haja vista que os processos básicos de interação entre elétrons, ou entre o elétron e sua antipartícula - o pósitron - são importantes em diversos ramos de aplicação da Física, como por exemplo a Física das Radiações [10], onde a aniquilação e produção de pares são utilizadas para o diagnóstico e tratamento de doenças. Este é o princípio da tomograﬁa por emissão de pósitrons (PET). Em par- ticular, vamos nos dedicar ao estudo de um processo que inclua estes dois aspectos: o processo Bhabha, que pode ser interpretado como uma aniquilação entre elétron e pósitron seguida da produção do par. LDirac = ¯ψ(iγµ∂µ −m)ψ. DOI: http://dx.doi.org/10.1590/1806-9126-RBEF-2017-0395 Revista Brasileira de Ensino de Física, vol. 40, nº 3, e3318, 2018 2. Introdução à Eletrodinâmica Quântica (1) (1) A transformação de gauge local (dependência de coorde- nadas tipos espaço em α(x)) ocorre da seguinte forma: ψ ′(x) →eiα(x)ψ(x) e ¯ψ ′(x) →¯ψ(x)e−iα(x) (2) (2) Vale lembrar que existem outros tipo de transforma- ções de gauge, como a global. Em que α, desta vez, não depende de coordenadas tipo espaço. Retornando a de- rivação, substituindo (2) em (1) e tomando a derivada parcial, obtemos: ∂µψ ′ →eiα(x)∂µψ(x) + ieiα(x)ψ(x)∂µα(x) (3) (3) O segundo termo de (3) quebra a invariância de LDirac. Uma forma de contornar esse problema, é modiﬁcar a derivada parcial para derivada covariante de forma a satisfazer a invariância de gauge local Por esse motivo, este texto básico se destina a es- tudantes de graduação interessados na fenomenologia dos processos da Física de Partículas, como uma leitura introdutória que apresenta as grandezas fundamentais envolvidas nestes. Para tanto, na seção 2, apresentaremos o princípio de Gauge aplicado a QED e sua aplicação ao processo Bhabha. No entanto, não vamos derivar o D ′ µψ ′ →eiα(x)Dµψ, (4) (4) 2”As equações de movimento são invariantes sob uma transforma- ção de Gauge local”. Revista Brasileira de Ensino de Física, vol. 40, nº 3, e3318, 2018 e3318-3 e3318-3 Nóbrega e Mackedanz em que deﬁnimos em que deﬁnimos que ilustra com bastante clareza as 4 componentes, di- ferenciadas pelo sinal negativo ante das coordenadas espaciais. Por conta deste sinal, dizemos que este espaço- tempo possui uma métrica diagonal, ou seja, a matriz que representa tal espaço-tempo é representada por uma matriz apenas com elementos não nulos na diagonal prin- cipal. Esta é chamada de métrica de Minkowski com diag(1, −1, −1, −1). D ′ µ ≡∂µ −ieA ′ µ, (5) (5) onde Aµ é um campo vetorial que transforma-se como: onde Aµ é um campo vetorial que transforma-se como: A ′ µ →Aµ + 1 e∂µα(x). (6) (6) (6) Refazendo o cálculo anterior, exceto por termos agora Dµ, obtemos Refazendo o cálculo anterior, exceto por termos agora Dµ, obtemos Na análise dinâmica de um espalhamento, a grandeza generalizada será o 4-vetor momentum (cf. [19–22]), de- notado por D ′ µψ ′ = Dµ(eiα(x)ψ) = (∂µ −ieA ′ µ)(eiα(x)ψ). (7) Substituindo (6) em (7), vem (7) pµ = (p0, p1, p2, p3) = (E, px, py, pz), (11) (11) Substituindo (6) em (7), vem D ′ µψ ′ = [∂µ −ie(Aµ + 1 e∂µα(x) ) ](eiα(x)ψ). 2. Introdução à Eletrodinâmica Quântica (8) a qual respeitar as transformações de Lorentz entre re- ferenciais inerciais. Comumente os referenciais mais uti- lizados são o de laboratório (LAB) onde as partículas envolvidas têm seus momenta medidos em relação a algo externo à colisão e o referencial de centro de massa (CM), usado para facilitar as considerações das leis de conser- vação (momentum e energia). Deste último, falaremos um pouco mais pela sua praticidade para analisar os espalhamentos. Lembramos que, para estes processos, a conservação do 3-vetor momentum é garantida. (8) Desta forma, encontramos D ′ µψ ′ = eiα(x)(∂µ −ieAµ)ψ = eiα(x)Dµψ. (9) (9) Logo, a equação (9) satisfaz à condição (4) tornando-se, assim, invariante de gauge local. Para podermos entender o processo de espalhamento Bhabha, bem como estender os conceitos para demais pro- cessos, precisamos inicialmente ter alguns conhecimentos e deﬁnições básicos sobre referenciais e grandezas pró- prias da Física de partículas. A próxima seção é dedicada a apresentar estes fundamentos, de forma breve. Eles podem ser encontrados, para quem desejar aprofundar seus estudos em [11–15]. No referencial de centro de massa, vamos considerar o processo esquematizado na Fig. 1 como uma colisão elástica, ou seja, Etotal = E1 + E2 também é conservada. Neste referencial, o momentum total inicial é nulo, ou seja, (Ptotal = P1 + P2) = 0. Isto signiﬁca que P1 = −P2). Além disso, como estamos trabalhando com feixes de mesma energia, com partículas de massa similar (pó- sitrons e elétrons), consideramos E1 = E2 = Ecm e P1 = P2 = P. DOI: http://dx.doi.org/10.1590/1806-9126-RBEF-2017-0395 Revista Brasileira de Ensino de Física, vol. 40, nº 3, e3318, 2018 Espalhamento Bhabha: Um Estudo Detalhado e3318-4 medida em unidades de área. Pelas dimensões envolvidas na Física de Partículas, deﬁnimos uma unidade especial chamada barn, em que 1 barn = 10−28 metros2. o que mostra que a massa é uma grandeza invariante. O último passo usa as deﬁnições para energia e momentum relativísticos. A partir destas relações entre os momenta iniciais, podemos construir relações úteis no referencial CM, também invariantes. Para determinar corretamente a seção de choque, deve- mos lembrar que a representação ocorre num espaço de transferência de momentum, chamado de espaço de fase do processo. Considerando um processo 1 + 2 →3 + 4, existe uma taxa de transição por unidade de volume deste espaço de fase Wfi. Em outras palavras, esta é a densidade de probabilidade do processo ocorrer. A seção de choque é proporcional a esta densidade, considerando o ﬂuxo de partículas incidentes e o número de estados detectados ao ﬁnal do processo. A forma como os 4-momenta são transferidos entre os estados inicial e ﬁnal permitem deﬁnir grandezas inva- riantes, denominadas variáveis de Mandelstam. Elas apresentam relações que envolvem a energia, o momen- tum e o ângulo de espalhamento das partículas iniciais e ﬁnais do processo. Tomando uma das partículas (e seu momentum) como base, podemos ter três combinações, vistas como canais de transferência de momentum: ani- quilação (canal s) e espalhamento (canais t e u), deﬁnidos como: Considerando cada um destes termos em separado, iniciamos com o número de estados ﬁnais. Para deﬁ- nir o número total destes, vamos considerar uma porção ﬁnita do espaço de fase, uma caixa cúbica de lado L. Em cada dimensão linear, o número de estados possíveis é L 2π∆p, onde o fator 2π surge das condições de contorno periódicas do problema. Como este espaço considerado é isotrópico, o número total de estado disponíveis dentro desta caixa é V (2π)3 ∆3p. O número de partículas em cada unidade de volume é deﬁnido como 2E, de forma que o número de estados permitidos por partícula é s = (P1 + P2)2 = (P3 + P4)2 t = (P1 −P3)2 = (P2 −P4)2 (15) u = (P1 −P4)2 = (P2 −P3)2 (15) No limite relativístico, a massa invariante das par- tículas envolvidas no espalhamento Bhabha pode ser considerada como nula (P 2 i = m2 i ≈0) e, aplicando a relação energia-momento 13, obtemos V (2π)3 d3p 2E . (19) V (2π)3 d3p 2E . Espalhamento Bhabha: Um Estudo Detalhado (19) s = P 2 1 + P 2 2 + 2P1P2 ≈2P1P2 = 2(E2 −⃗P1 · ⃗P2) s = 2(E2 −\|⃗P\|2cos θ) = 4E2 (16) (19) (16) O próximo passo é o cálculo do ﬂuxo inicial de partì- culas, onde podemos interpretar uma delas como projétil e outra como alvo. O ﬂuxo relativo à partícula incidente, por unidade de volume é dado pelo produto entre sua velocidade e a densidade volumétrica; logo, \|v\| 2E V . Para o alvo, apenas nos interessa sua densidade 2E V . onde o ângulo entre os vetores momentum das partículas corresponde a 180 graus (pela conservação do momentum linear). As demais variáveis de Mandelstam podem também ser deﬁnidas em termos do ângulo de espalhamento θ, dadas por: Para ﬁnalizar, precisamos determinar a densidade de probabilidade de ocorrência da colisão. Ela está associada à amplitude de espalhamento M, em es- pecial seu quadrado, tendo em vista que a densidade é um número real. Além disso, ela carrega um termo de conservação do 4-momentum, a função delta de Dirac, δ(4)(p). Assim, pode ser expressa como s = 4E2 t = −2\|⃗P\|2(1 −cos θ) = −4E2sen2(θ 2) (17) u = −2\|⃗P\|2(1 + cos θ) = −4E2cos2(θ 2) (17) Com isto, recuperamos, no limite relativístico (m ≈0), uma relação básica entre as variáveis de Mandelstam (garantida pela conservação do 4-momentum) Wfi = (2π)4δ(4)(pC + pD −pA −pB) V 4 \|M\|2, (20) (20) onde temos a normalização por unidade de volume. Con- siderando o processo A+B →C +D, podemos relacionar cada partícula a seu 4-momentum e temos s + t + u = 4m2. (18) (18) Na próxima seção, vamos deﬁnir uma quantidade funda- mental na Física de Partículas, uma vez que trabalhamos com espalhamentos e estes devem ter uma relação geo- métrica. ∆σ = 1 \|vA\|2EA2EB 1 (2π)3 d3pC 2EC 1 (2π)3 d3pD 2ED (2π)4 × δ(4)(pC + pD −pA −pB)\|M\|2, (21) (21) onde deﬁnimos a seção de choque pela letra grega σ. Como o volume do espaço é arbitrário, ele é cancelado nesta expressão. Podemos identiﬁcar aqui três elementos, genericamente denotados por Revista Brasileira de Ensino de Física, vol. 40, nº 3, e3318, 2018 DOI: http://dx.doi.org/10.1590/1806-9126-RBEF-2017-0395 3. Parâmetros em Física de Partículas Em termos dos 4-momenta, P µ 1 = (E1, P) e P µ 2 = (E2, −P). Para calcularmos um invariante associado ao 4- vetor momentum, utilizamos a regra de soma de Einstein Para estudarmos os processo básicos da Física de Partí- culas, em que no nosso caso é o espalhamentos, devemos conhecer a cinemática relativística, que descreve as gran- dezas dinâmicas e suas relações em altas energias. Nessa seção apresentaremos os principais parâmetros físicos necessários para compreendermos os processos simples, como o espalhamento Bhabha. Durante todo o trabalho, usaremos as unidades naturais: c=1 e ℏ=1, da forma como usualmente é trabalhado na Física de Partículas. Isso permite que tratemos todas as grandezas cinemáti- cas em termos de unidades de energia, que na área é o elétron-Volt (eV) e seus múltiplos. P 2 = P µPµ, (12) (12) onde a mudança na posição do índice simboliza a troca da matriz que representa o 4-vetor por sua transposta. O produto escalar entre dois 4-vetores momentum (também invariante de Lorentz) nos leva a relação de energia- momentum, P1 · P2 = P µ 1 · Pµ2 = (E, P) · (E, −P)T (13) = E2 −\|⃗P\|2 = m2, (14) Figura 1: Representação do espalhamento 2 →2 (13) (14) (14) Uma vez que os processos investigados envolvem partí- culas em altas velocidades, comparáveis à da luz (v ≈c), devemos utilizar a Relatividade Restrita( [16–18]). Nesse contexto, precisamos deﬁnir inicialmente grandezas que sejam invariantes de referencial, ou seja, grandezas que tem um valor único, não importando o referencial no qual as computemos. Uma rápida inspeção em textos básicos sobre Relatividade Restrita nos mostra que estas grandezas podem ser relacionados como um 4-vetor, com 1 componente temporal adicionada às três espaciais. O exemplo clássico é o intervalo deﬁnido no espaço-tempo Figura 1: Representação do espalhamento 2 →2 (∆s)2 = (∆t)2 −(∆x)2 −(∆y)2 −(∆z)2, (10) (10) Revista Brasileira de Ensino de Física, vol. 40, nº 3, e3318, 2018 Espalhamento Bhabha: Um Estudo Detalhado Espalhamento Bhabha: Um Estudo Detalhado e3318-5 e3318-5 Figura 2: Regras de Feynman para QED [23] onde F = \|vA\|2EA2EB e o elemento do espaço de fase invariante de Lorentz dLips = (2π)4δ(4)(pC + pD −pA − pB) d3pC (2π)32EC d3pD (2π)32ED . Estes dois termos podem ser cal- culados e demonstram ser manifestamente invariantes. A informação física sobre o processo reside apenas na amplitude de espalhamento. No referencial de centro de massa, onde os vetores mo- mentum são simétricos, os fatores invariantes são simpli- ﬁcados em termos de ângulo de espalhamento e variáveis de Mandelstam dLips = 1 (4π2) \|⃗pf\| 4√sdΩ (23) F = 4\|⃗pi\|√s, (24) (23) Figura 2: Regras de Feynman para QED [23] (24) onde \|⃗pi\| = \|⃗p1,2\| e \|⃗pf\| = \|⃗p3,4\|.Assim, a seção de choque diferencial (dependente do ângulo de espalhamento θ) para o processo é dada por [24]: dσ = \|⃗pf\| \|⃗pi\| < \|M\|2 > 64π2s dΩ (25) (25) Como esta colisão é elástica (e a conservação de momen- tum linear nos garante que \|⃗pf\| = \|⃗pi\|), temos E1 = E2. Logo, s = (E1 + E2)2 e: dσ dΩ= < \|M\|2 > 64π2(2E)2 . (26) (26) Figura 2: Regras de Feynman para QED [23] Para obtermos a predição dada pela teoria, resta-nos determinar o termo \|M\|2, associado à amplitude do es- palhamento, calculado pela teoria e que nos informa a intensidade da interação. contribuição negativa devido ao Princípio da Exclusão de Pauli. Richard Phillips Feynman3 mostrou como calcular es- tas amplitudes a partir de representações gráﬁcas, com regras deﬁnidas na Teoria Quântica de Campos para estas interações entre partículas, tratando estas repre- sentações não no espaço de posições, mas no espaço de momentum, ou seja, ilustrando graﬁcamente o ﬂuxo do momentum. Partindo dos diagramas de Feynman de um dado processo e usandos essas regras, apresentadas na Figura (2), conseguimos obter a amplitude invariante de Lorentz. Na próxima seção, utilizaremos estas regras para determinar a amplitude M. Vamos utilizar as regras apresentadas na ﬁgura 2 a ﬁm de obter a amplitude de Lorentz referente ao diagrama 1. Para analisar o gráﬁco devemos perceber que as linhas contínuas representam os férmions (elétrons ou pósitrons) e a seta indicativa do momentum diferencia ambos. Ou seja, considerando o tempo a partir da esquerda, P1 e P3 representam elétrons e P2 e P4 representam pósitrons. A leitura deste diagrama, usando as regras ilustradas na Fig. 2, nos fornece M1 = −e2 s [¯u(3)(γµ)v(4)][¯v(2)(γµ)u(1)] (27) (27) 3http://www-history.mcs.st-and.ac.uk/Biographies/Feynman. html 4. Seção de choque A seção de choque é um parâmetro que informa a proba- bilidade de uma dada reação ocorrer, sendo este medido experimentalmente. Além disso, ela pode ser interpre- tada como uma área efetiva de interação e, portanto, dσ = \|M\|2 F dLips, (22) (22) DOI: http://dx.doi.org/10.1590/1806-9126-RBEF-2017-0395 Revista Brasileira de Ensino de Física, vol. 40, nº 3, e3318, 2018 Nóbrega e Mackedanz Nóbrega e Mackedanz Revista Brasileira de Ensino de Física, vol. 40, nº 3, e3318, 2018 5. O Processo Bhabha e sua conjugada e sua conjugada Conforme mencionado na introdução, o processo estu- dado por Bhabha [25] é o espalhamento elétron-pósitron (e−+ e+ →e−+ e+). Para este processo temos a contri- buição de dois diagramas, conforme podemos ver na Fig. (3). O diagrama 1, corresponde ao processo de aniquila- ção, e o diagrama 2 ao de espalhamento. M∗ 1 = −e2 s [¯v(4)(γµ)u(3)][¯u(1)(γµ)v(2)] (28) (28) A troca de momentum desse diagrama ocorre no canal s, ou seja, s = (P1 + P2)2. A amplitude total de Lorentz para este processo con- sidera as contribuições dos dois diagramas, bem como de um termo de interferência entre eles. Este termo tem A média quadrática da amplitude invariante de Lorentz é deﬁnida por: < \|M\|2 >= 1 4\|M\|\|M†\|, (29) (29) DOI: http://dx.doi.org/10.1590/1806-9126-RBEF-2017-0395 Revista Brasileira de Ensino de Física, vol. 40, nº 3, e3318, 2018 318-6 Espalhamento Bhabha: Um Estudo Detalh Figura 3: Canais com as contribuições de aniquilação (canal s) e espalhamento (canal t) para o processo Bhabha e3318-6 Espalhamento Bhabha: Um Estudo Detalhado : Canais com as contribuições de aniquilação (canal s) e espalhamento (canal t) para o processo Bhabha Figura 3: Canais com as contribuições de aniquilação (canal s) e espalhamento (canal t) para o onde o fator 1 4 surge devido à média sobre todas as possíveis conﬁgurações de spins das partículas incidentes4 Podemos veriﬁcar que os dois diagramas têm expressões similares ﬁnais, apenas mediante a troca s ↔t. Esta observação é importante para que possamos veriﬁcar que os dois termos presentes na interferência terão este mesmo comportamento. Com isso, e utilizando as relações de completeza pre- sente no apêndice A1.1 e A1.2, tanto para o pósitron quanto para o elétron5, obtemos Para concluir o cálculo deste processo, devemos obter s termo de interferência. Na primeira parte dele, juntamos as expressões já deﬁnidas acima e temos < \|M1\|2 >= 8e4 (P1 + P2)4 [(p1·p3)(p2·p4)+(p1·p4)(p2·p3)], (30) i ã l ti í ti t tili d R (30) M1M∗ 2 = e4 ts [¯u(3)(γµ)v(4)] \| {z } a [¯v(2)(γµ)u(1)] \| {z } b × [¯u(1)(γν)u(3)] \| {z } c [¯v(4)(γν)v(2)] \| {z } d na aproximação relativística que estamos utilizando. Re- escrevendo esta expresão em termos das variáveis de Mandelstam (Eqs. 15), encontramos < \|M1\|2 >= 2e4 t2 + u2 s2 (31) (31) Como a,b,c e d são números, vamos reordená-los de maneira conveniente. Revista Brasileira de Ensino de Física, vol. 40, nº 3, e3318, 2018 DOI: http://dx.doi.org/10.1590/1806-9126-RBEF-2017-0395 ísica, vol. 40, nº 3, e3318, 2018 DOI: http://dx.doi.org/10.1590/1806-9126-RBEF-2017-0395 5. O Processo Bhabha Logo: No caso do diagrama 2, onde a troca de momentum desse diagrama ocorre no canal t, ou seja, t = (P1−P3)2, temos M1M∗ 2 = e4 ts [a · d · b · c] = e4 ts [¯u(3)(γµ)v(4)] \| {z } a × [¯v(4)(γν)v(2)] \| {z } d [¯v(2)(γµ)u(1)] \| {z } b [¯u(1)(γν)u \| {z c M2 = −e2 k2 [¯u(3)(γν)u(1)][¯v(2)(γν)v(4)] (32) (32) e sua conjugada e sua conjugada M∗ 2 = −e2 t [¯u(1)(γν)u(3)][¯v(4)(γν)v(2)] (33) Somando sobre todos os spins e sobre a média dos spins das partículas incidentes, temos: (33) Somando sobre todos os spins e sobre a média dos spins das partículas incidentes, temos: (33) Então, de forma similar ao cálculo que levou a Eq. 31, obtemos < \|M1M∗ 2\| >= X s X s X s X s 1 4\|M1M∗ 2\| Então, de forma similar ao cálculo que levou a Eq. 31, obtemos < \|M2\|2 >= 8e4 (P1 −P3)4 [(p1·p4)(p2·p3)+(p1·p2)(p3·p4)] (34) Como estamos trabalhando com números, conforme mos- tramos acima para rearranjá-los, esta expressão pode ser substituída usando a deﬁnição de traço de matriz (apêndice A.2.3) e a relação de completeza (apêndice A.1.1 e A.1.2) ( ) ou, em termos das variáveis de Mandelstam ( ) ou, em termos das variáveis de Mandelstam < \|M2\|2 >= 2e4 u2 + s2 t2 . (35) (35) < \|M1M∗ 2\| > = 1 4 e4 ts Tr[u(3)¯u(3) \| {z } P3+m γµ v(4)¯v(4) \| {z } P4−m γν × v(2)¯v(2) \| {z } P2−m γµ u(1)¯u(1) \| {z } P1+m γν] 4A média é dada por 1 (2S1+1)(2S2+1) onde Si é o spin da partícula incidente i. Neste caso em particular, o spin do elétron é 1/2. O spin do pósitron é idêntico ao do elétron. Logo, S1 = S2 = 1/2, resultando no fator 1/4. / 5Os espinores u e v satisfazem a equação de Dirac no espaço de momento. Tendo as seguintes características: são ortogonais ( ¯u1u2 = 0 e ¯v1v2 = 0) e normalizados( ¯u1u1 = 2mc e ¯v1v1 = −2mc). onde usamos a propriedade cíclica do traço para deslocar u(3) para o início do termo. No limite relativístico, que DOI: http://dx.doi.org/10.1590/1806-9126-RBEF-2017-0395 Revista Brasileira de Ensino de Física, vol. 5. O Processo Bhabha < \|M1M∗ 2\| >= 1 4 e4 ts (−2)Tr[ P3 γν P1 P4γν \| {z } D P2] Fazendo uso da propriedade das matrizes gama(apêndice A.3.7) em D6 na equação acima: < \|M1M∗ 2\| >= 1 4 e4 ts (−8(P1 · P4)Tr[ P3 P2] Por ﬁm, aplicando o teorema do traço (apêndice A.4.4), obtemos < \|M1M∗ 2\| >= 1 4 e4 ts (−32)(P1 · P4 · P3 · P2) Para obtermos a ﬁgura 4, os dados experimentais de- vem ser dividos por s (energia de centro de massa ao quadrado), pois é desta forma que os dados experimentais estão no artigo experimental. ou, em termos das variáveis de Mandelstam ou, em termos das variáveis de Mandelstam −< \|M1M∗ 2\| >= 2e4 u2 ts (37) (37) Além disso, integrando a equação(35) no ângulo sólido \|cos(θ)\| < 0.84, obtemos a seção de choque total em relação a energia de centro de massa, como é mostrado na ﬁgura 5. Como a troca do diagrama 1 pelo 2 levou a resultados similares em suas amplitudes de espalhamento, exceto pela troca s ↔t, podemos considerar a mesma troca para a segunda parte do termo de interferência, o que leva a um valor igual ao obtido no primeiro. Assim, a soma deles resulta em: Este resultado, confrontando teoria e experimento, é a principal avaliação de nosso cálculo. Antes de aﬁrmarmos a boa descrição dos dados experimentais, é importante ressaltar que esta etapa nos permite grandes avanços na teoria e na fenomenologia da Física de Partículas. Esta comparação deve ser vista, então, como o principal resultado de qualquer cálculo de processos na Física de Altas Energias. −(< \|M1M∗ 2\| > + < \|M ∗ 1 M2\| >) = 4e4 u2 ts (38) (38) Com isso, a amplitude total de Lorentz é: < \|M\|2 >= 2e4 t2 + u2 s2 + u2 + s2 t2 + 2u2 ts (39) (39) 5. O Processo Bhabha 40, nº 3, e3318, 2018 Nóbrega e Mackedanz e3318-7 e3318-7 Nóbrega e Mackedanz Usando as relações trigonométricas e a substituição x = cos θ, podemos reescrever como utilizamos, m ≈0 e temos: < \|M1M∗ 2\| >= 1 4 e4 ts Tr[( P3γµ P4γν P2γµ P1γν)] < \|M\|2 >= 2e4 (1 + x)2 + 4 (1 −x)2 + (1 + x2) 2 −(1 + x)2 (1 −x) Aplicando novamente a propriedade do traço (apêndice A.2.3) na equação anterior, temos: Aplicando a expressão acima na Eq. 26, obtemos < \|M1M∗ 2\| > = 1 4 e4 ts Tr[( P3γµ P4γν P2 \| {z } A γµ P1γν \| {z } B )] = 1 4 e4 ts Tr[γµ P1γν \| {z } B P3γµ P4γν P2 \| {z } A ] < \|M1M∗ 2\| > = 1 4 e4 ts Tr[γµ P1γν P3γµ \| {z } C P4γν P2] (36) dσ dΩ= 2e4 64π2(2E)2 (1 + x)2 + 4 (1 −x)2 + (1 + x2) 2 −(1 + x)2 (1 −x) usando a deﬁnição da constante de estrutura ﬁna(α = e2 4π ≈ 1 137) e de energia de centro de massa (√s = 2Efeixe): usando a deﬁnição da constante de estrutura ﬁna(α = e2 4π ≈ 1 137) e de energia de centro de massa (√s = 2Efeixe): < \|M1M∗ 2\| > = 1 4 e4 ts Tr[γµ P1γν P3γµ \| {z } C P4γν P2] (36) dσ dΩ= α2 2s (1 + x)2 + 4 (1 −x)2 + (1 + x2) 2 −(1 + x)2 (1 −x) (40) (40) Aplicando a propriedade da notação slash(apêndice A.3.8’) em C da Eq 36: A ﬁgura 5 mostra a seção de choque diferencial calcu- lada através da Eq. 40. Nesta ﬁgura foi comparada a curva teórica com os dados obtidos pela Colaboração Tasso7. Ainda, se a energia de centro de massa fosse da ordem da massa do bóson Z (mZ = 91.1876 GeV) [24], teríamos um diagrama idêntico ao do canal s exceto pela troca de γ por Z. O bóson Z(neutro) e W ±(carregado) são bósons massivos descritos pela teoria Eletrofraca. Sendo esta responsável pelo decaimentos radioativos. Desta forma, teríamos 3 diagramas mais as suas respectivas interferên- cias. 7Neste mesmo experimento foi descoberto o glúon [26] em 1979, o mediador da força forte, descrito pela cromodinâmica quân- tica(Quantum Cromodynamics - QCD). DOI: http://dx.doi.org/10.1590/1806-9126-RBEF-2017-0395 Revista Brasileira de Ensino de Física, vol. 40, nº 3, e3318, 2018 8Ou crossing symmetry. 6 Lembrando que agora P1P2 são escalares e, portanto, saem do traço. 5.1. Simetria de Cruzamento Substituindo as variáveis de Mandelstam (Eq. 15) por sua relação com os ângulos de espalhamento, a Eq. (39) torna-se A simetria de cruzamento8 pode ser usada para se obter a amplitude de espalhamento através de outro processo, ou seja, trocando a partícula P pela sua anti-partícula ¯P. Além disso, também permite a obtenção de um decai- mento do processo A →¯B + C + D a partir de A + B → C + D. Cabe salientar que o número leptônico sempre < \|M\|2 > = 2e4 " cos4( θ 2) + 1 sen4( θ 2) + (1 + cos2(θ)) 2 − 2 cos4( θ 2) sen2( θ 2) # 6 Lembrando que agora P1P2 são escalares e, portanto, saem do traço. 6 Lembrando que agora P1P2 são escalares e, portanto, saem do traço. DOI: http://dx.doi.org/10.1590/1806-9126-RBEF-2017-0395 Revista Brasileira de Ensino de Física, vol. 40, nº 3, e3318, 2018 e3318-8 Espalhamento Bhabha: Um Estudo Detalhado Espalhamento Bhabha: Um Estudo Detalhado Figura 4: Seção de choque diferencial em termos do cos θ. Figura 5: Seção de choque total em termos da energia de centro de massa (√s). rvado. Um exemplo prático da simetria de 6. Considerações Experimentais Figura 4: Seção de choque diferencial em termos do cos θ Figura 4: Seção de choque diferencial em termos do cos θ. Figura 4: Seção de choque diferencial em termos do cos θ. Figura 5: Seção de choque total em termos da energia de centro de massa (√s). Figura 5: Seção de choque total em termos da energia de centro de massa (√s). Revista Brasileira de Ensino de Física, vol. 40, nº 3, e3318, 2018 DOI: http://dx.doi.org/10.1590/1806-9126-RBEF-2017-0395 7. Considerações Finais [4] S. Tomonaga, Prog. Theo. Phys. 1, 27 (1948). [5] J. Schwinger, Phys. Rev. 73, 416 (1948). Neste trabalho, buscamos apresentar algumas das técni- cas utilizadas na Física de Partículas de forma didática, visando fornecer aos estudantes dos últimos anos dos cursos superiores uma visão da física e da matemática envolvidas nos tópicos mais avançados. Com a entrada em funcionamento do LHC, em 2010, e com o anúncio da descoberta do Bóson de Higgs, em 4 de Julho 2012, mui- tos acadêmicos trazem suas curiosidade ao ingressarem na graduação. Aqui, mostramos através do cálculo de um processo especíﬁco, como os físicos tratam as colisões num experimento, e a partir desse tratamento, fazem as predições sobre resultados para cada processo. [6] J. Schwinger, Phys. Rev. 74, 1439 (1948) [7] R. P. Feynman, Phys. Rev. 76, 749 (1949). [8] R. P. Feynman, Phys. Rev. 76, 769 (1949). [9] R. P. Feynman, Phys. Rev. 80, 440 (1950) [10] W. B. de Sousa, Física das Radiações: uma proposta para o Ensino Médio. Dissertação de Mestrado, Universidade de São Paulo, 2009. [11] J. M. F. Bassalo, Eletrodinâmica Quântica (Livraria da Física, São Paulo, 2006). [12] B. R. Martin e G. Shaw, Particle Physics (Wiley, Chi- chester, 2008), 4 ed. [13] M. Thompson, Modern Particle Physics (Cambridge University Press, Cambridge, 2013), 1 ed. A escolha do processo Bhabha deu-se, em primeiro lu- gar, por trabalhar com muitos dos aspectos fundamentais do cálculo de processos, porém numa interação limpa, que é a eletromagnética, descrita pela Eletrodinâmica Quântica. Além de seu caráter didático para o cálculo de amplitudes, o processo Bhabha é frequentemente usado como um monitoramento da luminosidade [1] para ex- perimentos envolvendo espalhamentos elétron-pósitron. Medidas precisas da luminosidade são necessárias para diminuir os erros experimentais na medição das seções de choque. [14] A. Bettini, Introduction to Elementary Particle Physics (Cambridge University Press, Cambridge, 2012), 2 ed. [15] D. Griﬀths, Introduction to Elementary Particles (Wiley, Weinheim, 2008), 2 ed. [16] H. V. Fagundes, Teoria da Relatividade no Nível Mate- mático do Ensino Médio (Editora Livraria da Física, São Paulo, 2009). [17] R. D’Auria e M. Trigiante, From Special Relativity to Feynman Diagrams: A Course in Theoretical Particle Physics for Beginners (Springer, Switzerland, 2016), 2 ed. [18] J. H. Smith, Introduction to Special Relativity (Dover Publications, Dover, 2015), 1 ed. 7. Considerações Finais Finalmente, entendemos que apresentar tópicos mais avançados da Física, cujo caráter se aproxima mais da pesquisa do que do ensino, em um nível suﬁcientemente claro para ser lido e estudado por acadêmicos nos últimos anos, é uma estratégia para despertar o talento e a curio- sidade precoces nestes, fomentando as oportunidades de formação nesta área. Os scripts (Mathematica, Gnuplot e Python) e o código em FORTRAN estão disponíveis online9. [19] Y. Nagashima, Elementary Particle Physics: Quantum Field Theory and Particles (Wiley, Weinheim, 2010), v. 1, p. 964. [20] M. E. Peskin e D. V. Schroeder, An Introduction to Quantum Field Theory (Perseus, Reading, 1995), 1 ed. [21] M. D. Schwartz, Quantum Field Theory and the Standard Model (Cambridge University Press, Cambridge, 2014), 1 ed. [22] T. Lancaster e S. J. Blundell, Quantum Field Theory for the Gifted Amateur (Oxford University Press, Oxford, 2014). Agradecimentos [23] F. Halzen e A. D. Martin, Quarks and Leptons: An Introductory Course in Modern Particle Physics (Wiley, New York, 1984), 1 ed. F. K. N. e L. F. M. agradecem ao revisor deste artigo pelas apropriadas recomendações. Por ﬁm, F.K.N. agradece ao CNPq pelo suporte ﬁnanceiro. [24] C. Patrignani et al. (Particle Data Group), Chin. Phys. C 40, 100001 (2016). 9https://github.com/fabiokopp Referências tigo [27]. Obtemos χ2=23.89 com número de graus de liberdade(ndf=ν) igual a 19. Para o χ2/ν=1.25. Um bom ajuste é aquele em que o (χ2/ν) aproximasse de 1. A pro- babilidade de obtermos o mesmo resultado ou uma resul- tado maior numa amostra qualquer é de P(χ2, ν) = 20%. Onde o ideal é quando P(χ2, ν) aproximasse de 50 % [28]. [1] I. Bozovic-Jelisavcic, S. Lukic, G. Milutinovic Dumbelo- vic, M. Pandurovic e I. Smiljanic, Journal of Instrumen- tation 8, P08012 (2013). [2] A.C.B. Machado, V. Pleitez e M.C. Tijero, Rev. Bras. Ens. Fís. 28, 407 (2006). [3] G. Brockington e M. Pietrocola, Inv. Ens. Cien. 10, 387 (2005). 6. Considerações Experimentais deve ser conservado. Um exemplo prático da simetria de crossing é a obtenção da amplitude de e−µ−→e−µ−a partir da amplitude do Bhabha no canal S. Neste pro- cesso temos e−(P1)+e+(P2) →e+(P3)+e−(P4). Por não termos aniquilação de partículas e por serem de sabores diferentes, teremos somente a contribuição no canal t. Para tanto, substituindo e+(P2) →µ−(−P3) na eq. 31, obteremos a amplitude da eq. 35, a qual corresponde a amplitude do processo e−µ−→e−µ−. Para checarmos a qualidade da teoria contra experimento, utilizamos o teste do qui-quadrado (χ2) e chi-quadrado reduzido (χ2/ν). As deﬁnições destes podem ser vistas no apêndice A.5. Para realizar esse teste, usamos a energia de centro de massa (√s= 34.8 GeV), com luminosidade integrada ( R Ldt = 174.5 pb−1) correspondendo a 166348 eventos, sendo este o maior número de eventos do ar- DOI: http://dx.doi.org/10.1590/1806-9126-RBEF-2017-0395 Revista Brasileira de Ensino de Física, vol. 40, nº 3, e3318, 2018 Nóbrega e Mackedanz e3318-9 e3318-9 Referências Revista Brasileira de Ensino de Física, vol. 40, nº 3, e3318, 2018 DOI: http://dx.doi.org/10.1590/1806-9126-RBEF-2017-0395 Material Suplementar 25] H. J. Bhabha, Proc. Roy. Soc. A154, 195 (1935) [26] J. Ellis, Int. J. Mod. Phys. A29, 1430072 (2014). [27] W. Braunschweig et al. (TASSO Coll.), Z. Phys. C37, 171 (1988). O seguinte material suplementar está disponível online: Apêndice A Apêndice A [28] P. R. Bevington e D. K. Robinson, Data Reduction and Error Analysis for the Physical Sciences (McGraw-Hill, New York, 2002), 3 ed. Revista Brasileira de Ensino de Física, vol. 40, nº 3, e3318, 2018
https://openalex.org/W4362483518	https://figshare.com/articles/journal_contribution/Supplementary_Table_7_from_Proteomic_Profiling_Identifies_Dysregulated_Pathways_in_Small_Cell_Lung_Cancer_and_Novel_Therapeutic_Targets_Including_PARP1/22528410/1/files/39991419.pdf	English	null	Supplementary Table 5 from Proteomic Profiling Identifies Dysregulated Pathways in Small Cell Lung Cancer and Novel Therapeutic Targets Including PARP1	null	2,023	cc-by	1,626	Supplemental Table 7. Antibodies used in reverse-phase protein microarrays Antibody (standard name) Source Cat. # PI3K Pathway Host Akt Cell signaling 9272 Phospho-Akt (Ser473) Cell signaling 9271 R Phospho-Akt (Thr308) Cell signaling 9275 R GSK-3α/β (P-Ser21/9) Cell signaling 9331 R GSK-3α/β Santa Cruz SC-7291 R mTOR Cell signaling 2983 M Phospho-p70 S6 Kinase(Thr389) Cell signaling 9205 R p70 S6 Kinase Epitomics EP1494-1 R Phospho-S6 Ribosomal Protein (Ser235/236) Cell signaling 2211 R Phospho-S6 Ribosomal Protein (Ser240/242) Cell signaling 2215 R S6 Ribosomal Protein Cell signaling 2217 R FoxO3a Cell signaling 9467 R Phospho-FoxO3a (Ser318/321) Cell signaling 9465 R PTEN Cell signaling 9552 R PI3-kinase p110α Epitomics EP1683 R PI3 Kinase p85 Cell signaling 4292 R Phospho-IRS 1 (Ser307) Upstate 07-247 R IRS1 Millipore 06-248 R Phopho-PDK1(Ser241) Cell signaling 3061 R PDK1 Cell signaling 3062 R Phospho-SGK (Ser78) Cell signaling 3271 R SGK Cell signaling 3272 R Phospho-4E-BP1 (P-Thr70) Cell signaling 9455 R Phospho-4E-BP1 (Thr37/46) Cell signaling 9459 R Phospho-4E-BP1 (Ser65) Cell signaling 9456 R 4E-BP1 Cell signaling 9452 R eIF4E Cell signaling 9742 R YAP Cell signaling 4912 R YB1(Val) SDI GA1725 R MAPK Pathway Phospho-MEK1/2 (Ser217/221) Cell signaling 9121 R MEK1 Epitomics EP1235-1 R Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) Cell signaling 9101 R Phospho-p44/42 MAPK(Erk1/2)(Thr202/Tyr204) (197G2) Cell signaling 4377 R ERK2 Santa Cruz sc-154 R Phospho-p38 MAPKinase (Thr180/Tyr182) Cell signaling 9211 R p38 MAP Kinase Cell signaling 9212 R JNK2 Cell signaling 4672 R c-Jun Cell signaling 9165 R Phospho-cJun (Ser73) Cell signaling 9164 R Phospho-p90RSK (Thr359/Ser363) Cell signaling 9344 R p90RSK Cell signaling 9347 R Raf-1 Millipore 05-739 R HSP27 Cell signaling 2402 M Supplemental Table 7. Antibodies used in reverse-phase protein microarrays Antibody (standard name) Source Cat. # PI3K Pathway Host Akt Cell signaling 9272 Phospho-Akt (Ser473) Cell signaling 9271 R Phospho-Akt (Thr308) Cell signaling 9275 R GSK-3α/β (P-Ser21/9) Cell signaling 9331 R GSK-3α/β Santa Cruz SC-7291 R mTOR Cell signaling 2983 M Phospho-p70 S6 Kinase(Thr389) Cell signaling 9205 R p70 S6 Kinase Epitomics EP1494-1 R Phospho-S6 Ribosomal Protein (Ser235/236) Cell signaling 2211 R Phospho-S6 Ribosomal Protein (Ser240/242) Cell signaling 2215 R S6 Ribosomal Protein Cell signaling 2217 R FoxO3a Cell signaling 9467 R Phospho-FoxO3a (Ser318/321) Cell signaling 9465 R PTEN Cell signaling 9552 R PI3-kinase p110α Epitomics EP1683 R PI3 Kinase p85 Cell signaling 4292 R Phospho-IRS 1 (Ser307) Upstate 07-247 R IRS1 Millipore 06-248 R Phopho-PDK1(Ser241) Cell signaling 3061 R PDK1 Cell signaling 3062 R Phospho-SGK (Ser78) Cell signaling 3271 R SGK Cell signaling 3272 R Phospho-4E-BP1 (P-Thr70) Cell signaling 9455 R Phospho-4E-BP1 (Thr37/46) Cell signaling 9459 R Phospho-4E-BP1 (Ser65) Cell signaling 9456 R 4E-BP1 Cell signaling 9452 R eIF4E Cell signaling 9742 R YAP Cell signaling 4912 R YB1(Val) SDI GA1725 R MAPK Pathway Phospho-MEK1/2 (Ser217/221) Cell signaling 9121 R MEK1 Epitomics EP1235-1 R Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) Cell signaling 9101 R Phospho-p44/42 MAPK(Erk1/2)(Thr202/Tyr204) (197G2) Cell signaling 4377 R ERK2 Santa Cruz sc-154 R Phospho-p38 MAPKinase (Thr180/Tyr182) Cell signaling 9211 R p38 MAP Kinase Cell signaling 9212 R JNK2 Cell signaling 4672 R c-Jun Cell signaling 9165 R Phospho-cJun (Ser73) Cell signaling 9164 R Phospho-p90RSK (Thr359/Ser363) Cell signaling 9344 R p90RSK Cell signaling 9347 R Raf-1 Millipore 05-739 R HSP27 Cell signaling 2402 M Supplemental Table 7. Antibodies used in reverse-phase protein microarrays PI3K Pathway MAPK Pathway Phospho-MEK1/2 (Ser217/221) Cell signaling 9121 R MEK1 Epitomics EP1235-1 R Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) Cell signaling 9101 R Phospho-p44/42 MAPK(Erk1/2)(Thr202/Tyr204) (197G2) Cell signaling 4377 R ERK2 Santa Cruz sc-154 R Phospho-p38 MAPKinase (Thr180/Tyr182) Cell signaling 9211 R p38 MAP Kinase Cell signaling 9212 R JNK2 Cell signaling 4672 R c-Jun Cell signaling 9165 R Phospho-cJun (Ser73) Cell signaling 9164 R Phospho-p90RSK (Thr359/Ser363) Cell signaling 9344 R p90RSK Cell signaling 9347 R Raf-1 Millipore 05-739 R HSP27 Cell signaling 2402 M LKB Pathway LKB1 Abcam ab15095 M Phospho-LKB1 (Ser428) Cell signaling 3051 R Phospho-AMPKα(Thr172) Cell signaling 2535 R AMPKα Cell signaling 2532 R Phospho-Tuberin/TSC2(Thr1462) Cell signaling 3611 R TSC2/Tuberin Epitomics EP1613-1 R Phospho-Acetyl-CoA Carboxylase (Ser79) Cell signaling 3661 R Acetyl-CoA Carboxylase 1 (ACC 1) Epitomics EP1768-1 R Tyrosine kinase mediated Pathways Phospho-EGFR (Tyr1173) Upstate 05-483 M Phospho-EGF Receptor (Tyr992) Cell signaling 2235 R EGFR(1005) Santa Cruz sc-03 R IGF-I Receptor β Cell signaling 3027 R Phospho-IGF-I Receptor β (Tyr1135/1136)/Insulin Receptor β (Tyr1150/1151) Cell signaling 3024 R IGFRBP2 Cell signaling 3922 R HER2_pY1248 Upstate 06-229 R HER2/Erb2 Cell signaling 2242 R Phospho-Src (Tyr527) Cell signaling 2105 R Phospho-Src Family (Tyr416) cell signaling 2101 R Src Cell signaling 2109 R FGFR1 Cell signaling 3472 R PDGFR a Cell signaling 3164 R c-Kit Epitomics EP1522-1 R Jak/Stat Pathway Phospho-Stat3(Ser727) Cell signaling 9134 R Phospho-Stat3 (Tyr705) Cell signaling 9131 R Stat3 Upstate 06-596 R Phospho-Stat5 (Tyr694) Cell signaling 9151 R Stat5-α Epitomics EP1289-1 R Phospho-Stat6 (Tyr641) Cell signaling 9161 R Cell Cycle Regulator p21 Santa Cruz sc-397 R p27 Santa Cruz sc-528 R p53 Cell signaling 9282 R Phospho-Rb (Ser807/811) Cell signaling 9308 R Rb Cell signaling 9309 M cyclin B1 Epitomics EP1495-1 R cyclin D1 Santa Cruz SC-718 R cyclin E Santa Cruz sc-247 M E2F1 Santa Cruz sc-251 M PCNA Abcam ab29 M EZH2 Cell signaling 4905 R Apoptosis Pthway Cleaved PARP Cell signaling 9541 R PARP Ab-2 Thermo scientific RB-1516 R PARP Ab-3 Thermo scientific RB-1680 R Tyrosine kinase mediated Pathways Phospho-EGFR (Tyr1173) Phospho-EGF Receptor (Tyr992) EGFR(1005) IGF-I Receptor β Phospho-IGF-I Receptor β (Tyr1135/1136)/Insulin Receptor β (Tyr1150/1151) IGFRBP2 HER2_pY1248 HER2/Erb2 Phospho-Src (Tyr527) Phospho-Src Family (Tyr416) Src FGFR1 PDGFR a c-Kit Tyrosine kinase mediated Pathways Jak/Stat Pathway Phospho-Stat3(Ser727) Phospho-Stat3 (Tyr705) Stat3 Phospho-Stat5 (Tyr694) Stat5-α Phospho-Stat6 (Tyr641) Bcl-2 Dako M0887 M Bim Epitomics EP1036 R Active Caspase-3 Epitomics EP1476 R Cleaved Caspase -7 Cell signaling 9491 R Bax Cell signaling 2772 R XIAP Cell signaling 2042 R Thymidylate synthase Abcam ab3145 M DNA Repair ATM Abcam ab32420 R ATR Cell signaling 2790 R ATRIP Cell signaling 2737 R Chk1 Cell signaling 2345 R Chk2 Cell signaling 3440 M DNA PKcs Abcam ab32566 R Ku80 Cell signaling 2180 R Mre11 Cell signaling 4847 R MSH2 Cell signaling 2850 M Phospho-ATR Cell signaling 2853 R Phospho-Chk1 (Ser345) Cell signaling 2348 R Phospho-Chk2 (Thr68) Cell signaling 2197 R RAD51 Calbiochem NA 71 M XRCC 1 Cell signaling 2735 R Other (1 ) Phospho-PKCα (Ser657) Upstate 06-822 R PKCα Upstate 05-154 M E-Cadherin Cell signaling 4065 R Phospho-β-Catenin(Ser33/37/Thr41) Cell signaling 9561 R β-Catenin Cell signaling 9562 R Caveolin Cell signaling 3238 R CD31 Dako M0823 M Phospho-ER α (pS118) Epitomics EP1091-1 R Phospho-Estrogen Receptor α (Ser167) Cell signaling 2514S R ER Neomarker RM-9101S R Rab25 Covance Custom Ab450 R PAI-1 BD Biosciences 612024 M Progesterone Receptor Epitomics EP1483-1 R VEGF Receptor 2 Cell signaling 2479 R Other (2) 14-3-3 β Santa Cruz sc-628 R 14-3-3 ζ Santa Cruz sc-1019 R AIB-1 BD Biosciences 611105 M Androgen Receptor Epitomics EP1852 R BRCA1 Upstate 07-434 R BRCA2 Cell signaling 9012 R BRF2 Abcam ab17011 G CA IX Novus Biologicals NB100-417 R CREB Cell signaling 9197 R Phospho-CREB S133 Cell signaling 9198 R N_Cadherin Cell signaling 4061 R FAK Epitomics 1107-1 R Bcl-2 Dako M0887 M Bim Epitomics EP1036 R Active Caspase-3 Epitomics EP1476 R Cleaved Caspase -7 Cell signaling 9491 R Bax Cell signaling 2772 R XIAP Cell signaling 2042 R Thymidylate synthase Abcam ab3145 M DNA Repair ATM Abcam ab32420 R ATR Cell signaling 2790 R ATRIP Cell signaling 2737 R Chk1 Cell signaling 2345 R Chk2 Cell signaling 3440 M DNA PKcs Abcam ab32566 R Ku80 Cell signaling 2180 R Mre11 Cell signaling 4847 R MSH2 Cell signaling 2850 M Phospho-ATR Cell signaling 2853 R Phospho-Chk1 (Ser345) Cell signaling 2348 R Phospho-Chk2 (Thr68) Cell signaling 2197 R RAD51 Calbiochem NA 71 M XRCC 1 Cell signaling 2735 R Bcl-2 Bim Active Caspase-3 Cleaved Caspase -7 Bax XIAP Thymidylate synthase DNA Repair ATM ATR ATRIP Chk1 Chk2 DNA PKcs Ku80 Mre11 MSH2 Phospho-ATR Phospho-Chk1 (Ser345) Phospho-Chk2 (Thr68) RAD51 XRCC 1 Other (1 ) Phospho-PKCα (Ser657) Upstate 06-822 R PKCα Upstate 05-154 M E-Cadherin Cell signaling 4065 R Phospho-β-Catenin(Ser33/37/Thr41) Cell signaling 9561 R β-Catenin Cell signaling 9562 R Caveolin Cell signaling 3238 R CD31 Dako M0823 M Phospho-ER α (pS118) Epitomics EP1091-1 R Phospho-Estrogen Receptor α (Ser167) Cell signaling 2514S R ER Neomarker RM-9101S R Rab25 Covance Custom Ab450 R PAI-1 BD Biosciences 612024 M Progesterone Receptor Epitomics EP1483-1 R VEGF Receptor 2 Cell signaling 2479 R Other (2) 14-3-3 β Santa Cruz sc-628 R 14-3-3 ζ Santa Cruz sc-1019 R AIB-1 BD Biosciences 611105 M Androgen Receptor Epitomics EP1852 R BRCA1 Upstate 07-434 R BRCA2 Cell signaling 9012 R BRF2 Abcam ab17011 G CA IX Novus Biologicals NB100-417 R CREB Cell signaling 9197 R Phospho-CREB S133 Cell signaling 9198 R N_Cadherin Cell signaling 4061 R FAK Epitomics 1107-1 R Fibronectin Epitomics 1574 R Phospho-cMyc (Thr58/Ser62) Cell signaling 9401 R cMyc Cell Signaling 9402 R COL6A1 Santa Cruz sc-20649 R Cox2 Cell signaling 4842 R EN1 Strategic Diagnostics Inc.(SDI) GA2104 R GATA3 BD Biosciences 558686 M MGMT Milipore MAB16200 R MACC-1 Sigma HPA020081 R NCKIPS1 Strategic Diagnostics Inc.(SDI) GA2217 R NF-κB p65 Cell Signaling 3034 R Notch 3 Santa Cruz sc-5593 R PTCH Strategic Diagnostics Inc.(SDI) GA2113 R Phospho Smad3 (pS423/425) Epitomics 1880-1 R Smad3 Epitomics EP1735-1 R Stathmin (Val) Epitomics EP1972 R Tau Upstate 05-348 M TAZ Abcam ab3961 R Telomerase Strategic Diagnostics Inc.(SDI) 1706 R VASP Cell signaling 3112 R * R: Rabbit M: Mouse G: Goat Epitomics 1574 R Cell signaling 9401 R Cell Signaling 9402 R Santa Cruz sc-20649 R Cell signaling 4842 R Strategic Diagnostics Inc.(SDI) GA2104 R BD Biosciences 558686 M Milipore MAB16200 R Sigma HPA020081 R Strategic Diagnostics Inc.(SDI) GA2217 R Cell Signaling 3034 R Santa Cruz sc-5593 R Strategic Diagnostics Inc.(SDI) GA2113 R Epitomics 1880-1 R Epitomics EP1735-1 R Epitomics EP1972 R Upstate 05-348 M Abcam ab3961 R Strategic Diagnostics Inc.(SDI) 1706 R Cell signaling 3112 R
https://openalex.org/W4287395000	https://zenodo.org/record/4132617/files/3213ijbb02.pdf	English	null	Application of three graph Laplacian based semisupervised learning methods to protein function prediction problem	Zenodo (CERN European Organization for Nuclear Research)	2,021	cc-by	6,436	Keywords: semi-supervised learning, graph Laplacian, yeast, protein, function Application of three graph Laplacian based semi- supervised learning methods to protein function prediction problem Loc Tran University of Minnesota tran0398@umn.edu International Journal on Bioinformatics & Biosciences (IJBB) Vol.3, No.2, June 2013 International Journal on Bioinformatics & Biosciences (IJBB) Vol.3, No.2, June 2013 Abstract: Protein function prediction is the important problem in modern biology. In this paper, the un-normalized, symmetric normalized, and random walk graph Laplacian based semi-supervised learning methods will be applied to the integrated network combined from multiple networks to predict the functions of all yeast proteins in these multiple networks. These multiple networks are network created from Pfam domain structure, co-participation in a protein complex, protein-protein interaction network, genetic interaction network, and network created from cell cycle gene expression measurements. Multiple networks are combined with fixed weights instead of using convex optimization to determine the combination weights due to high time complexity of convex optimization method. This simple combination method will not affect the accuracy performance measures of the three semi-supervised learning methods. Experiment results show that the un-normalized and symmetric normalized graph Laplacian based methods perform slightly better than random walk graph Laplacian based method for integrated network. Moreover, the accuracy performance measures of these three semi-supervised learning methods for integrated network are much better than the best accuracy performance measures of these three methods for the individual network. 1. Introduction Protein function prediction is the important problem in modern biology. Identifying the function of proteins by biological experiments is very expensive and hard. Hence a lot of computational methods have been proposed to infer the functions of the proteins by using various types of information such as gene expression data and protein-protein interaction networks [1]. First, in order to predict protein function, the sequence similarity algorithms [2, 3] can be employed to find the homologies between the already annotated proteins and theun-annotated protein. Then the annotated proteins with similar sequences can be used to assign the function to the un-annotated protein. That’s the classical way to predict protein function [4]. Second, to predict protein function, a graph (i.e. kernel) which is the natural model of relationship between proteinscan also be employed. In this model, the nodes represent proteins. The edges represent for the possible interactions between nodes. Then, machine learning methods such as Support Vector Machine [5], Artificial Neural Networks [4], un-normalized graph Laplacian based semi-supervised learning method [6,14], or neighbor counting method [7] can be applied to this graph to infer the functions of un-annotated protein. The neighbor counting method labels the protein with the function that occurs frequently in the protein’s adjacent nodes in the protein- protein interaction network. Hence neighbor counting method does not utilize the full topology of the network. However, the Artificial Neural Networks, Support Vector Machine, andun- normalized graph Laplacian based semi-supervised learning method utilize the full topology of 11 DOI: 10.5121/ijbb.2013.3202 International Journal on Bioinformatics & Biosciences (IJBB) Vol.3, No.2, June 2013 the network.Moreover, the Artificial Neural Networks and Support Vector Machine are supervised learning methods. the network.Moreover, the Artificial Neural Networks and Support Vector Machine are supervised learning methods. work.Moreover, the Artificial Neural Networks and Support Vector Machine are sed learning methods. While the neighbor counting method, the Artificial Neural Networks, and the un-normalized graph Laplacian based semi-supervised learningmethod are all based on the assumption that the labels of two adjacent proteins in graph are likely to be the same, SVM do not rely on this assumption. Graphs used in neighbor counting method, Artificial Neural Networks, and the un- normalized graph Laplacian based semi-supervised learningmethod are very sparse.However, the graph (i.e. kernel) used in SVM is fully-connected. Third, the Artificial Neural Networks method is applied to the single protein-protein interaction network only. 1. Introduction However, the SVM method and un-normalized graph Laplacian based semi- supervised learning method try to use weighted combination of multiple networks(i.e. kernels) such as gene co-expression networkand protein-protein interaction network to improve the accuracy performance measures. While [5] (SVM method) determines the optimal weighted combination of networks by solving the semi-definite problem, [6,14] (un-normalized graph Laplacian based semi-supervised learning method) uses a dual problem and gradient descent to determine the weighted combination of networks. In the last decade, the normalized graph Laplacian [8] and random walk graph Laplacian [9] based semi-supervised learning methods have successfully been applied to some specific classification tasks such as digit recognition and text classification. However, to the best of my knowledge, the normalized graph Laplacian and random walk graph Laplacian based semi- supervised learning methods have not yet been applied to protein function prediction problem and hence their overall accuracy performance measure comparisons have not been done. In this paper, we will apply three un-normalized, symmetric normalized, and random walk graph Laplacian based semi-supervised learning methods to the integrated network combined with fixed weights.These five networksused for the combination are available from [6]. The main point of these three methods is to let every node of the graph iteratively propagates its label information to its adjacent nodes and the process is repeated until convergence [8]. Moreover, since [6] has pointed out that the integrated network combined with optimized weights has similar performance to the integrated network combined with equal weights, i.e. without optimization, we will use the integrated network combined with equal weights due to high time-complexity of these optimization methods. This type of combination will be discussed clearly in the next sections. We will organize the paper as follows: Section 2 will introduce random walk and symmetric normalized graph Laplacian based semi-supervised learning algorithms in detail.Section 3will show how to derive the closed form solutions of normalized and un-normalized graph Laplacian based semi-supervised learning from regularization framework. In section 4, we will apply these three algorithms to the integrated network of five networks available from [6]. These five networks are network created from Pfam domain structure, co-participation in a protein complex, protein-protein interaction network, genetic interaction network, and network created from cell cycle gene expression measurements. Section 5 will conclude this paper and discuss the future directions of researches of this protein function prediction problem utilizing hypergraph Laplacian. International Journal on Bioinformatics & Biosciences (IJBB) Vol.3, No.2, June 2013 We do not claim that the accuracy performance measures of these two methods will be better than the accuracy performance measure of the un-normalized graph Laplacian based semi- supervised learning method (i.e. the published method) in this protein function prediction problem. We just do the comparisons. To the best of my knowledge, no theoretical framework have been given to prove that which graph Laplacian method achieves the best accuracy performance measure in the classification task. In the other words, the accuracy performance measures of these three graph Laplacian based semi-supervised learning methods depend on the datasets we used. However, in [8], the author have pointed out that the accuracy performance measure of the symmetric normalized graph Laplacian based semi-supervised learning method are better than accuracy performance measures of the random walk and un-normalized graph Laplacian based semi-supervised learning methods in digit recognition and text categorization problems. Moreover, its accuracy performance measure is also better than the accuracy performance measure of Support Vector Machine method (i.e. the known best classifier in literature) in two proposed digit recognition and text categorization problems. This fact is worth investigated in protein function prediction problem. Again, we do not claim that our two proposed random walk and symmetric normalized graph Laplacian based semi-supervised learning methods will perform better than the published method (i.e. the un-normalized graph Laplacian method)in this protein function prediction problem. At least, the accuracy performance measures of two new proposed methods are similar to or are not worse than the accuracy performance measure of the published method (i.e. the un-normalized graph Laplacian method). 1. Introduction Claim: Random walk and symmetric normalized graph Laplacians have been widely used not in classification but also in clustering [8,13]. In this paper, we will focus on the application of these two graph Laplacians to the protein function prediction problem. The accuracy performance measures of these two methods will be compared to the accuracy performance measure of the un- normalized graph Laplacian based semi- 12 International Journal on Bioinformatics & Biosciences (IJBB) Vol.3, No.2, June 2013 2. Algorithms Given networks in the dataset, the weights for individual networks used to combine to integrated network are . Given a set of proteins{ , … , , , … , } where = + is the total number of proteins in the integrated network, define c bethe total number of functional classes and the matrix ∈ ∗be the estimated label matrix for the set of proteins { , … , , , … , }, where the point is labeled as sign( ) for each functional class j (1 ≤ ≤). Please note that { , … , } is the set of all labeled points and { , … , } is the set of all un-labeled points. Let ∈ ∗the initial label matrix for n proteins in the network be defined as follow = 1 1 ≤ ≤ −1 1 ≤ ≤ 0 + 1 ≤ ≤ = 1 1 ≤ ≤ −1 1 ≤ ≤ 0 + 1 ≤ ≤ 0 + 1 ≤ ≤ Our objective is to predict the labels of the un-labeled points , … , . We can achieve this objective by letting every node (i.e. proteins) in the network iteratively propagates its label information to its adjacent nodes and this process is repeated until convergence. These three algorithms are based on three assumptions: Our objective is to predict the labels of the un-labeled points , … , . We can achieve this objective by letting every node (i.e. proteins) in the network iteratively propagates its label information to its adjacent nodes and this process is repeated until convergence. These three algorithms are based on three assumptions: - local consistency: nearby proteins are likely to have the same function - global consistency: proteins on the same structure (cluster or sub-manifolds) are likely to have the same function - these protein networks contain no self-loops Let ( ) represents the individual network in the dataset. 13 International Journal on Bioinformatics & Biosciences (IJBB) Vol.3, No.2, June 2013 Random walk graph Laplacian based semi-supervised learning algorithm In this section, we slightly change the original random walk graph Laplacian based semi- supervised learning algorithm can be obtained from [9]. The outline of the new version of this algorithm is as follows 1. Form the affinity matrix ( ) (for each k such that 1 ≤ ≤ ): ( ) = exp −\|\| − \|\| 2 ∗ ≠ 0 = 2. Construct = ∑ ( ) ( ) where ( ) = diag( ( ), … , ( )) and ( ) = ∑ ( ) 2. Construct = ∑ ( ) ( ) where ( ) = diag( ( ), … , ( )) and ( ) = ∑ ( ) 3. Iterate until convergence ( ) = ( ) + (1 − ) , where α is an arbitrary parameter belongs to [0,1] 3. Iterate until convergence ( ) = ( ) + (1 − ) , where α is an arbitrary parameter belongs to [0,1] [ , ] 4. Let ∗be the limit of the sequence { ( )}. For each protein functional class j, label each protein ( + 1 ≤ ≤ + ) as sign( ∗) 4. Let ∗be the limit of the sequence { ( )}. For each protein functional class j, label each protein ( + 1 ≤ ≤ + ) as sign( ∗) Next, we look for the closed-form solution of the random walk graph Laplacian based semi- supervised learning. In the other words, we need to show that Thus, by induction, Since is the stochastic matrix, its eigenvalues are in [-1,1]. Moreover, since 0<α<1, thus Since is the stochastic matrix, its eigenvalues are in [-1,1]. Moreover, since 0<α<1 thus thus 14 International Journal on Bioinformatics & Biosciences (IJBB) Vol.3, No.2, June 2013 International Journal on Bioinformatics & Biosciences (IJBB) Vol.3, No.2, June 2013 International Journal on Bioinformatics & Biosciences (IJBB) Vol.3, No.2, June 2013 Therefore, Now, from the above formula, we can compute ∗directly. The original random walk graph Laplacian based semi-supervised learning algorithm developed by Zhu can be derived from the modified algorithm by setting = 0, where 1 ≤ ≤ and = 1,where + 1 ≤ ≤ + . Random walk graph Laplacian based semi-supervised learning algorithm In the other words, we can express ( ) in matrix form as follows ( ) = ( ) + ( − ) , where I is the identity matrix and = 0 … 0 ⋮ ⋮ 0 … 0 0 0 1 0 ⋮ ⋮ 0 … 1 ( ) ( ) = ( ) + ( − ) , where I is the identity matrix and = 0 … 0 ⋮ ⋮ 0 … 0 0 0 1 0 ⋮ ⋮ 0 … 1 ( ) International Journal on Bioinformatics & Biosciences (IJBB) Vol.3, No.2, June 2013 Thus, by induction, Since for every integer k such that 1 ≤ ≤ , ( ) ( ) ( ) is similar to ( ) ( ) which is a stochastic matrix, eigenvalues of ( ) ( ) ( ) belong to [-1,1]. Moreover, for every k, ( ) ( ) ( ) is symmetric, then ∑ ( ) ( ) ( ) is also symmetric. Therefore, by using Weyl’s inequality in [10] and the references therein, the largest eigenvalue of ∑ ( ) ( ) ( ) is at most the sum of every largest eigenvalues of ( ) ( ) ( ) and the smallest eigenvalue of ∑ ( ) ( ) ( ) is at least the sum of every smallest eigenvalues of ( ) ( ) ( ) . Thus, the eigenvalues of = ∑ ( ) ( ) ( ) belong to [-1,1]. Moreover, since 0<α<1, thus lim →∞ = 0 lim →∞ ( ) = ( − ) lim →∞ = 0 lim →∞ ( ) = ( − ) Therefore, ∗= lim →∞ ( ) = (1 − )( − ) Now, from the above formula, we can compute ∗directly. Normalized graph Laplacian based semi-supervised learning algorithm Normalized graph Laplacian based semi-supervised learning algorithm Next, we will give the brief overview of the original normalized graph Laplacian based semi- supervised learning algorithm can be obtained from [8]. The outline of this algorithm is as follows 1. Form the affinity matrix ( ) (for each 1 ≤ ≤ ): ( ) = exp −\|\| − \|\| 2 ∗ ≠ 0 = atrix ( ) (for each 1 ≤ ≤ ): ( ) = exp −\|\| − \|\| 2 ∗ ≠ 0 = 2. Construct = ∑ ( ) ( ) ( ) where ( ) = diag( ( ), … , ( )) and ( ) = ∑ ( ) 2. Construct = ∑ ( ) ( ) ( ) where ( ) = diag( ( ), … , ( )) and ( ) = ∑ ( ) 2. Construct = ∑ ( ) ( ) ( ) where ( ) = diag( ( ), … , ( )) and ( ) = ∑ ( ) 3. Iterate until convergence ( ) = ( ) + (1 − ) , where α is an arbitrary parameter belongs to [0,1] 3. Iterate until convergence ( ) = ( ) + (1 − ) , where α is an arbitrary parameter belongs to [0,1] [ , ] 4. Let ∗be the limit of the sequence { ( )}. For each protein functional class j, label each protein ( + 1 ≤ ≤ + ) as sign( ∗) 4. Let ∗be the limit of the sequence { ( )}. For each protein functional class j, label each protein ( + 1 ≤ ≤ + ) as sign( ∗) Next, we look for the closed-form solution of the normalizedgraph Laplacian based semi- supervised learning. In the other words, we need to show that ∗= lim →∞ ( ) = (1 − ) − Suppose ( ) = , then Suppose ( ) = , then 15 International Journal on Bioinformatics & Biosciences (IJBB) Vol.3, No.2, June 2013 3. Regularization Frameworks In this section, we will develop the regularization framework for the normalized graph Laplacian based semi-supervised learning iterative version. First, let’s consider the error function 16 International Journal on Bioinformatics & Biosciences (IJBB) Vol.3, No.2, June 2013 International Journal on Bioinformatics & Biosciences (IJBB) Vol.3, No.2, June 2013 In this error function ( ), and belong to . Please note that c is the total number of protein functional classes, ( ) = ∑ ( ), and is the positive regularization parameter. Hence In this error function ( ), and belong to . Please note that c is the total number of protein functional classes, ( ) = ∑ ( ), and is the positive regularization parameter. Hence = ⋮ = ⋮ = ⋮ = ⋮ Here ( ) stands for the sum of the square loss between the estimated label matrix and the initial label matrix and the smoothness constraint. Hence we can rewrite ( ) as follows Hence we can rewrite ( ) as follows ( ) = − + (( − ) ( − )) Our objective is to minimize this error function. In the other words, we solve Our objective is to minimize this error function. In the other words, we solve = 0 This will lead to This will lead to Let = . Hence the solution ∗of the above equations is Let = . Hence the solution ∗of the above equations is ∗= (1 − )( − ) Also, please note that = ∑ ( ) ( ) is not the symmetric matrix, thus we cannot develop the regularization framework for the random walk graph Laplacian based semi- supervised learning iterative version. Also, please note that = ∑ ( ) ( ) is not the symmetric matrix, thus we cannot develop the regularization framework for the random walk graph Laplacian based semi- supervised learning iterative version. Next, we will develop the regularization framework for the un-normalized graph Laplacian based semi-supervised learning algorithms. First, let’s consider the error function ( ) = 1 2 ( ) − , + ‖ − ‖ In this error function ( ), and belong to . Please note that c is the total number of protein functional classes and is the positive regularization parameter. Hence In this error function ( ), and belong to . 3. Regularization Frameworks Please note that c is the total number of protein functional classes and is the positive regularization parameter. Hence = ⋮ = ⋮ Here ( ) stands for the sum of the square loss between the estimated label matrix and the initial label matrix and the smoothness constraint. Here ( ) stands for the sum of the square loss between the estimated label matrix and the initial label matrix and the smoothness constraint. 17 International Journal on Bioinformatics & Biosciences (IJBB) Vol.3, No.2, June 2013 Hence we can rewrite ( ) as follows nce we can rewrite ( ) as follows ( ) = 1 ( ) + (( − ) ( − )) Please note that un-normalized Laplacian matrix of the networkis ( ) = ( ) − ( ). Our objective is to minimize this error function. In the other words, we solve Please note that un-normalized Laplacian matrix of the networkis ( ) = ( ) − ( ). Our objective is to minimize this error function. In the other words, we solve = 0 = 0 = 0 This will lead to 1 ( ) + ( − ) = 0 ∑ ( ) + = This will lead to This will lead to This will lead to 1 ( ) + ( − ) = 0 ∑ ( ) + = 1 ( ) + ( − ) = 0 ∑ ( ) + = ∑ ( ) + = Hence the solution ∗of the above equations is ∗= (1 ( ) + ) Similarly, we can also obtain the other form of solution ∗of the normalized graph Laplacian based semi-supervised learning algorithm as follows (note normalized Laplacian matrix of networkis ( ) = − ( ) ( ) ( ) ) Similarly, we can also obtain the other form of solution ∗of the normalized graph Laplacian based semi-supervised learning algorithm as follows (note normalized Laplacian matrix of networkis ( ) = − ( ) ( ) ( ) ) ∗= (1 ( ) + ) 4. Experiments and results The Dataset The three symmetric normalized, random walk, and un-normalized graph Laplacian based semi- supervised learning are applied to the dataset obtained from [6]. This dataset is composed of 3588 yeast proteins from Saccharomyces cerevisiae, annotated with 13 highest-level functional classes from MIPS Comprehensive Yeast Genome Data (Table 1). This dataset contains five networks of pairwise relationships, which are very sparse.These five networks are network created from Pfam domain structure ( ( )), co-participation in a protein complex ( ( )), protein-protein interaction network ( ( )), genetic interaction network ( ( )), and network created from cell cycle gene expression measurements ( ( )). The first network, ( ), was obtained from the Pfam domain structure of the given genes. At the time of the curation of the dataset, Pfam contained 4950 domains. For each protein, a binary vector of this length was created. Each element of this vector represents the presence or absence of one Pfam domain. The value of ( ) is then the normalization of the dot product between the domain vectors of proteins i and j. The fifth network, ( ), was obtained from gene expression data collected by [12]. In this network, an edge with weight 1 is created between two proteins if their gene expression profiles are sufficiently similar. 18 International Journal on Bioinformatics & Biosciences (IJBB) Vol.3, No.2, June 2013 The remaining three networks were created with data from the MIPS Comprehensive Yeast Genome Database (CYGD). ( ) is composed of binary edges indicating whether the given proteins are known to co-participate in a protein complex. The binary edges of ( ) indicate known protein-protein physical interactions. Finally, the binary edges in ( ) indicate known protein-protein genetic interactions. The protein functional classes these proteins were assigned to are the 13 functional classes defined by CYGD at the time of the curation of this dataset. A brief description of these functional classes is given in the following Table 1. 4. Experiments and results The Dataset Table 1: 13 CYGD functional classes Classes 1 Metabolism 2 Energy 3 Cell cycle and DNA processing 4 Transcription 5 Protein synthesis 6 Protein fate 7 Cellular transportation and transportation mechanism 8 Cell rescue, defense and virulence 9 Interaction with cell environment 10 Cell fate 11 Control of cell organization 12 Transport facilitation 13 Others Results Table 1: 13 CYGD functional classes Classes 1 Metabolism 2 Energy 3 Cell cycle and DNA processing 4 Transcription 5 Protein synthesis 6 Protein fate 7 Cellular transportation and transportation mechanism 8 Cell rescue, defense and virulence 9 Interaction with cell environment 10 Cell fate 11 Control of cell organization 12 Transport facilitation 13 Others Results From the above table 3, we recognized that the symmetric normalized and un-normalized graph Laplacian based semi-supervised learning methods slightly perform better than the random walk graph Laplacian based semi-supervised learning method. Results In this section, we experiment with the above three methods in terms of classification accuracy performance measure. All experiments were implemented in Matlab 6.5 on virtual machine. For the comparisons discussed here, the three-fold cross validation is used to compute the accuracy performance measures for each class and each method. The accuracy performance measure Q is given as follows True Positive (TP), True Negative (TN), False Positive (FP), and False Negative (FN) are defined in the following table 2 19 International Journal on Bioinformatics & Biosciences (IJBB) Vol.3, No.2, June 2013 Table 2: Definitions of TP, TN, FP, and FN Predicted Label Positive Negative Known Label Positive True Positive (TP) False Negative (FN) Negative False Positive (FP) True Negative (TN) In these experiments, parameter is set to 0.85 and = 1. For this dataset, the third table shows the accuracy performance measures of the three methods applying to integrated network for 13 functional classes Table 2: Definitions of TP, TN, FP, and FN In these experiments, parameter is set to 0.85 and = 1. For this dataset, the third table shows the accuracy performance measures of the three methods applying to integrated network for 13 functional classes Table 3: Comparisons of symmetric normalized, random walk, and un-normalized graph Laplacian based methods using integrated network Functional Classes Accuracy Performance Measures (%) Integrated Network Normalized Random Walk Un-normalized 1 76.87 76.98 77.20 2 85.90 85.87 85.81 3 78.48 78.48 77.56 4 78.57 78.54 77.62 5 86.01 85.95 86.12 6 80.43 80.49 80.32 7 82.02 81.97 81.83 8 84.17 84.14 84.17 9 86.85 86.85 86.87 10 80.88 80.85 80.52 11 85.03 85.03 85.92 12 87.49 87.46 87.54 13 88.32 88.32 88.32 From the above table 3, we recognized that the symmetric normalized and un-normalized graph Laplacian based semi-supervised learning methods slightly perform better than the random walk graph Laplacian based semi-supervised learning method. Results the published) and normalized graph Laplacian based semi-supervised learning methods slightly perform better than the random walk graph Laplacian based semi-supervised learning method using network ( ) and ( ). For ( ), ( ), and ( ), the random walk and the normalized graph Laplacian based semi- supervised learning methods slightly perform better than the un-normalized (i.e. the published) graph Laplacian based semi-supervised learning method. ( ), ( ), and ( ) are all three networks created with data from the MIPS Comprehensive Yeast Genome Database (CYGD). From the above tables, we easily see that the un-normalized (i.e. the published) and normalized graph Laplacian based semi-supervised learning methods slightly perform better than the random walk graph Laplacian based semi-supervised learning method using network ( ) and ( ). For ( ), ( ), and ( ), the random walk and the normalized graph Laplacian based semi- supervised learning methods slightly perform better than the un-normalized (i.e. the published) graph Laplacian based semi-supervised learning method. ( ), ( ), and ( ) are all three networks created with data from the MIPS Comprehensive Yeast Genome Database (CYGD). From the above tables, we easily see that the un-normalized (i.e. the published) and normalized graph Laplacian based semi-supervised learning methods slightly perform better than the random walk graph Laplacian based semi-supervised learning method using network ( ) and ( ). For ( ), ( ), and ( ), the random walk and the normalized graph Laplacian based semi- supervised learning methods slightly perform better than the un-normalized (i.e. the published) graph Laplacian based semi-supervised learning method. ( ), ( ), and ( ) are all three networks created with data from the MIPS Comprehensive Yeast Genome Database (CYGD). Moreover, the accuracy performance measures of all three methods for ( ), ( ), ( ), and ( ) are un-acceptable since they are worse than random guess. Again, this fact occurs due to the sparseness of these four networks. Moreover, the accuracy performance measures of all three methods for ( ), ( ), ( ), and ( ) are un-acceptable since they are worse than random guess. Again, this fact occurs due to the sparseness of these four networks. For integrated network and every individual network except ( ), we recognize that the symmetric normalized graph Laplacian based semi-supervised learning method performs slightly better than the other two graph Laplacian based methods. Results Table 3: Comparisons of symmetric normalized, random walk, and un-normalized graph Laplacian based methods using integrated network 20 International Journal on Bioinformatics & Biosciences (IJBB) Vol.3, No.2, June 2013 Next, we will show the accuracy performance measures of the three methods for each individual network ( ) in the following tables: Next, we will show the accuracy performance measures of the three methods for each individual network ( ) in the following tables: Next, we will show the accuracy performance measures of the three methods for each individual network ( ) in the following tables: Table 4: Comparisons of symmetric normalized, random walk, and un-normalized graph Laplacian based methods using network ( ) Functional Classes Accuracy Performance Measures (%) Network ( ) Normalized Random Walk Un-normalized 1 64.24 63.96 64.30 2 71.01 71.07 71.13 3 63.88 63.66 63.91 4 65.55 65.41 65.47 5 71.35 71.46 71.24 6 66.95 66.69 67.11 7 67.89 67.70 67.84 8 69.29 69.29 69.31 9 71.49 71.40 71.52 10 65.30 65.47 65.50 11 70.09 70.04 70.12 12 72.71 72.66 72.63 13 72.85 72.77 72.85 Table 5: Comparisons of symmetric normalized random walk and un-normalized graph 21 network ( ) in the following tables: Table 4: Comparisons of symmetric normalized, random walk, and un-normalized graph Laplacian based methods using network ( ) Functional Classes Accuracy Performance Measures (%) Network ( ) Normalized Random Walk Un-normalized 1 64.24 63.96 64.30 2 71.01 71.07 71.13 3 63.88 63.66 63.91 4 65.55 65.41 65.47 5 71.35 71.46 71.24 6 66.95 66.69 67.11 7 67.89 67.70 67.84 8 69.29 69.29 69.31 9 71.49 71.40 71.52 10 65.30 65.47 65.50 11 70.09 70.04 70.12 12 72.71 72.66 72.63 13 72.85 72.77 72.85 Table 5: Comparisons of symmetric normalized, random walk, and un-normalized graph Laplacian based methods using network ( ) Functional Classes Accuracy Performance Measures (%) Network ( ) Normalized Random Walk Un-normalized 1 24.64 24.64 24.64 2 27.84 27.84 27.79 3 23.16 23.16 23.08 ble 4: Comparisons of symmetric normalized, random walk, and un-normalized graph Laplacian based methods using network ( ) ble 5: Comparisons of symmetric normalized, random walk, and un-normalized graph Laplacian based methods using network ( ) 21 International Journal on Bioinformatics & Biosciences (IJBB) Vol.3, No.2, June 2013 4 22.60 22.60 22.52 5 26.37 26.37 26.23 6 24.39 24.39 24.19 7 26.11 26.11 26.37 8 27.65 27.65 27.62 9 28.43 28.43 28.34 10 25.81 25.81 25.22 11 27.01 27.01 25.98 12 28.43 28.43 28.40 13 28.54 28.54 28.54 Table 6: Comparisons of symmetric normalized, random walk, and un-normalized graph Laplacian based methods using network ( ) Functional Classes Accuracy Performance Measures (%) Network ( ) Normalized Random Walk Un-normalized 1 29.63 29.57 29.40 2 34.11 34.11 33.95 3 27.93 27.90 27.70 4 28.51 28.48 28.57 5 34.03 34.03 33.92 6 30.57 30.55 30.04 7 32.08 32.08 32.02 8 33.05 33.03 32.92 9 33.78 33.78 33.75 10 30.18 30.18 29.99 11 32.64 32.64 32.53 International Journal on Bioinformatics & Biosciences (IJBB) Vol.3, No.2, June 2013 International Journal on Bioinformatics & Biosciences (IJBB) Vol.3, No.2, June 2013 4 22.60 22.60 22.52 5 26.37 26.37 26.23 6 24.39 24.39 24.19 7 26.11 26.11 26.37 8 27.65 27.65 27.62 9 28.43 28.43 28.34 10 25.81 25.81 25.22 11 27.01 27.01 25.98 12 28.43 28.43 28.40 13 28.54 28.54 28.54 International Journal on Bioinformatics & Biosciences (IJBB) Vol.3, No.2, June 2013 Table 6: Comparisons of symmetric normalized, random walk, and un-normalized graph Laplacian based methods using network ( ) Functional Classes Accuracy Performance Measures (%) Network ( ) Normalized Random Walk Un-normalized 1 29.63 29.57 29.40 2 34.11 34.11 33.95 3 27.93 27.90 27.70 4 28.51 28.48 28.57 5 34.03 34.03 33.92 6 30.57 30.55 30.04 7 32.08 32.08 32.02 8 33.05 33.03 32.92 9 33.78 33.78 33.75 10 30.18 30.18 29.99 11 32.64 32.64 32.53 22 International Journal on Bioinformatics & Biosciences (IJBB) Vol.3, No.2, June 2013 International Journal on Bioinformatics & Biosciences (IJBB) Vol.3, No.2, June 2013 12 34.53 34.53 34.45 13 34.48 34.48 34.31 Table 7: Comparisons of symmetric normalized, random walk, and un-normalized graph Laplacian based methods using network ( ) Functional Classes Accuracy Performance Measures (%) Network ( ) Normalized Random Walk Un-normalized 1 18.31 18.28 18.26 2 20.93 20.90 20.88 3 18.09 18.06 18.09 4 18.39 18.39 18.39 5 21.07 21.07 21.04 6 18.98 18.98 18.90 7 18.73 18.73 18.67 8 19.90 19.90 19.62 9 20.04 20.04 19.93 10 17.31 17.28 17.17 11 19.18 19.18 19.09 12 20.54 20.54 20.57 13 20.54 20.54 20.48 Table 8: Comparisons of symmetric normalized, random walk, and un-normalized graph Laplacian based methods using network ( ) Functional Classes Accuracy Performance Measures (%) Network ( ) Normalized Random Walk Un-normalized Table 8: Comparisons of symmetric normalized, random walk, and un-normalized graph Laplacian based methods using network ( ) Functional Classes Accuracy Performance Measures (%) Network ( ) Normalized Random Walk Un-normalized 1 26.45 26.45 26.51 2 29.21 29.21 29.21 23 International Journal on Bioinformatics & Biosciences (IJBB) Vol.3, No.2, June 2013 International Journal on Bioinformatics & Biosciences (IJBB) Vol.3, No.2, June 2013 3 25.89 25.78 25.92 4 26.76 26.62 26.76 5 29.18 29.18 29.18 6 27.42 27.23 27.42 7 28.21 28.18 28.01 8 28.51 28.54 28.54 9 29.71 29.68 29.65 10 26.81 26.95 27.01 11 28.79 28.82 28.85 12 30.16 30.13 30.16 13 30.18 30.16 30.18 International Journal on Bioinformatics & Biosciences (IJBB) Vol.3, No.2, June 2013 From the above tables, we easily see that the un-normalized (i.e. Results Finally, the accuracy performance measures of these three methods for the integrated network are much better than the best accuracy performance measure of these three methods for individual network. Due to the sparseness of the networks, the accuracy performance measures for individual networks W2, W3, W4, and W5 are unacceptable. They are worse than random guess. The best accuracy performance measure of these three methods for individual network will be shown in the following supplemental table. 24 International Journal on Bioinformatics & Biosciences (IJBB) Vol.3, No.2, June 2013 Supplement Table:Comparisons of un-normalized graph Laplacian based methods using network ( ) and integrated network Functional Classes Accuracy Performance Measures (%) Integrated network (un-normalized) Best individual network ( ) (un-normalized) 1 77.20 64.30 2 85.81 71.13 3 77.56 63.91 4 77.62 65.47 5 86.12 71.24 6 80.32 67.11 7 81.83 67.84 8 84.17 69.31 9 86.87 71.52 10 80.52 65.50 11 84.92 70.12 12 87.54 72.63 13 88.32 72.85 5. Conclusion The detailed iterative algorithms and regularization frameworks for the three normalized, random walk, and un-normalized graph Laplacian based semi-supervised learning methods applying to the integrated network from multiple networks have been developed. These three methodsare successfully applied to the protein function prediction problem (i.e. classification problem). Moreover, the comparison of the accuracy performance measures for these three methods has been done. These three methods can also be applied to cancer classification problems using gene expression data. Moreover, these three methods can not only be used in classification problem but also in ranking problem. In specific, given a set of genes (i.e. the queries) making up a protein complex/pathways or given a set of genes (i.e. the queries) involved in a specific disease (for e.g. leukemia), these three methods can also be used to find more potential members of the complex/pathway or more genes involved in the same disease by ranking genes in gene co-expression network (derived 25 International Journal on Bioinformatics & Biosciences (IJBB) Vol.3, No.2, June 2013 from gene expression data) or the protein-protein interaction network or the integrated network of them. The genes with the highest rank then will be selected and then checked by biologist experts to see if the extended genes in fact belong to the same complex/pathway or are involved in the same disease. These problems are also called complex/pathway membership determination and biomarker discovery in cancer classification. In cancer classification problem, only the sub- matrix of the gene expression data of the extended gene list will be used in cancer classification instead of the whole gene expression data. Finally, to the best of my knowledge, the normalized, random walk, and un-normalized hypergraph Laplacian based semi-supervised learning methods have not been applied to the protein function prediction problem. These methods applied to protein function prediction are worth investigated since [11] have shown that these hypergraph Laplacian based semi- supervisedlearning methods outperform the graph Laplacian based semi-supervised learning methods in text-categorization and letter recognition. References 1. Shin H.H., Lisewski A.M.and Lichtarge O.Graph sharpening plus graph integration: a synergy that improves protein functional classificationBioinformatics23(23) 3217-3224, 2007 1. Shin H.H., Lisewski A.M.and Lichtarge O.Graph sharpening plus graph integration: a synergy that improves protein functional classificationBioinformatics23(23) 3217-3224, 2007 2. Pearson W.R. and Lipman D.J. Improved tools for biological sequence comparison Proceeding the National Academy of Sciences of the United States of America, 85(8), 2444–2448, 1998 3. Lockhart D.J., Dong H., Byrne M.C., Follettie M.T., Gallo M.V., Chee M.S., Mittmann M., Wang C., Kobayashi M., Horton H., and Brown E.L. Expression monitoring by hybridization to high- density oligonucleotide arrays Nature Biotechnology, 14(13), 1675–1680, 1996 y g y gy ( ) 4. Shi L., Cho Y., and Zhang A. Prediction of Protein Function from Connectivity of Protein Interaction Networks International Journal of Computational Bioscience, Vol.1, No. 1, 2010 5. Lanckriet G.R.G., Deng M., Cristianini N., Jordan M.I., and Noble W.S. Kernel-based data fusion and its application to protein function prediction in yeastPacific Symposium on Biocomputing (PSB), 2004 ( ) 6. Tsuda K., Shin H.H, and Schoelkopf B. Fast protein classification with multiple networks Bioinformatics (ECCB’05), 21(Suppl. 2):ii59-ii65, 2005 7. Schwikowski B., Uetz P., and Fields S. A network of protein–protein interactions in yeast Nature Biotechnology, 18(12), 1257–1261, 2000 gy 8. Zhou D., Bousquet O., Lal T.N., Weston J. and Schölkopf B. Learning with Local and Global Consistency Advances in Neural Information Processing Systems (NIPS) 16, 321-328. (Eds.) S. Thrun, L. Saul and B. Schölkopf, MIT Press, Cambridge, MA, 2004 p g 9. Xiaojin Zhu and Zoubin Ghahramani. Learning from labeled and unlabeled data with label propagation Technical Report CMU-CALD-02-107, Carnegie Mellon University, 2002 10. Knutson A. and Tao T. Honeycombs and sums of Hermitian matrices Notices Amer. Math. Soc. 48, no. 2, 175–186, 2001 11. Zhou D., Huang J. and Schölkopf B. Learning with Hypergraphs: Clustering, Classification, and Embedding Advances in Neural Information Processing System (NIPS) 19, 1601-1608. (Eds.) B. Schölkopf, J.C. Platt and T. Hofmann, MIT Press, Cambridge, MA, 2007. 12. Spellman P., Sherlock G., and et al. Comprehensive identification of cell cycle-regulated genes of the yeast saccharomyces cerevisiae by microarray hybridization Mol. Biol. Cell, 9:3273– 3297, 1998 13. Luxburg U. A Tutorial on Spectral Clustering Statistics and Computing 17(4): 395-416, 2007 14. Shin H., Tsuda K., and Schoelkopf B.Protein functional class prediction with a combined graphExpert Systems with Applications, 36:3284-3292, 2009 26
https://openalex.org/W3114014189	https://jobrc.org/index.php/jobrc/article/download/293/290	English	null	Detection of Perforin in suspension of leukemia lymphocyte culture	Maǧallaẗ Markaz buḥūṯ al-taqniyyaẗ al-aḥyāʾiyyaẗ	2,014	cc-by	3,227	2014 2014 Journal of Biotechnology Research Center (Special edition) Vol. 8 No. 1 Abstract This work aimed to study the concentrations and hemolytic assay of perforin in serum and culture suspension of lymphocyte for acute lymphocyte leukemia ALL; chronic lymphocyte leukemia CLL, and control. To achieve this goal, blood samples were collected from 20 leukemic cases 10 ALL and 10 CLL, and isolation of lymphocyte done then proliferation of leukemia lymphocyte in culture for the detection of perforin concentration by ELISA kit and hemolytic activity of perforin in suspension lymphocyte culture and serum were impacted. The results, showed significant increase in the level of concentrations of perforin in both CLL and ALL as compared with the control, however, showed significant lower in level of hemolytic activity of perforin in these groups as compared to the control. The conclusion is that expression of perforin is strongly associated with leukemia patient in Iraqi population. Key words: Perforin, leukemia lymphocyte انًسرخهص ذهذف انذساسح قُاس ذشكُز وفعانُح تشوذٍُ انثُشفىسٍَ انثاقة فٍ يصم انذو ويزسعح انخالَا انرائُح انهًفاوَح نهًصاتٍُ تاتُضاض انذو انحاد وانًزيٍ وانسُطشج ، ونرحقُق هزا انهذف جًعد20 عُُه يٍ انذو10 عُُه يٍ اتُضاض انذو انحاد و10 عُُه يٍ اتُضاض انذو انًزيٍ وعزل انخالَا انًفاوَه وذى ذحذَذ ذشكُز ا نثاقة وانفعانُح انرحهُهُح نه فٍ انًزسعح وانًصم ار أظهشخ اسذفاع ذشكُز انثاقة نًشضً اتُضاض انذو انهًفٍ انحاد وانًزيٍ تانًقاسَه يع انسُطشج تًُُا اَخفضد انفعانُه نهثاقة نهًجًىعرٍُ تانًقاسَح يع انسُطشج . االسرُراجاخ ، ذعثُش انثاقة نه عالقح قىَح يع يشض اتُضاض انذو نهًج ذىٍع انعشاق . انكهًاخ انًفراحُح : ٌانثُشفىسٍَ ، اتُضاض انذو انهًفاو Introduction Key words: Perforin, leukemia lymphocyte Key words: Perforin, leukemia lymphocyte انًسرخهص ذهذف انذساسح قُاس ذشكُز وفعانُح تشوذٍُ انثُشفىسٍَ انثاقة فٍ يصم انذو ويزسعح انخالَا انرائُح انهًفاوَح نهًصاتٍُ تاتُضاض انذو انحاد وانًزيٍ وانسُطشج ، ونرحقُق هزا انهذف جًعد20 عُُه يٍ انذو10 عُُه يٍ اتُضاض انذو انحاد و10 عُُه يٍ اتُضاض انذو انًزيٍ وعزل انخالَا انًفاوَه وذى ذحذَذ ذشكُز ا نثاقة وانفعانُح انرحهُهُح نه فٍ انًزسعح وانًصم ار أظهشخ اسذفاع ذشكُز انثاقة نًشضً اتُضاض انذو انهًفٍ انحاد وانًزيٍ تانًقاسَه يع انسُطشج تًُُا اَخفضد انفعانُه نهثاقة نهًجًىعرٍُ تانًقاسَح يع انسُطشج . االسرُراجاخ ، ذعثُش انثاقة نه عالقح قىَح يع يشض اتُضاض انذو نهًج ذىٍع انعشاق . انكهًاخ انًفراحُح : ٌانثُشفىسٍَ ، اتُضاض انذو انهًفاو Introduction Detection of Perforin in suspension of leukemia lymphocyte culture ٍَانكشف عٍ انثُشفىس(انثاقة ) فٍ عانق انًزسعح اتُضاض انذو انهًفاوَح Najwa Sh. Ahmed Amal G.S. Fayyad * Wan Mohter Yusoff * Subhi Jawad Hamza Abdul Hussen Al-Faisal* Adeeb A. AL-Shami**** Biotechnology Research Center/ Al-Nahrain University University Kebangsaan Malaysia * Science college/ Baghdad University *Institute of Genetic Engineering and Biotechnology for Postgraduate/ Baghdad University ** National Center of Hematology/ Al Mustanisyria University َجىي شهاب احًذ آيال فُاض* واٌ يخراس َىسف* صثحٍ جىاد حًزج عثذ انحسٍُ يىَد انفُصم* ٍأدَة عثاس انشاي**** مركز بحىث التقنيات االحيائية /جامعة النهرين الجامعه الماليزية* كلية العلىم /جامعة بغداد معهد الهندسة الىراثية والتقنيات االحيائية للدراسات العليا / جامعة بغداد * ال مركز الىطني ال مراض الدم/ الجامعة المستنصرية*** * Sample Collection Five ml of blood was collected by vein puncture from 20 cases ALL and CLL who were admitted to the National Center of Haematology/ Al Mustanisyria University from May 2010 till March 2011. The disease was diagnosed by the consultant medical staff at the centre. In addition, 5 healthy looking subjects (controls) were also included. Isolation of Lymphocytes Preparation of solutions and media were done according to the methods described by [9; 10]. The lymphocytes were isolated from the peripheral heparinized whole blood as follows: three ml of blood was centrifuged at 1000rpm for 15min. The plasma was collected for perforin estimation, Buffy coat was collected in a 10 ml centrifuge tubes and diluted with 5ml RPMI 1640 (cell suspension), five ml of the diluted cell suspension was layered on 3ml of ficoll-isopaque separation fluid, the tubes were centrifuged at 2000rpm for 30min in a cooled centrifuge at 4ºC. After centrifugation, the mononuclear cells were visible as cloudy band between the RPMI1640 and lymphoprep layers. The band was collected in a 10ml test tube and the cells were suspended in 5 ml RPMI 1640. The tube was centrifuged at 2000rpm for 5min (first wash), the supernatant was discarded and the cells were resuspended in 5 ml RPMI 1640 (repeated twice). The suspension was centrifuged at 1000rpm for 10min, the supernatant was discarded. The precipitated cells were resuspended in 1ml RPMI solution and used in the planned experiments [11]. Counting the cells was performed before experiment [12], the numbers of lymphocytes were counted by improved neuber chamber and the cells concentration was adjusted to 1X106 cell/ml. The isolated cells were grown in a flask containing 10ml RPMI 1640 medium supplemented with 10% BSA (Bovine serum albumin) and incubated at 37ºC for 48h in CO2 incubator [11]. Using a microtiter plate (96 wells) cell culture technique 4X105 cells/ml and the complete RPMI 1640 was used as a negative control and complete RPMI1640 with PBS as positive control. The exposure time was 48hr. Each plate was designed to contain three replications of each concentration and 12 wells for negative control and 12 wells for positive control [11]. Perforin Detection Assay The perforin concentration was detected using perforin enzyme linked immuno sorbent assay (ELISA) for in vitro quantitative determination of perforin in supernatant, buffered solutions, serum and plasma samples. The perforin kit is a solid phase sandwich ELISA, a monoclonal antibody specific for perforin has been coated onto the wells of the microtiter plate. Standard Perforin (Cell science company/ USA), two nanogram powder of perforin was dissolved in 1ml of standard diluents to give 2000pg/ml and stored at 4ºC until used then diluents were prepared from 2000 to 62.5 pg/ml and the absorbance read on a spectrophotometer at 450nm. Introduction Perforin is a cytolytic, channel-forming protein expressed in cytolytic T cells and natural killer cells. It plays a crucial role in the killing cell-mediated machining of virally infected host cells, tumor cells, and allotransplant and endocytic parasitic manifestation [1]. The protein is encoded by the PRF1 gene which is mapped to 10q22 in human [2]. Its locus is not linked to any genes of the terminal complement system. Two-thirds of perforin sequence is homologous to the lytic, channel-forming complement proteins C6, C7, C8, and C9. The protein attack complex of complement produces pores of up to 20 nm in diameter on target membranes [3]. When the CTLs binds to its target, the contents of the granules are discharged by exocytose and perforin molecules insert themselves into the plasma of target cells forming a pore that enables granzymes to enter the cell [4]. Perforin is a 555 amino acid protein that has a 21 amino terminal signal sequence, an approximately 300 amino acid region that shares homology to the C9 complement protein, a 36 amino acid epidermal growth factor-like domain (EGF), and a 132 amino acid domain homologous to the C2 domain of protein kinase C [5, 2]. Patients with mutations in the perforin gene (PRF1) have been found to express low level or even absence of perforin in NK cells and diminished lymphocyte cytotoxicity [6]. Missense mutations in PRF1 have also been described in an adult with chronic active Epstein barr virus EBV infection [7], in children and adults with bone marrow malignancies [8], and in healthy individuals. The phenotypic expression of PRF1 mutations is variable, and the spectrum of perforin-related disease may include fatal immune dysregulation in early childhood, 20 Journal of Biotechnology Research Center (Special edition) 2014 Vol. 8 No. 1 nonfatal, inflammatory reactions at any age, and impaired tumor surveillance in children and adults [8]. The present study aimed to investigate the apoptosis profile of ALL and CLL through the following parameters: Perforin in terms of level and activity in serum and supernatant of cultured T-cytotoxic lymphocyte. nonfatal, inflammatory reactions at any age, and impaired tumor surveillance in children and adults [8]. The present study aimed to investigate the apoptosis profile of ALL and CLL through the following parameters: Perforin in terms of level and activity in serum and supernatant of cultured T-cytotoxic lymphocyte. Perforin Detection Protocol Standard perforin 200µl was added into wells A1 and A2. Standard diluents 100µl was added to standard wells B1, B2, C1, C2, D1, D2, E1, E2, F1, F2. Serial transfers of 100µl solutions were made in accordance to alphabet and numbers orders, 100µl of standard diluents was added to the blank well G1, G2 and 100µl of sample was added to sample well. The Covered plate was incubated for 1h at room temperature 25ºC. The plate was washed as follow: The liquid was aspirated from each well, 0.3ml of washing solution was dispensed into well then the content of well was aspirated (washing was repeated twice). Diluted biotinylated anti-perforin 50µl was added to all wells, the wells were covered and incubated for 1h at room temperature then washed as in up step. Horseradish peroxidase (HRP) solution 100µl was added into all wells including the blank wells, incubated at room temperature for 20min then wells were emptied and washed as in up step. TMB (tetra methyl benzidine) substrate solution 100µl was added into all wells; the plate was wrapped with aluminum foil and incubated in 21 Journal of Biotechnology Research Center (Special edition) 2014 Vol. 8 No. 1 the dark for 10-15min at room temperature. H2SO4 100µl was added into each well as a stop reagent, the absorbance of each well were read on a ELISA micro plate reader (Olympus/ Japan) at 450nm. H l ti A f P t i A ti it the dark for 10-15min at room temperature. H2SO4 100µl was added into each well as a stop reagent, the absorbance of each well were read on a ELISA micro plate reader (Olympus/ Japan) at 450nm. Hemolytic Assay for Protein Activity Perforin activity was determined depending on the hemoglobin released from lysed erythrocytes after treatment with perforin according to the method described by [13,14], 2% erythrocyte prepared by mouse blood sample was centrifuged at 1500rpm for 10 min, the serum was removed and the cells were resuspended in 1ml PBS saline then 2% erythrocyte was prepared by adding Tris-buffer saline. 100µl samples including purified perforin, blood serum from patients; healthy and cell culture suspension were added to 100µl of 2% erythrocyte. Perforin Detection Protocol The mixture was incubated for 30 min at 37ºC, then diluted to 2ml Tris HCl/NaCl, the mixture was centrifuged at 1500rpm for 10min and the supernatant was subjected to hemoglobin assay using spectrophotometer at 420nm, the following formula: Experimental - Spontaneous hemolytic Hemolytic activity = × p p y Maximal hemolytic – Spontaneous Hemolytic Statistical analysis The statistical analysis is a very important final step in the research to analyze and evaluate the obtained results. Medical statistics of this study was conducted via computer based statistical program which was: 1- SPSS for Windows computer package Programmer 11.5. 2- Microsoft Excel 2003. Th i i l l i hi h 2- Microsoft Excel 2003. c oso t ce 003. The statistical analysis tests which used in this were as follows: Duncan test is non-parametric test which used to determine whether there is a significant difference between the expected frequencies with respect to two variables. It is a well used test for the medical statistics. P value <0.05 is considered a significant correlation. Duncan test is non-parametric test which used to determine whether there is a significant difference between the expected frequencies with respect to two variables. It is a well used test for the medical statistics. P value <0.05 is considered a significant correlation. Detection of Perforin and Hemolytic Activity in Suspension of Cultured Leukemia Cells It was found that concentration of perforin was highly significant in the ALL and CLL groups (820.0±193.06 and 1510.4± 263.07) respectively than its controls (243.5±25.59). Perforin concentrations produced from CTLs in both group ALL and CLL have differed significantly from their controls P≤ 0.001. However, there was significant difference between the CLL and ALL groups, after 24h growth of CTLs P≤0.01, but after 48h perforin concentration highly significantly in ALL and CLL groups (1300.6±274.40 and (2057.2±357.67) respectively than the controls 299.3± 21.94 and have differed significantly from their controls P≤0.001. Hemolytic activity after 24 h for growth of CTLs lowered significantly in the ALL and CLL groups (0.806±0.129 and 0.484±0.006) respectively than its controls (1.065±0.152). Hemolytic activity produced from CTLs in CLL groups has differed significantly from their controls P≤0.001, but there was no significant difference between ALL and control. However, a significant difference between the CLL and ALL group after 24h for growth of CTLs P≤ 0.01 was found, but after 48h period hemolytic activity lowered significantly in the ALL and CLL groups (0.971±0.14 and 0.704±0.055) respectively than the control group (1.58±0.065). Hemolytic activity of perforin released from CTLs in both group ALL and CLL have differed significantly from their controls P≤0.01 and there was a significant difference between the CLL and ALL group P≤ 0.001 as shown in Table (1). rcentage frequency of perforin concentration and hemolytic assay in leukemia patients (CLL and ALL) and l Table (1): Percentage frequency of perforin concentration and hemolytic assay in leukemia patients (CLL and ALL) and controls. Table (1): Percentage frequency of perforin concentration and hemolytic assay in leukemia patients (CLL and ALL) and controls. Groups No. Mean ±S.E. Perforin Pg/ 24h. Hemolytic activity Perforin Pg/ 48h. Hemolytic activity CLL 10 1510.4±263.07A 0.484± 0.006 B 2057.2±357.60 A 0.704±0.055C ALL 10 820.0±193.06 B 0.806 ±0.129 A 1300.6±274.40 B 0.971±0.14 B Control 5 243.50±25.58 C 1.065± 0.152 A 299.3 ± 21.94C 1.58± 0.06 A *Different letters: Significant difference (P ≤ 0.001) between means of columns (Duncan test). The mean ± standard error was calculated [15] for perforin contents in the natural killer cells and cytotoxic T-cells of controls and found to be 3.561±1.157, but the contents were reduced in individual Reference 1. Trapani, JA, and Smyth, MJ. (2002). Functional significance of the perforin/granzyme cell death pathway. NatRev Immunol. 2:735-747. 1. Trapani, JA, and Smyth, MJ. (2002). Functional significance of the perforin/granzyme cell death pathway. NatRev Immunol. 2:735-747. 2. Lichtenheld, MG., Olsen, KJ. and Lu, P.(1988). Structure and function of human perforin. Nature. 335:448-51. 3. Anderson, DH. (2003). Granulysin crystal structure and a structure-derived lytic mechanism. J. Mol. Biol. 325, 355–365. 3. Anderson, DH. (2003). Granulysin crystal structure and a structure-derived lytic mechanism. J. Mol. Biol. 325, 355–365. 4. Joza, N., Susin, SA., Daugas, E., Stanford, WL., Cho, SK. and Li, CY. (2001). Essential role of the mitochondrial apoptosis-inducing factor in programmed cell death. Nature. 410:549–54. 4. Joza, N., Susin, SA., Daugas, E., Stanford, WL., Cho, SK. and Li, CY. (2001). Essential role of the mitochondrial apoptosis-inducing factor in programmed cell death. Nature. 410:549–54. 5. Uellner, R. (1997). Perforin is activated by a proteolytic cleavage during biosynthesis which reveals a phospholipid binding C2domain. EMBOJ. 16:7287-7296. 5. Uellner, R. (1997). Perforin is activated by a proteolytic cleavage during biosynthesis which reveals a phospholipid binding C2domain. EMBOJ. 16:7287-7296. 6. Katano, H. and Cohen, JI. (2005). Perforin and lymphohistiocytic proliferative disorders. Br J Haematol. 128:739-750. 6. Katano, H. and Cohen, JI. (2005). Perforin and lymphohistiocytic proliferative disorders. Br J Haematol. 128:739-750. 7. Karapinar, B., Yilmaz, D., Aydinok, Y., Turkoglu, E., Hekimgil, M. and Kavakli K. (2007). Intense myelofibrosis in a child: unusual result of EBV associated hemophagocytic lymphohistiocytosis. Turk J. Hematol. 24:32–5. 8. Henter, JI., Horne, A., Arico, M., Egeler, RM., Filipovich, AH., Imashuku, S. (2007). HLH. 2004, Diagnostic and therapeutic guidelines for hemophagocytic lymphohistiocytosis. Pediatr Blood Cancer. 48:124–31. 9. Marlise, M. (1997). Chromosome sensitivity to bleomycin in G2 lymphocytes from Down’s syndrome patients. Brazelian Journal of Genetics. 1: 310-319. 10. Bottran, RF. and Vetvicka, V. (2001). Advanced methods in cellular immunology. Boca Raton, New York, Washington, D.C. 11. Boyum, A. (1968). Separation of lymphocyte from blood and bone marrow. Scand .J. clin. lab. invent. 21. suppt.97. 12. Porakishvili, V., Kardava, L., Jewell, A., Yang, K., Glennie, M., Akbar, A. and Lydyard, P. (2004). Cytotoxic CD4 Tcells in patients with Bcell chronic lymphocytic leukemia kill via aperforin mediated pathway. Haematological. 89: 435-443. 13. Winkler, U., Pickett, TM and Hudig, D. (1996). Fractionation of perforin and granzymes by immobilized metal affinity chromatography (IMAC). J.Immunol.Methods.191:11. 14. Hudig, D., Allison, NJ., Pickett, TM., Winkler, U., Kam, C-M. Detection of Perforin and Hemolytic Activity in Suspension of Cultured Leukemia Cells The mean ± standard error was calculated [15] for perforin contents in the natural killer cells and cytotoxic T-cells of controls and found to be 3.561±1.157, but the contents were reduced in individual The mean ± standard error was calculated [15] for perforin contents in the natural killer cells and cytotoxic T-cells of controls and found to be 3.561±1.157, but the contents were reduced in individual 22 Journal of Biotechnology Research Center (Special edition) 2014 Vol. 8 No. 1 with heterozygous and homozygous perforin deficiency family hemophagocytic lymphohistocytosis and were found to be 2260 and 2120 rMol of anti perforin antibodies per NK cells. [16] refer to higher level expression of perforin from NK cells 94±2.5% of B-CLL patient compared with 75.5± 14.7% control and higher expression of perforin from CD8+cells 56.5±17 compared with healthy 27.9±15.6. On the other hand, [17; 18] referred to lower expression of perforin from hemophagocytic lymphohistiocytosis patients and detected partial or complete perforin deficiency because missense and nonsense mutations in exon 2 (del 207C) and exon 3 (del 1090-91CT) in coding region of perforin gene. [19] refer to loss of perforin protein or production of nonfunctional protein lead to lacks perforin expression in patient with familial hemophagocytic lymphohistocytosis FHLH and found mutation A91V in perforin gene. [20] referred to reduce and absent expression of cytotoxic T-lymphocyte and NK cells activity in FHLH patient from Iran because of two mutations were found at codon 273 (GCC>GCT) and 299 (CAC>CAT). In conclusion, the results appeared significantly elevated levels of concentration of perforin in both CLL and ALL as compared with control. However, the detected hemolytic activity of perforin in these groups at lower levels as compared to control. Reference and Powers, JC. (1991). The function of lymphocyte proteases: inhibition and restoration of granule-mediated lysis with isocoumarin serine protease inhibitors. J Immunol.147:1360-1368. 15. Maher, K., Klimas, N., Hurwitz, B., Schiff, R. and Fletcher, M. (2002). Quantitative fluorescence measures for determination of intracellular perforin content. Clin. Diagn. Lab. Immunol. 9:1248- 1252. 23 Journal of Biotechnology Research Center (Special edition) Vol. 8 No. 1 2014 16. Porakishvili, N., Roschupkina, T., Kalber, T., Jewell, AP., Patterson, KY. and Lydyard, P.M. (2001). Expansion of CD4+ Tcells with acytotoxic phenotype in patients with B-chronic lymphocytic leukemia (B-CLL). Clin. Exp. Immunol.126: 29–36. 17. Kogawa, K., Lee, S., Villanueva, J., Marmer, D., Sumegi, J. and Filipovich, A. (2002). Perforin expression in cytotoxic lymphocytes from patients with hemophagocytic lymphohistiocytosis and their family members. Blood. 99:61-66. 18. Yanai, F., Ishii, E., Kojima, K., Hasegawa, A., Azuma, T., Hirose, S. (2003). Essential roles of perforin in antigen specific cytotoxicity mediated by human CD4+T-lymphocytes: analysis using the combination of hereditary perforin deficient effector cells and Fas-Deficient target cells. The Journal of Immunology.170:2205-2213. 19. Trambas, C., Gallo, F., Pende, D., Marcenaro, S., Moretta, L., De Fusco, C. (2005). A single aminoacid change, A91V, leads to conformational changes that can impair processing to the active form of perforin. Blood.106:932–937. 20. Galehdari, H., Mohammadi, E., Andashti, B., Nader, A. and Molavi, M. (2007). Perforin gene analysis in an Iranian family with familial hemophagocytic lymphohistiocytosis. Iran. J. Immunol. 4:122-126. 24 24
https://openalex.org/W2464947758	https://www.matec-conferences.org/articles/matecconf/pdf/2016/29/matecconf_ibcc2016_00071.pdf	English	null	Energy Prediction versus Energy Performance of Green Buildings in Malaysia. Comparison of Predicted and Operational Measurement of GBI Certified Green Office in Kuala Lumpur	MATEC web of conferences	2,016	cc-by	4,200	DOI: 10.1051/matecconf/20166600071 DOI: 10.1051/matecconf/20166600071 00071 (2016) , MATEC Web of Conferences 66 IBCC 2016 Energy Prediction versus Energy Performance of Green Buildings in Malaysia. Comparison of Predicted and Operational Measurement of GBI Certified Green Office in Kuala Lumpur Suzaini M. Zaid1 and Amir Kiani1 ,a 1Department of Building Surveying, Faculty of Built Environment, University of Malaya, Malaysia Abstract. Forward from the sustainability agenda of Brundtland in 1987 and the increasing demand for energy efficient buildings, the building industry has taken steps in meeting the challenge of reducing its environmental impact. Initiatives such as ‘green’ or ‘sustainable’ design have been at the forefront of architecture, while green assessment tools have been used to predict the energy performance of building during its operational phase. However, there is still a significant hap between predicted or simulated energy measurements compared to actual operational energy consumption, or is more commonly referred as the ‘performance gap’. This paper tries to bridge this gap by comparing measured operational energy consumption of a Green Building Index (GBI) certified office building in Kuala Lumpur, with its predicted energy rating qualification. © 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/). 1 Introduction however it does very little to measure actual performance of the so-called green buildings. Therefore, there is a need to fill in the gap of predicted and operational performance of buildings – known as the ‘performance gap’ [6] that can truly be a testament of real energy and CO2 reduction. Global warming is one of the biggest issues of this century, and according to National Aeronautics and Space Administration (NASA), in the past few years the number of carbon dioxide (CO2) parts in the atmosphere exceeded an amount that it had never pass in the last 650,000 years [1]. Buildings play a big role in producing CO2 gases; in fact 30% of greenhouse gas emission come from building sector, and between 80-90% of CO2 emissions from the building sector come from the operational phase of already built buildings [2]. Consequently, mitigation of CO2 emissions has affected the building industry in a number of countries in the South East Asian region like Indonesia, Singapore, the Philippines, Thailand and Vietnam are already implementing energy efficiency legislation on newly built buildings [3]. However, it cannot be said the same about the building industry in Malaysia, where such legislation does not exist for both new built and existing buildings. a Corresponding author: suzaini_zaid@um.edu.my 2.1 Green Rating Tool and Simulation Many countries have developed their own green rating tool based on one another, mostly adapted from the pioneering BREEAM and LEED rating tools. Examples of green building rating tools available in the South East Asian region are Green Mark from Singapore, Building for Ecologically Responsive Design Excellence (BERDE) from Philippines and the Green Building Index (GBI) from Malaysia [12-14]. Some of the assessment criteria in these green building rating tools are similar, for example energy efficiency (EE), indoor environmental quality (EQ), and water efficiency (WE) [12-14]. However, the assessment measurements and scoring system may differ between these green building rating tools according to local context. Table 1 briefly highlights the assessment criteria for Green Mark, BERDE and GBI for Non-Residential New Construction category, and the maximum scoring points [12-14]. As the focus of this paper is to review the Malaysian building performance, a brief comparison between the energy efficiency criteria of the GBI with its other South East Asian counterpart Green Mark and BERDE is described also in Table 2. Table 2. Energy Requirements for BCA Green Mark, BERDE and GBI. Sources: [12-14]. BCA Green Mark (Building Energy Performance) BERDE (Energy) GBI (Energy Efficiency) 1. Energy Efficiency 2. Air- Conditioning Total System Efficiency 3. Lighting Efficiency 4. Energy Effectiveness 5. Building Energy 6. Car Park Energy 7. Receptacle Energy 8. Renewable Energy 9. Feasibility Study 10. Solar Ready Roof 11. Replacement Energy 1. Energy Sub- Metering 2. Energy Efficient Lighting 3. Natural Ventilation 4. On-Site Renewable Energy Generation 5. Energy Efficiency Improvement 6. Energy Efficiency Building Envelope 7. Energy Efficient Equipment 8. Building Automation Systems 1. Minimum EE Performance 2. Lighting Zoning 3. Electrical Sub- Metering 4. Advanced or Improved EE Performance – BEI 5. Enhanced Commissioni ng 6. Post Occupancy Commissioni ng 7. EE Verification 8. Sustainable Maintenance Table 2. Energy Requirements for BCA Green Mark, BERDE and GBI. Sources: [12-14]. Table 1. Assessment criteria of BCA Green Mark, BERDE and GBI. Sources: [12-14]. BCA Green Mark – 120 points BERDE – 120 points GBI – 100 points 1.Climatic Responsive Design – 30 2.Building Energy Performance – 30 3.Resource Stewardship – 30 4.Smart & Healthy Building – 30 5.Advanced Green Efforts – 20 1.Management – 14 2.Land Use and Ecology – 20 3.Water – 7 4.Energy – 9 5.Transportation – 18 6.Indoor Environmental Quality – 7 7.Materials – 6 8.Emissions – 4 9.Waste – 11 10. 2 Green Revolution There has been large body of work that has been done to enhance how a building performs in its operational phase, through simulation and ‘green’ design or architecture [7]. The term ‘green’ architecture/building refers to how environmental friendly the building is, in terms of the design, techniques and technology used in the building [8]. The green building revolution is being observed internationally [9], through energy standards such as the European Energy Performance of Building Directive (EBPD), the French Low Energy Building Decree and the Norwegian Standard for Residential Passive House [10] and is even starting to gain awareness locally in Malaysia with the birth of GBI. Other than building energy efficiency legislation, other strategies or approaches however has been done sporadically in the Malaysian building landscape, such as the introduction of Green Building Index (GBI) to rate how ‘green’ buildings are designed – not operated. The GBI has taken heed from pioneering green building rating tools such as Building Research Establishment Environmental Assessment Method (BREEAM) in The United Kingdom [4] and Leadership in Energy & Environmental Design (LEED) in The United States [5]. These green building rating tools are predicted models of how buildings would perform in operational phase, The believed benefits of green building practices will possibly provide the triple-bottom-line benefits of sustainable development, in terms of environmental, economic and social [9] [11] Some of the environmental benefits include: biodiversity and ecosystems enhancement; air and water quality improvement; waste reduction; natural resources conservation; and minimizing global warming effects. Other possible economic benefits could include: reduction of operation and maintenance costs; creation of green products and 00071 (2016) , MATEC Web of Conferences 66 00071 (2016) , MATEC Web of Conferences 66 IBCC 2016 DOI: 10.1051/matecconf/20166600071 11.Innovation - 20 services; life-cycle economic performance optimization; image improvement; and reduction of cost for civil infrastructure [9]. Meanwhile, perceived social benefit include: occupant productivity improvement; and occupant absenteeism minimization [9]. 2.1 Green Rating Tool and Simulation Heritage Conservation – 4 1.Energy Efficiency (EE) – 35 2.Indoor Environmental Quality (EQ) – 21 3. Sustainable Site Planning & Management (SM) - 16 4.Material & Resources (MR) – 11 5.Water Efficiency (WE) – 10 6.Innovation (IN) - 7 Table 1. Assessment criteria of BCA Green Mark, BERDE and GBI. Sources: [12-14]. The GBI Minimum EE Performance is for building envelope and installations like Building Energy Intensity Tool (BEIT) to hoped to minimize the energy consumption, hence reducing CO2 emission [14]. Lighting Zoning; as the name suggests it is allocated for lighting and zonings of lightings and the flexibility of the system. In this part, GBI’s requirements are to install motion sensors to turn off the light in the absence of a user, to install auto-sensors to automatically adjust the level of artificial lighting according the level of natural lighting, and to provide switches for every enclosed area individually to increase the flexibility of the system [14]. Electrical Sub-metering & Tenant Sub-metering is for installing different sub-meters for all energy use more than 100 kilovolt amps (kVa) as well as an individual sub-metering for lighting [14]. Renewable Energy is to promote the use of renewable energies and points are given according the amount of electricity provided by the renewable sources, so the more renewable energy, the more points, and a 2 00071 (2016) , MATEC Web of Conferences 66 00071 (2016) , MATEC Web of Conferences 66 IBCC 2016 DOI: 10.1051/matecconf/20166600071 intended as a CO2 emissions compliance tool [18] rather than energy simulation software. building can get as high as 5 points [14]. Points are given in Advanced or Improved EE Performance if a building manages to consume less than 150kWh/m2/year (this number may vary according to the type of building, for more information please refer to GBI) using BEI software or any other software recognized by GBI or to save at least 20% of the normal energy consumption over 3 years using BEI [14]. Enhanced Commissioning is used to ensure that every energy using system is working at its best potential and to ensure the minimal energy used by these systems [14]. However, these simulation tools are not able to predict the biggest variable in the calculations, as user behaviour, presence and activities can differ considerably to personal and group preference [6,7]. 2.1 Green Rating Tool and Simulation Building occupants can considerably effect energy consumption by influencing internal conditions, such as individual controls on air-conditioning and heat, window openings - passive ventilation and cooling methods, and shading mechanisms [6]. Furthermore, building occupants also have personal control over various electrical equipment and appliances – or known as ‘unregulated loads’, such as computers, laptops, printers, and so forth [6]. Points on On-going Post Occupancy Commissioning will be given for regular Post Occupancy Evaluation (POE) and more points will be given if they are assessed by professional engineers [14]. EE Monitoring & Improvement is for the use of Energy Management System (EMS) to monitor all the energy usage in the building as well as the sub-metering. If a building is not equipped with EMS then they should submit BEI, fuel and water consumption of the building on annual basis to GBI for the 3 years of validity period [14]. Sustainable Maintenance focuses on the maintenance of the energy related systems and points are given is the maintenance is planned properly and at least 75% of maintenance team participate in carrying out of maintenance [14]. Conversely during the operational stage of a building, energy and facility management can significantly effect how energy is consumed in a building. Good management and control can enhance operational efficiency of building services while the opposite reaction can occur through inappropriate strategies that result in unnecessary energy wastage [19]. Other significant parameters to evaluate operational energy performance of buildings include energy efficiency services and fittings, thermal performance of materials and material efficiency [20]. A study done in New York City found that one forth of the LEED Energy Star buildings actually consume more energy than predicted, and some was even consuming more than the national baseline (Refer Figure 1) [21]. The discrepancy is likely due to differences in operational practices and schedules, equipment, construction changes and other issues not predicted in the energy modelling process [21]. 2.2 Predicted and Simulated Building Performance Other than green building rating tools, simulation tools like EnergyPlus and Simplified Building Energy Model (SBEM) are also used as strategies to reduce the building sector’s environmental impact and climate change effect. Simulation and modelling tools are used as a design tool to simulate and predict how the said building would perform in its operational phase. An open source software called EnergyPlus was developed under the prevue of the United States Department of Energy Building Technologies Office to simulate energy performance of heating, cooling, ventilation, lighting, plug and process loads and water consumption of buildings [15]. Some of the EnergyPlus features include simulated: thermal zone conditions for heating, ventilation and air-conditioning (HVAC) systems; heat balance-based solution; combined heat and mass transfer; luminance and glare, and built-in HVAC and lighting control strategies [15]. Other than green building rating tools, simulation tools like EnergyPlus and Simplified Building Energy Model (SBEM) are also used as strategies to reduce the building sector’s environmental impact and climate change effect. Figure 1: Predicted Energy Savings Versus Actual Performance. Source: [21]. Figure 1: Predicted Energy Savings Versus Actual Performance. Source: [21]. The Simplified Building Energy Model (SBEM) is a software developed by the Building Research Establishment (BRE) to model building’s CO2 emission and energy consumption rates for new buildings, in compliance with United Kingdom Building Regulations [16]. SBEM predicts monthly energy use and CO2 emissions of a building, using variables such as building geometry, construction, function and usage, HVAC systems and lighting [16]. The SBEM uses a steady-state model for its building energy calculation, which is highly simplified and highly computational – as it largely ignores any dynamic characteristic [17], and is mainly Similar results were found in United Kingdom, through a free online energy data-sharing platform called CarbonBuzz (refer Figure 2). CarbonBuzz was launched in 2008 by the Royal Institute of British Architects (RIBA) and the Chartered Institution of Building Services Engineers (CISBE) in United Kingdom, published its analysis of shared energy consumption data in three building sectors, i.e. schools, general offices and university buildings [6] [22]. 3 00071 (2016) , MATEC Web of Conferences 66 00071 (2016) , MATEC Web of Conferences 66 IBCC 2016 DOI: 10.1051/matecconf/20166600071 modelled [6]. Consequently, this paper helps to bridge the gap by collecting real operational energy consumption data of a GBI rated building, and comparing it with a conventional non-GBI rated building to develop a baseline study. 3 Methodology The methodology used in this study is comparative case study. This method is used when two or more similar case studies are subjected and in depth comparison is required between them [24]. Case studies for this research have been chosen firstly from GBI Non- Residential New Construction (NRNC) list of certified buildings, the second conventional baseline building is chosen carefully from the commercial buildings, which have the similar number of floors and floor area for a fair and valid comparison. There are total number of 116 NRNC buildings in Malaysia which have been awarded by GBI certification, 54 of which have been awarded by GBI Certified certification, 18 have been awarded GBI Silver certification, 39 have been awarded Gold and only 5 are awarded GBI Platinum certification [25]. Figure 2: CarbonBuzz median electricity consumption per- sector – predicted vs. actual. Source: [22]. In an energy audit done by CarbonBuzz in 2013, it was largely found that the actual energy performance of all the building sectors was higher than its predicted performance, approximately about 70% to 89% higher than the predicted energy performance, or a Factor of Change (from Design to Actual) – Performance Gap between 1.71 to 1.90 (refer Table 3) [23]. The typical variable that affects operational energy performance of a building largely lies in the underestimating the ‘unregulated’ energy use from occupant-related usage, such as personalized computer/laptop, electronic plug-in appliances, heating and cooling, operating time, number of occupant and so forth [23]. Consequently, electricity bills were collected from the case studies and the data are quantitative and the monthly average electricity consumption is calculated for each building. The selected GBI certified building is categorized under the Non-Residential New Construction category. The data is then categorized in Building Energy Index (BEI), in terms of kWh/m2/year. The findings are then compared to see how better (or worse) GBI certified buildings perform, in comparison to a baseline conventional non-GBI certified building. Table 3. CarbonBuzz median electricity consumption per-sector – predicted vs. actual. Source: [23]. Building Sector Median Electricity Consumption (kWh/m2/year) Factor Change Design to Actual – ‘Performance Gap’ Differen -ce (%) Predict- ed Actual Schools 71 121 1.71 70% General Office 56 106 1.90 89% Table 3. CarbonBuzz median electricity consumption per-sector – predicted vs. actual. Source: [23]. 2.2 Predicted and Simulated Building Performance Figure 2: CarbonBuzz median electricity consumption per- sector – predicted vs. actual. Source: [22]. 5 Discussion The electricity data collected for both the case study buildings were for a total duration of 12 months between 2013 and 2014. Table 5 represents the collated electricity data and energy performance of the baseline building and the GBI certified building. It was found that the energy performance of the baseline building was lower than the GBI certified building, at 69 kWh/m2/year and 111 kWh/m2/year, respectively. As presented in Table 5, there is a stark difference in total electricity consumed by both buildings, where the annual electricity consumption by the baseline was at 236,984 kWh and the GBI building was at 407,131 kWh. This paper has highlighted the need to bridge the ‘performance gap’ between simulated and operational measured energy performance of buildings. Through the case studies selected and the data collected, it was found that the selected non-residential GBI certified building was performing better than the simulated or intended energy performance. However, through the comparative case study between a conventional non-GBI certified building and a GBI certified building, it was found that the conventional building was performing better than the GBI rated building over time. Identifying the factors that affect how energy is consumed in buildings will enable researched to bridge this performance gap to potentially predict energy performance and design building more accurately through simulated. Table 5: Electricity Consumption and Energy Performance of Case Studies Month Baseline Building GBI Building Floor Area: 41249 m2 Floor Area: 43943 m2 kWh kWh/m2 kWh kWh/m2 1 281,333 7 212,551 5 2 251,001 6 212,201 5 3 225,765 5 235,059 5 4 200,778 5 418,868 10 5 213,104 5 427,861 10 6 263,810 6 478,788 11 7 231,928 6 469,016 11 8 225,961 5 479,199 11 9 252,135 6 524,961 12 Table 5: Electricity Consumption and Energy Performance of Case Studies Researching how user behaviour affects energy performance of buildings is essential to bridging the performance gap, and can be done through Post- Occupancy Evaluation (POE) and sensor monitoring. POE is a common method to measure actual building performance, and now can be used to address to bridge the gap between simulated and operational building performance (Menezes et al., 2011). Green building rating tools like the GBI is an effective tool to reduce electricity consumption, but using this tool alone and certification does not guarantee energy saving. 4 Case Study and Findings The selected GBI certified building was designed to perform at 150 kWh/m2/year and has a total floor area of 43,943 m2, while the baseline building with 41,249 m2. Both buildings used double-glazed glass for its windows, and the GBI certified building has a total of 15 lifts, while the baseline building had 11 lifts. A brief description of the building characteristics of the selected case studies is presented in Table 4. Table 4: Building Characteristics of Selected Case Studies Item Non-Residential Baseline Building GBI Certified Building Number of Floors 36 35 Total Floor Area (m2) 41,249 43,943 Building Façade Double-glazed low E insulated glass Tempered Double- Glazed tinted glass Table 4: Building Characteristics of Selected Case Studies The ‘performance gap’ discrepancy can also be attributed to the lack of feedback to building designers after handover to enhance building performance [6] and therefore the inability to ensure the building performs as predicted. Other factors that could contribute to such discrepancies insufficient data and assumptions during the design stage, where the building function, use and future tenant are largely unknown or uncertain, which could lead to oversimplified and unrealistic performance [6]. Additionally, the modelling and simulation software used can contain fundamental errors in its calculation if the tools have not been extensively validates and do not consider the specific typology of buildings being 4 00071 (2016) , MATEC Web of Conferences 66 IBCC 2016 DOI: 10.1051/matecconf/20166600071 Lifts 4 high-speed lifts for low zone, 4 high-speed lifts for high zone, 2 carpark lifts and 1 VIP lift. Total of 11 lifts 6 high-speed lifts for low zone, 6 high- speed lifts for high zone and 3 car park lifts. Total of 15 lifts Air- Conditio ning System Variable Refrigerant Volume (VRV) system. Central water chilled air-conditioning system supported by high energy-efficient ‘green’ chiller and complemented by individual air handling unit (AHU) installed at every floor. Lighting System 28 Watt T5 tubes; 18 Watt T8 LED tubes; 9 Watt Plasma Lighting System (PLS); 18 and 36 Watt Fluorescent lights; 8, 15 and 20 Watt LED T5 Fluorescent tube equipped with high frequency electronic ballast. 4 Case Study and Findings 10 243,920 6 482,067 11 11 232,277 6 483,446 11 12 221,791 5 461,555 11 kWh/ year kWh/m2/ year kWh/year kWh/m2/ year Total 2,843,80 3 69 4,885,572 111 Aver- age 236,984 6 407,131 9 In comparison to the intended energy performance designated for the selected GBI building case study at 150 kWh/m2/year, it would seem that the GBI case study building was performing better than the intended or simulated with a lower energy performance of 111 kWh/m2/year. However, this does not negate from the significantly higher electricity consumption of the GBI certified building to the baseline non-GBI certified building. Further research needs to be done to investigate the factors of electricity consumption in non-residential building to understand why conventional non-GBI certified building consume lower electricity than the supposedly ‘green’ buildings that are GBI certified. Residential%20Tool%20V1.0.pdf 15. U.S Department of Energy, 2015. EnergyPlus Energy Simulation Software. Retrieved from http://apps1.eere.energy.gov/buildings/energyplus/ 3. Zaid, S., & Graham, P. (2015a). The Need for Energy Efficiency Legislation in Malaysian Building Sector. A Comparative Study of South East Asian Policies. Conference Proceedings of RICS COBRA Legal Research Symposium 2012. 16. BRE. (2016). Simplified Building Energy Model (SBEM). Building Research Establishment Ltd. Retrieved from https://www.bre.co.uk/page.jsp?id=706; http://www.uk-ncm.org.uk/ 4. BREEAM. (2013). What is BREEAM? Building Research Establishment (BRE) Ltd. Retrieved from: http://www.breeam.org/about.jsp?id=66 17. Wang, S., Yan, C., & Xiao, F. (2012). Quantitative energy performance assessment methods for existing buildings. Energy and Buildings, 55, 873-888. 5. U.S. Green Building Council. (2013). Leadership in Energy and Environmental Design (LEED). Retrieved from: http://www.usgbc.org/leed 18. Lee, W. L., & Burnett, J. (2008). Benchmarking energy use assessment of HK-BEAM, BREEAM and LEED. Building and Environment, 43(11), 1882- 1891. 6. Menezes, A. C., Cripps, A., Bouchlaghem, D., & Buswell, R. (2012). Predicted vs. actual energy performance of non-domestic buildings: Using post- occupancy evaluation data to reduce the performance gap. Applied Energy, 97, 355-364 19. Bordass B, Cohen R, Standeven M, Leaman A. Assessing building performance in use 3: energy performance of probe buildings. Build Res Inform 2001;29(2):114–28. 7. GhaffarianHoseini, A., Dahlan, N. D., Berardi, U., GhaffarianHoseini, A., Makaremi, N., & GhaffarianHoseini, M. (2013). Sustainable energy performances of green buildings: A review of current theories, implementations and challenges. Renewable and Sustainable Energy Reviews, 25, 1-17. 20. Mwasha A,Williams R G, Iwaro J. Modeling the performance of residential building envelope: The role of sustainable energy performance indicators. Energy and Buildings 2011;43 (9):2108–17. gy g ( ) 21. Turner, C., & Mark, F. (2008). Energy Performance of LEED for New Construction Buildings. Vancouver, WA: New Buildings Institute, 2008. 8. Love, P. E., Niedzweicki, M., Bullen, P. A., & Edwards, D. J. (2011). Achieving the green building council of Australia’s world leadership rating in an office building in Perth. Journal of Construction Engineering and Management, 138(5), 652-660. 22. Hamilton I, Steadman P, Bruhns H. CarbonBuzz – Energy Data Audit. UCL Energy Institute, July; 2011. 23. CarbonBuzz. (2013). Summary of Audits Performed on CarbonBuzz by the UCL Energy Insitute. CarbonBuzz Performance Gap. Retrieved from http://www.carbonbuzz.org/index.jsp#performancega p; http://www.carbonbuzz.org/downloads/Performance Gap.pdf 9. Nilashi, M., Zakaria, R., Ibrahim, O., Majid, M. Z. A., Zin, R. M., Chugtai, M. W., ... & Yakubu, D. A. (2015). A knowledge-based expert system for assessing the performance level of green buildings. 5 Discussion From the research it is understood that a well-managed and efficient building operations of a conventional non-GBI certified building can perform as well as or even better than a green building. 5 DOI: 10.1051/matecconf/20166600071 00071 (2016) , MATEC Web of Conferences 66 IBCC 2016 00071 (2016) , MATEC Web of Conferences 66 References 13. BERDE. (2013). BERDE for New Construction Commercial Buildings. Phillippine Green Building Council. Retrieved from http://philgbc.net/berde/berde-nc/1.1.0/BERDE-NC- COM-v110.pdf 1. NASA. (2013). Climate change: How do we know? The National Aeronautics and Space Administration (NASA). Retrieved from http://climate.nasa.gov/evidence 14. GBI. (2009). GBI Assessment Criteria for Non- Residential New Construction (NRNC). GreenBuildingIndex Sdn Bhd. Retrieved from http://new.greenbuildingindex.org/Files/Resources/G BI%20Tools/GBI%20NRNC%20Non- Residential%20Tool%20V1.0.pdf 2. UNEP-SBCI. (2010). Common Carbon Metric. United Nations Environmental Programme- Sustinable Building Climate Initiative. Retrieved from http://www.unep.org/sbci/pdfs/UNEPSBCICarbonM etric.pdf Residential%20Tool%20V1.0.pdf Knowledge-Based Systems, 86, 194-209. 10. Zaid, S. M., Myeda, N. E., Mahyuddin, N., & Sulaiman, R. (2015b). Malaysia’s Rising GHG Emissions and Carbon ‘Lock-In’Risk: A Review of Malaysian Building Sector Legislation and Policy. Journal of Surveying, Construction and Property, 6(1). p p 24. Mills, A. J., Durepos, G., & Wiebe, E. (Eds.). (2009). Encyclopedia of case study research (Vol. 2). Sage Publications. 25. Greenbuildingindex Sdn Bhd. (2014). GBI Certified Buildings. Retrieved from greenbuildingindex: http://www.greenbuildingindex.org/organisation- certified-buildings-NRNC-01.html 11. Ahn, Y. H. (2010). The development of models to identify relationships between first costs of green building strategies and technologies and life cycle costs for public green facilities (Doctoral dissertation, Virginia Polytechnic Institute and State University). 12. BCA Green Mark. (2015). BCA Green Mark for New Buildings (Non-Residential). Building and Construction Authority (BCA). Retrieved from http://www.bca.gov.sg/GreenMark/others/GM2015_ NewBuildings_NRB_Pilot.pdf 6
https://openalex.org/W2802977320	https://figshare.com/ndownloader/files/37058647	English	null	Front-of-package nutrition labels need to be assessed on their nutrition science rigour	Public health nutrition	2,018	cc-by	613	Letter to the Editor First published online 10 May 2018 Acknowledgements Financial support: This research received no speciﬁc grant from any funding agency in the public, commercial or not-for- proﬁt sectors. Conﬂict of interest: None. Authorship: M.L. conceived and prepared this letter and J.W. contributed to its drafting. Ethics of human subject participation: Not applicable. But these implementation ‘beneﬁts’ and ‘risks’ could more accurately be described as ‘risks’ and ‘beneﬁts’, i.e. inverted, if the nutrition science rigour underpinning the HSR is lacking. Our ﬁndings from the only compre- hensive evaluation of the impact of the voluntary HSR system in the marketplace shows that this is the case. The HSR is being exploited to market discretionary (energy- dense, nutrient-poor) foods, with 57 % of the HSR on these foods that have entered the marketplace since its launch displaying ≥2·5 (out of 5) so-called ‘health’ stars(2). Mark Lawrence and Julie Woods Mark Lawrence and Julie Woods Institute for Physical Activity and Nutrition (IPAN) School of Exercise and Nutrition Sciences Deakin University 221 Burwood Highway, Burwood, VIC 3125, Australia Email: mark.lawrence@deakin.edu.au Institute for Physical Activity and Nutrition (IPAN) School of Exercise and Nutrition Sciences Deakin University 221 Burwood Highway, Burwood, VIC 3125, Australia Email: mark.lawrence@deakin.edu.au Given that discretionary foods are not a part of a healthy diet and Australians need to eat less of them, we believe that encouraging the display of 3, 3·5 and 4 health stars on foods such as ice confections and chocolate-covered muesli bars misrepresents nutrition science and adds to public health nutrition confusion. Madam We read with interest the recent editorial by Kanter et al.(1) which provides a timely update on global front-of-pack (FOP) nutrition labelling activities. We note the editorial assesses the risks and beneﬁts of FOP, but only in relation to process issues rather than their nutrition science rigour. For instance, the editorial refers to the controversial Health Star Rating (HSR) system being implemented in Australia and New Zealand, saying its beneﬁts include that it is implemented well and contributes to food reformulation and its risks are that it is displayed on only 5 % of products. 1. Kanter R, Vanderlee L & Vandevijvere S (2018) Front-of- package nutrition labelling policy: global progress and future directions. Public Health Nutr. Published online: 21 March 2018. doi: 10.1017/S1368980018000010. Front-of-package nutrition labels need to be assessed on their nutrition science rigour These are important considerations, but they are a distraction until there can be conﬁdence in the rigour of the nutrition science that underpins the design of FOP schemes. From a nutrition science perspective, the FOP labelling research priority is to better align nutrient proﬁling-based schemes with food-based dietary guideline recommendations informed by evidence synthesised from studies of relationships between whole foods/dietary patterns and health outcomes. doi:10.1017/S1368980018001155 doi:10.1017/S1368980018001155 doi:10.1017/S1368980018001155 Letter to the Editor First published online 10 May 2018 Public Health Nutrition: 21(15), 2773 Public Health Nutrition: 21(15), 2773 2. Lawrence M, Dickie S & Woods J (2018) Do nutrient-based front-of-pack labelling schemes support or undermine food- based dietary guideline recommendations? Lessons from the Australian Health Star Rating System. Nutrients 10, 32, doi: 10.3390/nu10010032. References The editorial identiﬁes FOP labelling research priorities in terms of: The editorial identiﬁes FOP labelling research priorities in terms of: ∙consumer behaviour and industry reformulation; ∙label characteristics; 2. Lawrence M, Dickie S & Woods J (2018) Do nutrient-based front-of-pack labelling schemes support or undermine food- based dietary guideline recommendations? Lessons from the Australian Health Star Rating System. Nutrients 10, 32, doi: 10.3390/nu10010032. ∙equitable access to the information; ∙nutrition labelling use in real-world settings; and ∙implications of voluntary v. mandatory labelling. ∙nutrition labelling use in real-world settings; and ∙implications of voluntary v. mandatory labelling. © The Authors 2018
https://openalex.org/W4394062336	https://www.qeios.com/read/FBGVNZ/pdf	English	null	Review of: "Critical Research Thinking: A Recipe for Academic Writing Success and Publications"	null	2,024	cc-by	199	Qeios, CC-BY 4.0 · Review, April 7, 2024 Qeios ID: FBGVNZ · https://doi.org/10.32388/FBGVNZ Review of: "Critical Research Thinking: A Recipe for Academic Writing Success and Publications" Nishtha Khatri1 1 King Edward Memorial Hospital Nishtha Khatri1 1 King Edward Memorial Hospital Nishtha Khatri1 Potential competing interests: No potential competing interests to declare. Potential competing interests: No potential competing interests to declare. This seems like a well-chosen topic and a relevant subject for the present era of researchers. However, after reading it, I realised that there is a certain amount of repetition in terms of what the author wants to convey. Few sentences seem to convey the very same message but have been reframed to appear in different parts of the article. The major crux of the article is rested on the qualities of critical thinking, research thinking, and critical research thinking. However, all the qualities have a degree of overlap amongst them. Some of the references used are old, dating back to 1988. There should be a clear mention in the methodology of a review stating the duration of the literature search. On the whole, the topic is relevant and will benefit the readers. Qeios ID: FBGVNZ · https://doi.org/10.32388/FBGVNZ 1/1
https://openalex.org/W3119300370	https://zenodo.org/record/2018068/files/article.pdf	English	null	The Manufacture of Hydrogen from Water Gas	Scientific American	1,913	public-domain	6,482	By Henri Brot THE use of very low temperatures has not only furnished us with a valuable industrial process for the production of oxygen and nitrogen from the atmosphere, but has also given us an economical method of manu facturing hydrogen. fact that of late hydrogen has found increasing applica tion for aeronautical purposes, for the production of synthetic ammonia (see this SUPPLEMENT, July 26th, 1913, page 58), for the hydrogenation of oils to produce therefrom solid fats (see this SUPPLEMENT February 1st, 1913, page 71), etc. On the other hand, a description of this process is of peculiar interest owing to the very considerable technical difficulties which had to be over come in developing it. As will be seen, the temperatures employed in the processes for the manufacture of oxygen and nitrogen from air are not sufficiently low to effect the separation of hydrogen from water gas, and to achieve this result new apparatus had to be devised. The temperature employed is no less than 205 deg. Cent. below the freezing point of water, and is the lowest temperature reached in any industria: manufacturing process. fact that of late hydrogen has found increasing applica tion for aeronautical purposes, for the production of synthetic ammonia (see this SUPPLEMENT, July 26th, 1913, page 58), for the hydrogenation of oils to produce therefrom solid fats (see this SUPPLEMENT February 1st, 1913, page 71), etc. On the other hand, a description of this process is of peculiar interest owing to the very considerable technical difficulties which had to be over come in developing it. As will be seen, the temperatures employed in the processes for the manufacture of oxygen and nitrogen from air are not sufficiently low to effect the separation of hydrogen from water gas, and to achieve this result new apparatus had to be devised. The temperature employed is no less than 205 deg. Cent. below the freezing point of water, and is the lowest temperature reached in any industria: manufacturing process. in which the several ingredients are present depends on the temperature at which the gas was prepared. In the Linde, Frank and Caro process the raw material employed consists of water gas containing about 50 per cent of hydrogen. The first step is to eliminate the carbon dioxide by passing the gas over caustic potash. Splicing Transmis sion Rope* thllt wllPre the seyeral strands are rejoilled, each fast elling Ol' "tuck" should be made in ",uch mllnller as to prevellt its wearing aWIlY and the l'olle unstranding. There tire mally different splices !lOW ill use, but after a ;;;lreful stmly of each we are cOII\'illced that all the aboye features are combilled only in what is called the 1ùlIglish transmisl';ion slJlice, and experience has proyed it the best olle known. In describing this we take for our example a four strand rope, I%, inches diameter, as spliced on sheaves in the multiple system. In describing this we take for our example a four strand rope, I%, inches diameter, as spliced on sheaves in the multiple system. The rope is lirst placed around sheaves, and, with a tackle, stretched and hauled taut; the ends :;hould pass each othel' from six to seven feet, the passing point heing mal'ked with twine on each rope. The 1'Ope is then :;lipped from sheaves and allowed to rest on shafts, to give sufficient slack for making splice. There tire mally different splices !lOW ill use, but after a ;;;lreful stmly of each we are cOII\'illced that all the aboye features are combilled only in what is called the 1ùlIglish transmisl';ion slJlice, and experience has proyed it the best olle known. After the rope has been in service a few days, the projecting ends at locks wear away, and if tucl,s have been made carefully, and original twist of yarns pre served, the diameter of the rope will not be increased, 1101' can the splice be located when rope is in motion. Dnlay the strands in pairs as far back as the twines, M M', crotch the four pairs of strands thus opened (Fig. 1), cores having been drawn out together on the -'pper side. • By courtesy of the American Manufacturing Company. • By courtesy of the American Manufacturing Company. Fig. 4. Fig. 6 l'hen, having removed marking twine M, un lay the two strands 6 and 8, still in pairs, back a distance of two feet, to A; the strands 1 and 3, also in pairs, being carefully laid in their place. Fig. 4. Fig. 4. Fig. 5. Fig. 7. Fig. 5. Next unlay the strands 5 and 7 in pairs, to A', re placing them as before with 2 and 4. The rope is now as shown in l·'ig. 3. Splicing Transmis sion Rope* The pair of strands 6 and 8 are now separated, and 8 unlaid four feet back, to E, a distance of six feet from center, strand 6 being left at A. (See I;'ig. 2.) The pair of strands 1 and 3 having been separated, 3 is left at A, as companion for 6, strand 1 being care fully laid in place of strand 8 until they meet at point n. Fig. 4. Fig. 6. '1'he two pairs of strands 2-4 and 5-7 are now :;ep arated and laid in the same manner, every care being taken, while thus putting the rope together, that orig inal twist and lay of strand are maintained. Fig. 5. The protruding cores are now cut off so that the ends, when pushed back in rope, butt together. Fig. 7. The rope now appears as shown in Fig. 2, and after the eight strands have been cut to convenient working lengths (about two feet), the companion strands are ready to be fastelled together and "tucked." This operation is described for "trawls 2 and 7, the method being i(lentical for the other three pairs. llnlay 2 alld 7 for about ]:! to 14 inches, divide each strand in half by removing it::; cover yarns (see Fig. 4), whip with twine the ends of interior yarns 2' and' 7' ; Fig. 6. Fig. 7. SCIENTIFIC AMERICAN SUPPLEMENT SCIENTIFIC AMERICAN SUPPLEMENT October Ill, 1918 244 Fig. 2. Fig. 1. Fig. 3. Fig. 2. * Translated for the SCIENTIFIC AMERICAN SUPPLEMENT, from Le Gente CiŤtl. Splicing Transmis sion Rope* Fig. 3. Fig. 1. then, leaving coyer 2, relay 2' until Ilea l' 7 allll 7', here join with simple knot 2' and 7' (Fig. 5). WE ha ve had occasion in a previous issue to set forth some of the advantages of rope transmission. So far Ill'; the use of ropes fOl' power transmission has led to complaints, it may be said that most of the "troubles" complained of in rope drives are directly caused by lJad Sillicillg, although it is a simple matter to make n proller slJlice, ollce the idea has been graslJed. Divide cover yarns 7, and pass 2' through them, continuing on through the rope under the two adjacent l';trands, avoiding the core, thus locking 2' (Fig. 6). ln no event pass 2' over these 01' any other strands. Half strand 7' must now be taken care of; at the right of the knot made with 2' and 7', 2' is slightly raiHed with a marlin spike, and 7' passed or tucked around it two or three times, these two half strands forming in this way a whole strand. Half-strand 7' is tucked until cover two is reached, whose yarns are divided and 7' passed through them and drawn under the two adjacent strands, forming again the lock. The strand ends at both locks are now cut off, leaving about two inches, so that the yarns may draw slightly with out unlocking. This completes the joining of one pair of strands (Fig. 7). The three remaining pairs of strands are joined in the same manner. The essential points in a transmission splice are: First, that its diameter be absolutely the same as that of the original rope; second, that it be smooth and free from lumps; third, that the original lay of strands alld yarns be disturbed as little as possible; and fourth. thllt wllPre the seyeral strands are rejoilled, each fast elling Ol' "tuck" should be made in ",uch mllnller as to prevellt its wearing aWIlY and the l'olle unstranding. The essential points in a transmission splice are: First, that its diameter be absolutely the same as that of the original rope; second, that it be smooth and free from lumps; third, that the original lay of strands alld yarns be disturbed as little as possible; and fourth. COLD OF THE GASES. In the same way the cold gases, after separation, pass through heat exchangers in which they become reheated while themselves absorbing heat from the entering stream of gas. These heat exchangers are constructed, as in all Linde apparatus, of concentric tubes. The gases cir culate either through the central bore of the tubes or through the annular outer channel, and the heat ex change takes place through the walls of the tubes. For the sake of compactness the tubes are coiled up into spirals or worms. Proper care is of course taken to pro vide as perfect a heat insulation as possible, so as to prevent heat losses (or to be more exact, losses of cold) by heat conduction and radiation. .Air C'ol1l/Jl·es."lQr Fig. I.-Diagrammatic general view of a plant for manufacturing hydrogen. Fig. 4.-Diagrammatic plan view of a plant for manufacturing hydrogen It would seem at first sight as if the net balance of heat produced and consumed in apparatus per unit of time were zero, and as if, once the apparatus were started, there were no further need of producing any cold. Under these circumstances the preparation of the gases should consume no other energy than that small amount which is necessary to keep the gases flowing through the apparatus. a, steam boiler; b, gas purifier; 0, gas generator; d, ven· tllator; 6, gasometer for water gas; f, gasometer for car bon monolride; g, gas engine; h, air compressor; j, am· monia compressor. For the sake of simplicity only the conduits conveying the water gas, the carbon monolride and the hydrogen have been shown. a, steam boiler; b, gas purifier; 0, gas generator; d, ven· tllator; 6, gasometer for water gas; f, gasometer for car bon monolride; g, gas engine; h, air compressor; j, am· monia compressor. For the sake of simplicity only the conduits conveying the water gas, the carbon monolride and the hydrogen have been shown. .Air C'ol1l/Jl·es."lQr Fig. I.-Diagrammatic general view of a plant for manufacturing hydrogen. The question arises, where, in the course of the process with which we are concerned, do we find the equivalent of this expenditure of energy. Mr. Claude is of the opinion that the item in question appears in the ecessity of compressing the gases before treatment. Is this correct? By Henri Brot The gas thus purified is then compressed to about 30 atmospheres and cooled to a temperature in the neighbor hood of the normal boiling point of oxygen, say 200 degrees Cent. A partial liquefaction takes place, and two phases are formed, the one liquid, containing a higher percentage of liquefiable constituents than the original gas'inixture; the other gaseous, and containing a higher percentage of volatil{\ constituents than the original mixture. In the manufacture of nitrogen and oxygen from air, either of two general methods may be made use of: 1. Either the air is liquefied and is then subjected to a fractional distillation (rectification). This is the basis of the Linde process. 2. Or the air is subjected to partial liquefaction; the oxygen, being less volatile, is the principal product of condensation. The residual gas is then liquefied and fractionally distilled. This is the method followed in the Claude process. Hydrogen also can be very economically prepared by the use of very low temperatures by a somewhat similar process, due to Messrs. Linde, Frank and Caro. g The volatile constituent of the mixture is the hydrogen, whose critical point is -242 degrees Centigrade. The liquefiable constituents are the carbon monoxide and nitrogen, whose critical temperatures are respectively -136 degrees Centigrade and -146 degrees Centigrade. The conditions of pressure a.nd temperature selected PRINCIPLES OF THE LINDE, FRANK AND CARO PROCESS. Water gas consists essentially of hydrogen, carbon monoxide and nitrogen. It also contains a comparatively small percentage of carbon dioxide. The proportion o This process has gained special interest owing to the o This process has gained special interest owing to the asld © 1913 SCIENllFIC AMERICAN,INC. SCIENTIFIC.AMERICAN SUPPLEMENT October 18, 1913 245 are such that the liquid phase, very rich in carbon monox ide, contains but little hydrogen, and the gaseous phase rich in hydrogen, contains very little of the other constituents, only about 2.5 to 3 per cent. erator) at our disposal were water also at 100 degrees Cent. which was to be evaporated. 'Evidently all that would be required would be to cause the steam to pass through a worm tube immersed in the hot water. The liquifaction would take place within this tube ENERGY ABSORBED IN THE SEPARATION OF A PHYSICAL MIXTURE OF TWO GASES. The purely physical separation of two gases which are mechanically mixed together requires the expenditure of a certain amount of work. By Henri Brot A simple proof of this fact has been given by Mr. G. Claude, who has taken his clue from the work of Van der Waals. Let a mixture of two gases be placed in a cylinder in the bore of which two pistons can be moved, of which one is perfectly permeable to carbon monoxide, but impermeable to hydrogen, while the other on the contrary allows hydrogen to pass unhindered while being completely impermeable to carbon monoxide. Suppose that one piston is placed at each extremity of the cylinder, which is filled with the gas mixture at atmospheric pressure. Then let the two pistons be brought together until they touch, the distance traveled by each being proportional to the percentage of the particular gas to which it is permeable. We shall then have the two gases separated and contained re spectively in the two spaces comprised between the walls of the tube and the pistons. The gases will moreover be at atmospheric pressure. It will be seen that the minimum work required thus to separate the gases will be the sum of the work of isothermal compression of each gas from its partial pressure in the original mixture to atmospheric pressure. It is therefore impossible by any method to separate two gases without expend ing work. GENERAL DESCRIPTION OF THE PLANT. Prof. Linde's method, as employed in the preparation of liquid air, can not be used for the liquefaction of water gas. pv 1,0 0,8 D,S 0,4. 0.2 o .= F" - ' - ·'· H (·10S" ) fI (o'C) .Air :o'Cl = . .- o'(o'ri' .- f--.. - -' -11:::/"'03 ·,1 .ţ H( .. 2.1.2 .l _H"18;O o 50 100 150 200 Fig. 2.-Curves showing the variations in the product "pv" of different gases as functions of the pressure of "p" at different temperatures. This method, as is well known, consists in first com pressing the gas, thereby raising its temperature; thereĈ upon cooling it to the temperature of the surroundings; and finally extracting from the gas the heat correspond ing to internal work of expansion, by allowing it to escape through a valve (Joule-Thomson effect). f The water gas employed contains, as has been stated, 50 per cent of hydrogen, and therefore on the whole displays the physical properties of hydrogen. COLD OF THE GASES. At first sight it seems not to be so, for since we are now considering the matter from a purely theoretical point of view, we are justified in considering the wall of the worm tube as having an infinite conductivity. In this case the difference of temperature between its two faces may be made as small as desired, no matter how great the heat transfer through the tube. But it must not be forgotten that on the outside of the worm tube we have liquid carbon monoxide, and on the inside a gaseous mixture, and that it is necessary to establish the requisite conditions to ensure effective separation. But, as in all industrial operations, there is of course a certain loss, and the heat balance is not zero for the following reasons: expansion, is greater than Tlo the temperature before expansion. 1. To cause the separation of two gases, even if purely physically mixed, theoretically requires a certain amount of work to be done, so that a fortiori in practice such work must actually be furnished. Hence, when allowed to escape from an open valve, at the temperature of the surroundings, hydrogen does not cool, but on the contrary, rises in temperature. Hence, when allowed to escape from an open valve, at the temperature of the surroundings, hydrogen does not cool, but on the contrary, rises in temperature. Thus the Linde method, as applied in the liquefaction of air, is not applicable for cooling water gas, and another expedient must be resorted to, as in the liquefaction of hydrogen. The agent made use of is liquid air. By allowing it to evaporate it can be made to furnish the requisite degree of cold to reach the temperatures in question. n 2 However well designed and constructed the ap paratus may be it is not perfect and there are certain losses of cold which occur. Water6'as (( CO 11- .. -II Q.- ·c c_ Section et._. .®(l ·Ii " A Liquid co to rir-;===!J be evaporated Fig. 3.-Diagram of apparatus for manufactur ing hydrogen from water gas by the aid of liquid air according to the process of Linde, Frank and Caro. Water6'as (( CO 11- .. -II Q.- ·c c_ Section et._. .®(l ·Ii " A Liquid co to rir-;===!J be evaporated f IMPERFECTIONS OF THE APPARATUS However carefully constructed and designed tho heat exchangers may be, they are not perfect, and the gases which issue from the apparatus are in practice always of a temperature several degrees below that of the entering gases. Again, however carefully insulated the ap paratus may be, the heat conductivity of their insulating mantles is never quite zero, so that there is always a slight loss of cold which must be made good. In order to compensate for these losses, liquid air is continuously supplied to the apparatus after once operations are started, which, by its evaporation, furnishes the requisite amount of cold. By Henri Brot Now, it will be remembered that hydrogen presents a peculiarity, as compared with other gases (except helium, whiciJ in this respect resembles hydrogen), in that the product P v (pressure times volume) increases with the pressure, instead of decreasing as the pressure increases, as is the case with air for example. (See the curves, Fig. 1.) Fig. 2.-Curves showing the variations in the product "pv" of different gases as functions of the pressure of "p" at different temperatures. the case with air for example. (See the curves, Fig. 1.) On the other hand, the temperature change produced by the Joule Thomson effect is given by the relation: p ( , g ) On the other hand, the temperature change produced by the Joule-Thomson effect is given by the relation: with the liberation of heat, while at the same time, in the hot water jacket around the worm tube evaporation would occur with absorption of heat and the quantities liquefied and vaporized would be almost equal. We note that it would be necessary for the steam to be slightly compressed, so that it would become liquefied at a temperature just a little above that of the water, for in order that heat should pass from one surface of the worm tube to the other, there must be at least a slight temperature difference provided. by the Joule-Thomson effect is given by the relation: (T1-T2)Cv= (rl-r2) +A(P2V2-P1VI) where T is the tempecature, P the pressure, v the volume and r internal energy. The suffix 1 denotes the value of the correspond ing variable before expansion, and the suffix 2 similarly relates to values after expansion. The symbol Cv denotes as customary the specific heat a.t cOlJ.stant volume, while A....is the factor'by which the number of work units measuring a given quantity of energy is converted into the corresponding number of heat units. The curve shown in Fig. 2 rises sufficiently steeply to cause the term A (P V -P1Vl) to exceed (in absolutelvalue) the term (Vl-V2). Hence T1-T2 is ne.gative, :ior 3n:other+words,ĉjT2:)he temperature after - -·-·Ht]J-·_·_·- ! I I K' © L Fig. 4.-Diagrammatic plan view of a plant for manufacturing hydrogen a, steam boiler; b, gas purifier; 0, gas generator; d, ven· tllator; 6, gasometer for water gas; f, gasometer for car bon monolride; g, gas engine; h, air compressor; j, am· monia compressor. By Henri Brot For the sake of simplicity only the conduits conveying the water gas, the carbon monolride and the hydrogen have been shown. - -·-·Ht]J-·_·_·- ! I I K' © L Fig. 4.-Diagrammatic plan view of a plant for manufacturing hydrogen COLD OF THE LIQUID CARBON MONOXIDE. As the liquid carbon monoxide issuing from the process is vaporized, the heat necessary for this evaporation is imparted to it by the entering stream of carbon monoxide, which, in assuming the liquid form, absorbs the cold contained in the previously liquified carbon monoxide, according to the well-known counter-current principle. COLD-ECONOMIZER PRINCIPLE. In spite of the high price of "cold" (one frigorie between -10 and -15 deg. Cent. costs from twelve to fifteen times as much as one calorie), the constituents of the air are produced by liquefaction industrially at a very low figure, owing to the fact that the cold employed in the process is "recovered" after the manner of "heat recovery" in a heat exchanger. In fact, in the Linde oxygen and nitrogen process cold is merely a means to an end. The purpose is not to produce cold, but simply to furnish a temperature at which the two gases can be separated. The products themselves, oxygen and nitrogen, or hydrogen and carbon monoxide, as the case may be, are of course not collected at this low temperature, but at that of the room. Hence both the cold of the liquid carbon monoxide and that of the gaseous products is conserved in the apparatus. To do this it is necessary to compress the gases to be treated and there can be no doubt that it is at this point in the process that the expenditure of energy required to effect separation enters into the process. f IMPERFECTIONS OF THE APPARATUS. II. Here, then, is an apparent discrepancy between Stokes's law and the facts. The law informs us that the speed of subsidence decreases rapidly with decreas ing radius of the particles, but it does not lead us to ex pect the total absence of settling which presents itself when the average radius is 10 1"1-' or thereabout. Every one is familiar with the distinctions between solutions and suspensions. Suspensions are turbid in aspect, and the solid can be removed by letting it settle, or by filtration. Solutions are clear. dissolved matter does not subside and is unaffected by filtering. Col loidal solutions occupy an intermediate position. The explanation, of course, is moleeular motion, or, in other words, heat. The particles are hattered, on all sides, by a hailstorm of molecular impaets. If the par ticle is large, the blows of the molecules of the solvent in different directions neutralize each other. But when the particle is not so very much larger than the mole cules themselves a molecule striking, say, on the left, will give the particle a very perceptible push toward the right, "just as a cork follows better than a large ship the motion of the waves of the sea."2 As the di mensions of the particle approach the molecular dimen sions it begins to behave like a molecule and is swept along in the endless molecular movement. The cause which prevents the particles in a colloidal solution from settling is in no way different from the cause which pre vents the earth's atmosphere from subsiding to a snowy layer a few feet deep on the surface of the planet. However, the molecules of the sugar retain their struc ture intact, the action being limited to their dispersion. When salt, on the other hand, is dissolved in water, a further breakdown occurs, the molecule is separated and ions of sodium and of chlorine move about in the liquid. Both solutions freeze below 0 deg. C. and boil above 100 deg. C. The most important difference between them is that the salt solution conducts the electric cur rent, while the sugar solution is as poor a conductor as water itself. Consider for a moment the effect of increasing sub division on a suspension of finely-divided gold in water. II. So long as the diameter of the particles is much greater than a thousandth of a millimeter,l the system will be turbid and the gold will settle rapidly. But the waVe length of visible light ranges between 0.4" and 0.71-', and when the particles become smaller than this they can no longer reflect light and the liquid will appear clear. At the same time there will be a rapid falling off in the speed of settling. Stokes has derived a formula for the velocity of subsidence, v, of small spheres of radius R and density 8 falling in a liquid of density 8' and inter nal friction f under the force of gravity g: R2 A fourth possibility presents itself when glue or gel atin is treated with water. The gelatin absorbs water, swells up and, under the influence of heat, dissolves, but the liquid freezes and boils at practically the same temperatures as pure water. The study of the solution shows that the dispersion is not molecular. The parti cles of gelatin in it are composed of variable and rather large numbers of molecules. A system like this gelatin solution which presents a case of very fine but not mol ecular subdivision is called a colloidal solution. There are certain solids such as gelatin and dextrin (with water), rubber (with benzene and carbon disulphide), which, when they dissolve in liquids, are invariably dispersed in this way. Such solids may properly be referred to as colloids. They are all amorphous. Crystallized sub stances never yield colloidal solutions by mere spon· taneous solution in a liquid. They always produce molecular or ionic dispersions. However, the phenom enon of colloidal solution is perfectly general, and crystal lized substances can also be obtained in this condition, but not mere solution. 2 R2 V = 9 g(8-8') T l f Substituting the proper values for gold and water and assuming a radius of 1" for the particles, the value for V is about 14 centimeters per hour. This means, of course, that the system would be a coarse suspension and would clear up at once. But when R=lO I-' 1-'. V is only about a centimeter a month. This begins already to be fairly permanent. It must be remembered that the high density of gold (19.5) increases the rapidity of sub sidence. COST o' PHODUCTION. In figuring out the cost of production with ruferllm,e to a plant which produces only hydrogen, we have the following items: Hydrogen prepared in this manner is not absolutely pure; as has been stated above, it contains about 2.5 to 3 per cent of impurities. Nevertheless this is a very creditable performance, .as will be well understood by those who are familiar with the industrial manu facture of gases, and who know the difficulties of obtain ing the last degrees of purity in the product.l This dogree of purity is sufficient for most uses. Neverthe less in certain cases, especially for aeronautics, where density is a very important factor, it is desirable to increase as far as possible the hydrogen contents. A percent of 97 to 97.5 hydrogen corresponds to a density of 0.094, while when the percentage of hydrogen is raised to 99.2 or 99.4 percent, the density falls to 0.077. The lifting power of one cubic meter of the gas is then raised from 1,175 to 1,195 grams. Hourly production in cubic meters of hydrogen . . . . . . . . . . . . . . . .. [)O 100 200 500 Water gas consumed cubic meters per hour .. ....... .. ..... .. . . 125 250 5UO 1250 Coke consumed per hour (lbs.) .. 176 352 704 1720 Cooling water required cubic meters per hour ............. 3.8 7.6 13.5 32.5 i y }1Ľig. 4 is a plan view in which the reference letters denote the same features as in Fig. 1. In spite of the preliminary purification of the water gas, whereby water vapor and carbon dioxide are eliminated, traces of these bodies nevertheless pass into the separating apparatus, and, being deposited there in the solid form, ultimately obstruct it after the lapse of a certain time. This imevitable though regrettable condition is met by using two similar apparatus employed in rotation, say in weekly shifts. Whichever of the duplicate plants is out of operation can in the meantime be brought back into working shape by heating it and blowing gas through it. From these data it will be seen that for plants of the type considered, the consumption of materials is vory nearly in pcoportion to the output. 'rhis is due to the fact that the percentage composition of the gaHoous products is practically independent of the size of the apparatus. SCIENTIFIC AMERICAN SUPPLEMENT SCIENTIFIC AMERICAN SUPPLEMENT October 18, 1918 246 products from this step of the process. products from this step of the process. through valve C is vaporized, after having been separated in the separator D. E is a valve at which the hydrogen may be allowed to expand, before it is drawn from the apparatus. Finally, F represents diagrammatically the liquid air jacket which serves on the one hand for the preliminary cooling and on the other for the continued cooling during the process for the purpose of making good incidental losses. since it would become solid in the course of the process and stop up the pipes. Hence, after the first stage of the compression, the gas mixture is passed through purifying towers B, in which a current of water absorbs the greater part of the carbon dioxide Then, after issuing from the compressor, the stream of gas is passed over caustic potash contained in receptacle C, which arrests the last traces of car bon dioxide and also takes up any moisture present, which would of course cause the same trouble as that just pointed out with regard to carbon dioxide. D and E are the usual coolers for the compressor. F is the separating apparatus described above. G is the liquid air plant worked by the aid of liquid ammonia in the usual way. L is the soda lime purifier for the hydrogen. since it would become solid in the course of the process and stop up the pipes. Hence, after the first stage of the compression, the gas mixture is passed through purifying towers B, in which a current of water absorbs the greater part of the carbon dioxide Then, after issuing from the compressor, the stream of gas is passed over caustic potash contained in receptacle C, which arrests the last traces of car bon dioxide and also takes up any moisture present, which would of course cause the same trouble as that just pointed out with regard to carbon dioxide. D and E are the usual coolers for the compressor. F is the separating apparatus described above. G is the liquid air plant worked by the aid of liquid ammonia in the usual way. L is the soda lime purifier for the hydrogen. The carbon monoxide could be rectified and obtained pure. lIt is usual to employ the symbol" (the Greek letter mu) for the thouslUldth of a millimeter. In the S&IIle W&f "p. indie&tes 1ibe IUillionth of !\ milliIlleter, * Presented at the meeting of the SectIon of Physics and Chemistry of the Franklin Institute lUld pu bUs4e<l. ill tbe Journal of tbe Insmute, I It is interesting to note here that tho manufacture of cyanamide calls for the use of nitrogen of 99.8 per cent purity. The ap paratus devised by Lindo and Claude completely fulfills those requirements .. 'Thomas Graham introduced tbe tll!"Ţ sQI &s lUl abbreviitlQIl for coIloid&1 aQ.lu jiOIl. II. If we make the calculation for 8 =3, which is about the density of arsenious sulphide, V comes out only about a millimeter a month. It is worth remembering, also, that the particles of the dispersed phase ordinarily possess an electric charge, which is usually negative. The effect of the repulsion of these similar charges would be to preserve the dis tribution of the particles throughout the liquid. It is a fact that, when the charges are removed, the system becomes instable and subsidence-preceded by coalos ence of the small particles-readily, but not necessar ily, occurs. Colloids and Crystals* Two Worlds of Matter By Robert H. Bradbury By Robert H. Bradbury vision is maintained. Colloidal gold solutions have been preserved unehanged for years. I have a solution of arsenious sulphide w hieh has remained apparontly un changed for three years and whose countless particles can readily be seen, engaged in their incessant Brown ian movement, with an ordinary oil immersion lenľ. Whenever settling does occur, it is preceded hy the ag gregation of the particles into larger particles, whh·h finally attain a diameter of 11-' or over, and slowly subside. It is an interesting fact that a substance whieh yields a colloidal solution with one solvent may form an ordin ary molecular solution with another. Soap is an example. I ts concentrated solution in water boils at about 100 deg., freezes at about 0 deg., and exhibits the behavior of a col loidal solution in general. On the contrary, a soap solution in alcohol shows the normal change in freezing and boiling points corresponding to the molecular weight, and conducts itself in all respects like an ordinary mol ecular dispersion. WHEN a solidis brought into contact with a liquid the rosult depends upon the nature of both. There may be apparontly an entire absence of interaction, as when rosin is shaken up with water or chalk with alcohol. Or, as when sugar is agitated with water, the solid may disappear, entering into solution in the liquid. The study of sugar solution shows quite clearly that the con nection of the sugar molecules with each other has been completely destroyed. They are dispersed through the water very much as the molecules of a gas distribute themselves uniformly in a vacant space, and in b6th cases the permanence of the uniform dispersion is due to the incessant motion of the molecules. Were the molecules at rest, both the sugar and the gas would settle and form a layer on the bottom of the containing vessel. GENERAL DESCRIPTION OF THE APPARATUS. The apparatus employed is diagrammatically rep resented in Fig. 3 of our drawings. A is the heat exchanger with the three conduits a, b, c, which convey respectively the compressed water gas, the hydrogen, and the carbon monoxide and nitrogen. B is the vapor izer. Within the worm tube, the liquefiable constituents of the gas mixtures are condensed, while in the space around this coil, the carbon monoxide introduced Fig. 3.-Diagram of apparatus for manufactur ing hydrogen from water gas by the aid of liquid air according to the process of Linde, Frank and Caro. This corresponds exactly to what would occur if the problem were to liquify water vapor, (steam) at 100 degrees Centigrade, if the ouly source of cold (refrig- © 1913 SCIENTIFIC AMERICAN, INC. COST o' PHODUCTION. Nevertheless the advantages of large scale apparatus is felt in a reduction in the proportionate consumption of coke and cooling water. At the samo time the cost of labor is reduced. A gas containing a very high percentage of hydrogen l',an be obtained by passing the 97 per cent over gas soda lime. This yields a product containing as its .only impurity 0.6 to 0.8 percent of nitrogen. The cost of hydrogen, not including chargos for interest and sinking fund on the plant varies from ton to twelve centimes per cubic meter in the case of a plant of 200 cubic meters per hour capacity. With the special purification of which mention was made above, the prieo rises to about 12.5 to 14 centimes. It is interesting to compare these figures with the price of hydrogen as prepared by the old method of acting upon iron with sulphuric acid, where 75 centimes must be regarded as the minimum price attainable. At the same time there is little doubt that the process is open to further improvement and that ultimately the price of the product will be still further redueed. BY-PRODUCTS o' THE HYDHOGEN MANUFACTUHE. 'I'he accompanying illustrations Figs. 1 and 4, which are derived from the Zeitschrift des V creins, Deutscher Ingenieure and the Zeitschrift filr Angewandte Chemie give a rather more complete representation of the entire plant. In Fig. 1 A is a compound compressor for com pressing the water gas. This gas contains about three percent of carbon dioxide, which must first be eliminated, In this process of manufacture the principal product is of course the hydrogen; nevertheless it is possible to recover certain by-products which are not without value. Thus the carbon monoxide can be collected in a gaso meter in quantities sufficient to feed a gas motor which can be used to drive the compressors of the plant. Moreover we noted above that the cold lost by con duction, etc., must be made up for by a cold furnished from evaporating liquid air. Prof. Linde has succeeded in recovering separately oxygen and nitrogen as by- SCIENTIFIC AMERICAN SUPPLEMENT But hitherto it has been found more satisfactory to simply employ it as a fuel without preliminary purification. III. On the subject of the classification of colloid systems we must be very brief. One proposal subdivides them into suspensoids, such as the sols3 of gold and arsenious So much for calculation. N ow what are the facts? As a matter of fact, the dispersed substance in a colloi dal solution does not settle at all, so long as the subdi- 'Perrin. 'Perrin. * Presented at the meeting of the SectIon of Physics and Chemistry of the Franklin Institute lUld pu bUs4e<l. ill tbe Journal of tbe Insmute, © 1913 SCIENTIFIC AMERICAN, INC.
https://openalex.org/W2597680358	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0205120&type=printable	English	null	The crowding-out effect of tobacco expenditure on household spending patterns in Bangladesh	PloS one	2,018	public-domain	8,498	RESEARCH ARTICLE Editor: Rachel A. Nugent, RTI International, UNITED STATES Editor: Rachel A. Nugent, RTI International, UNITED STATES Muhammad Jami HusainID1, Biplab Kumar Datta1, Mandeep K. Virk-Baker2, Mark Parascandola2, Bazlul Haque Khondker3 1 Global Noncommunicable Diseases Branch, Division of Global Health Protection, Center for Global Health, Centers for Disease Control and Prevention, Atlanta, GA, United States of America, 2 National Cancer Institute, National Institutes of Health, Rockville, MD, United States of America, 3 Department of Economics, University of Dhaka, Dhaka, Bangladesh a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 MHusain@cdc.gov * MHusain@cdc.gov Methods Received: May 24, 2018 Accepted: August 22, 2018 Published: October 9, 2018 We analyzed data from the Bangladesh Household Income and Expenditure Survey 2010 to examine the differences in consumption expenditure pattern between tobacco user and non-user households. We further categorize tobacco user households in three mutually exclusive groups of smoking-only, smokeless-only, and dual (both smoking and smoke- less); and investigated the crowding-out effects for these subgroups. We compared the mean expenditure shares of different types of households, and then estimated the condi- tional Engel curves for various expenditure categories using Seemingly Unrelated Regres- sion (SUR) method. Crowding-out was considered to have occurred if estimated coefficient of the tobacco use indicator was negative and statistically significant. Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Data Availability Statement: The national Household Income Expenditure Survey (HIES) is conducted by Bangladesh Bureau of Statistics (BBS) with technical and financial assistance from the World Bank. The data is available publicly for purchase from the Bangladesh Bureau of Statistics. The authors purchased the data by contacting BBS; the authors did not have any special access privileges that others would not have. The researchers/institutions that intend to use the data need to contact the BBS for purchasing (www.bbs. gov.bd/). Background OPEN ACCESS Citation: Husain MJ, Datta BK, Virk-Baker MK, Parascandola M, Khondker BH (2018) The crowding-out effect of tobacco expenditure on household spending patterns in Bangladesh. PLoS ONE 13(10): e0205120. https://doi.org/10.1371/ journal.pone.0205120 Tobacco consumption constitutes a sizable portion of household consumption expenditure, which can lead to reduced expenditures on other basic commodities. This is known as the crowding-out effect. This study analyzes the crowding-out effect of tobacco consumption in Bangladesh, and the research findings have relevance for strengthening the tobacco control for improving health and well-being. The crowding-out effect of tobacco expenditure on household spending patterns in Bangladesh Muhammad Jami HusainID1*, Biplab Kumar Datta1, Mandeep K. Virk-Baker2, Mark Parascandola2, Bazlul Haque Khondker3 Introduction Tobacco use is among the leading causes of preventable premature deaths and disabilities, globally. Smoking tobacco harms nearly every organ of the body, impairs immune function, causes inflammation, and increases risk for deaths from all causes in men and women [1]. Smokeless tobacco use can cause cancer of mouth, esophagus, and pancreas, increase risks for early delivery and stillbirth during pregnancy, lead to nicotine addiction and nicotine poison- ing in children, and increase risk of deaths from cardiovascular diseases [2, 3]. The tobacco epidemic kills more than 7 million people each year globally; and more than two-thirds of the more than 1 billion smokers worldwide live in low and middle-income countries [4]. In addition to the deleterious health effects, the economic impacts of tobacco consumption at the individual, family, and national levels are also a major concern. Tobacco use causes enormous economic costs for individuals and families due to illness, disability, premature deaths, forgone consumption and investments, and bad consumption choices [2, 3, 5]. At the macro-level, tobacco use contributes to less healthy workforce, lost productivity, high health- care costs, strains in healthcare systems, degradation of natural environment, and health dis- parities [5, 6, 7]. Published studies on the nexuses among tobacco use, nutrition, human capital investments, and poverty demonstrate that expenditures on tobacco smoking constitute a significant por- tion of household budget, which can lead to reduced spending on other basic commodities like food, health, education, housing, transport, energy etc. [8, 9, 10, 11, 12, 13, 14]. This phe- nomenon is known as the crowding-out effect, which in turn, may exacerbate the effects of poverty, including the impact on nutritional status of children [15, 16, 17, 18]. Tobacco use is pervasive in Bangladesh, where 58% of men and 29% of women use tobacco in smoking or smokeless form [19]. On average, a tobacco-user household spent nearly 392 Bangladesh Taka (BDT) per month in 2010, constituting 4.1% of the household budget. This expenditure share is much higher than many other food consumption items, including fruits, legumes and beans, oil and fat, milk, sugar, and eggs [20]. Husain et al. [20] suggested that, in Bangladesh, the tobacco expenditure-shift could translate to an additional 4.6 to 7.7 million food-energy malnourished persons meeting their caloric requirements. Previously, Efroymson et al. The crowding-out effect of tobacco expenditure on household spending patterns in Bangladesh Policy measures that reduce tobacco use could reduce displacement of commodities by households with tobacco users, including those commodities that can contribute to human capital investments. Policy measures that reduce tobacco use could reduce displacement of commodities by households with tobacco users, including those commodities that can contribute to human capital investments. Competing interests: The authors have declared that no competing interests exist. Competing interests: The authors have declared that no competing interests exist. Results We find that tobacco user households on average allocated less in clothing, housing, educa- tion, energy, and transportation and communication compared to tobacco non-user house- holds. The SUR estimates also confirmed crowding-out in these consumption categories. Mean expenditure share of food and medical expenditure of tobacco user households, how- ever, are greater than those of tobacco non-user households. Albeit similar patterns observed for different tobacco user households, there were differences in magnitudes depending on the type of tobacco-use, rural-urban locations and economic status. 1 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0205120 October 9, 2018 Funding: The authors received no specific funding for this work. Funding: The authors received no specific funding for this work. Data We used the Bangladesh Household Income and Expenditure Survey (HIES, 2010). The HIES is a nationally representative survey, consisting of 12,240 households (7,840 rural and 4,400 urban), and the major source of socio-economic information at the household level in Bangla- desh. The HIES methodology and survey design details are available in published reports [21, 22]. HIES collects consumption information on over 300 food and non-food items, which were separated into 11 mutually exclusive subgroups (tobacco, food, clothing, housing, educa- tion, health, lifestyle and hygiene, energy and utility, transport and communication, entertain- ment, and miscellaneous). The daily food consumption quantities and expenditure values were collected for 14 days over seven visits with two days’ recall. There are some weekly food-items (e.g. various spices), information on which were collected for two weeks. Daily consumption of 122 food items under 14 broad categories, and weekly consumption of 11 food items under the spices category were reported in the survey. Expenditures on tobacco and tobacco products, including ciga- rettes, tobacco leaf, bidies, and gul were recorded in the daily-food-consumption section, whereas some other smokeless products (i.e. betel leafs and chew goods) were recorded in the weekly-food-consumption section. These tobacco products were grouped into smoking and smokeless categories (i.e. smoking only, smokeless only, and dual). The consumption module recorded expenditures on non-food items either as monthly expenditure (e.g. energy, life style and hygiene, transport and communication), or yearly expenditure (e.g. clothing, housing, education, health) [21, 22]. For our analysis, all consumption expenditures are converted in average monthly expenditures. The crowding-out effect of tobacco expenditure on household spending patterns in Bangladesh the tobacco use crowding-out literature by examining how the household expenditure patterns for smoking-, smokeless-, and dual- (both smoking and smokeless) user households differ from tobacco non-user households, from the recent data in Bangladesh. The findings of the study highlights the potential trade-offs between tobacco expenditure and all other different household expenditure categories, and provides further evidence for tobacco control as an essential component for improving health and well-being. Introduction [8] asserted that, in Bangladesh, the average male cigarette smoker spends more than twice as much on cigarettes as compared to combined expenditures on clothing, housing, health and education; that a typical poor smoker could add over 500 calories to the diet of one or two children by eliminating daily tobacco expenditure; and that an estimated 10.5 million malnourished people could have an adequate diet if money on tobacco were spent on food instead. Previous literature on the consumption displacement attributable to tobacco use primarily considered smoking tobacco (e.g. cigarettes, bidi, and other forms of smoking tobacco). This paper presents an analysis of household level tobacco use by different tobacco use types. Husain et al. [20] analyzed smokeless tobacco use that showed the potential trade-offs between nutrition intake and tobacco use. To our knowledge, this is the first study that contributes to 2 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0205120 October 9, 2018 The crowding-out effect of tobacco expenditure on household spending patterns in Bangladesh Table 1. Distribution of households by tobacco use type and household expenditure per capita quintiles. All (%) Bottom Quintile (%) Top Quintile (%) National Households do not consume tobacco 30.2 28.0 37.0 Households consume any tobacco 69.8 72.0 63.0 Smoking only tobacco use 17.3 19.7 15.1 Smokeless only tobacco use 26.0 24.7 28.2 Dual (both) tobacco use 26.5 27.6 19.8 Number of Households 33,028,016 6,605,682 6,604,107 Number of Households (Sample) 12,240 2,444 2,495 Urban Households do not consume tobacco 43.3 34.3 50.6 Households consume any tobacco 56.7 65.7 49.4 Smoking only tobacco use 19.2 22.9 18.6 Smokeless only tobacco use 20.9 20.2 21.7 Dual (both) tobacco use 16.6 22.5 9.0 Number of Households 8,860,148 1,773,390 1,769,080 Number of Households (Sample) 4,400 1,219 709 Rural Households do not consume Tobacco 25.4 28.5 24.4 Households consume any tobacco 74.6 71.5 75.6 Smoking only tobacco use 16.5 19.1 11.6 Smokeless only tobacco use 27.9 25.5 32.6 Dual (both) tobacco use 30.1 26.9 31.4 Number of Households 24,167,867 4,835,249 4,831,015 Number of Households (Sample) 7,840 1,563 1,580 Note: The distributions were derived applying relevant weights to take into account the complex survey design. Shares of smoking only-, smokeless only-, and dual (both)- tobacco use add up to “Households consuming any tobacco”. Note: The distributions were derived applying relevant weights to take into account the complex survey design. Shares of smoking only-, smokeless only-, and dual (both)- tobacco use add up to “Households consuming any tobacco”. In Table 1, the distribution of households by tobacco use type for the top and bottom con- sumption expenditure quintiles shows contrasting prevalence of smoking and smokeless household tobacco use. The pattern also differs by rural and urban populations. At the national level, 69.8% of the households reported spending money on tobacco, whereas the household level prevalence in urban areas is much lower (56.7%) than in rural areas (74.6%). Household level prevalence of smoking-only tobacco is higher in urban area than in rural area (19.2% vs. 16.5%); in contrast, prevalence of smokeless-only tobacco is higher in rural areas than in urban areas (27.9% vs. 20.9%). This contrasting pattern is more striking for the households in the top expenditure quintile. 18.6% of households in top quintile in urban areas reported spending on smoking-only tobacco compared to 11.6% of top quintile in rural areas. Household tobacco use A household was considered tobacco-user if that household reported any expenditure on tobacco. The expenditure can be incurred by a single person, or by multiple persons, and can include smoking-, smokeless-, or dual- tobacco user(s). Comparisons were made between tobacco non-user households and ‘any’ tobacco user households; and also with mutually exclu- sive smoking-only, smokeless-only, and dual (both smoking and smokeless) tobacco user households. HIES data allowed this unique categorization of household groups by tobacco use types. This kind of classification distinguishes our study from previous literature by highlight- ing differential tobacco use determinants associated with household characteristics, and in terms of econometric model specifications. According to the Bangladesh Global Adult Tobacco Survey (GATS 2009), at the individual level, tobacco use is more prevalent in rural areas (45.1%) than urban areas (38.1%), and in the lowest quintiles of socioeconomic status (SES) (55.6%). At the individual level, 23.0% (21.9 million persons) of adults aged 15 years or above smoke tobacco, and 27.2% (25.9 million) use smokeless tobacco. For males, 44.7% and 26.4% use smoking and smokeless tobacco, respec- tively. The corresponding prevalence for females are 1.5% and 27.9% for smoking and smoke- less tobacco, respectively [19]. 3 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0205120 October 9, 2018 PLOS ONE \| https://doi.org/10.1371/journal.pone.0205120 October 9, 2018 https://doi.org/10.1371/journal.pone.0205120.t001 The crowding-out effect of tobacco expenditure on household spending patterns in Bangladesh Table 2. Descriptive statistics on household socio-economic and demographic variables. Household tobacco use type Tobacco non-user Smoking-only tobacco user Smokeless-only tobacco user Dual (Smoking and smokeless) user Household income per capita (BDT) 3311 (3044, 3578) 2503 (2317, 2688) 2599 (2452, 2746) 2256 (2130, 2382) Food expenditure per capita (BDT) 1340 (1293, 1388) 1261 (1213, 1308) 1330 (1286, 1374) 1240 (1206, 1274) Non-food expenditure per capita (BDT) 1435 (1293, 1578) 1153 (1005, 1300) 1224 (1148, 1300) 943 (894, 992) Tobacco expenditure per adult (BDT) 0.0 109 (101, 117) 99 (91, 107) 193 (181, 205) Household Size (number of people) 4.0 (3.9, 4.1) 4.4 (4.3, 4.5) 4.5 (4.4, 4.6) 5.1 (5.0, 5.2) Proportion of male among adult (%) 42.9 (42.0, 43.8) 50.9 (50.3, 51.4) 43.2 (42.4, 44.0) 50.8 (50.3, 51.2) Proportion of children (%) 32.9 (32.0, 33.9) 34.2 (33.2, 35.3) 27.9 (27.0, 28.8) 31.9 (31.0, 32.9) Presence of elderly (proportion) 0.17 (0.16, 0.19) 0.13 (0.12, 0.15) 0.36 (0.34, 0.38) 0.28 (0.27, 0.30) Female-headed household (proportion) 0.23 (0.21, 0.25) 0.03 (0.02, 0.03) 0.20 (0.19, 0.22) 0.04 (0.04, 0.05) Household Head’s Education (Proportion) No education 0.45 (0.42, 0.48) 0.55 (0.52, 0.58) 0.50 (0.48, 0.53) 0.63 (0.61, 0.65) Primary 0.14 (0.12, 0.15) 0.17 (0.15, 0.19) 0.17 (0.16, 0.18) 0.16 (0.14, 0.17) Secondary 0.32 (0.30, 0.34) 0.25 (0.23, 0.28) 0.27 (0.25, 0.29) 0.19 (0.18, 0.21) Baccalaureate 0.05 (0.04, 0.06) 0.03 (0.02, 0.03) 0.04 (0.03, 0.05) 0.01 (0.01, 0.01) Graduate/ Professional 0.04 (0.03, 0.06) 0.01 (0.00, 0.01) 0.02 (0.01, 0.02) 0.01 (0.00, 0.01) Note: BDT: Bangladesh Taka. 95% confidence interval in the parenthesis. Table 2. Descriptive statistics on household socio-economic and demographic variables. Note: BDT: Bangladesh Taka. 95% confidence interval in the parenthesis. Table 2 presents descriptive statistics on some socio-economic and demographic variables. All the descriptive statistics were generated taking into account the complex survey design and using appropriate survey weights. It is evident that the proportion of male among adults in smoking-only tobacco user households are higher compared to smokeless-only tobacco user households (50.9% vs. 43.2%, respectively). Also, only 3.0% of the smoking-only households are headed by a female member, compared to 20.0% of smokeless-only households headed by a female member. The proportion of household heads with no education is higher among tobacco user households compared to non-user households (50–63% vs. 45%). For the smokeless-only tobacco use in the top quintile households the percentages are 21.7% and 31.4% for urban and rural areas, respectively. Comparison of the distribution of household tobacco use between bottom and top quintiles revealed that, at the national level, 72.0% of households in the bottom quintile reported spend- ing on any tobacco compared to 63.0% in the top quintile. This pattern is similar for smoking- only tobacco use at the national, urban, and rural populations; i.e. percentage of households in the bottom quintile is higher than the top quintile counterparts. However, this pattern is reversed for smokeless-only tobacco use at the national level (24.7% for bottom quintile vs. 28.2% for top quintile); and for urban (20.2% vs. 21.7%), and rural (26.9% vs. 31.4%) areas. 4 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0205120 October 9, 2018 Table 2 also suggests larger average household size and lower average per capita income for tobacco user households. The proportion of household heads with higher degrees is relatively less in tobacco user households; and larger percentage of smokeless and dual user households have elderly present in the household. To explore the determinants of tobacco use by types, we used a logistic regression model that estimated the probability of a household using any tobacco; and a multinomial logistic regression that estimated the probabilities of a household using smoking-only, smokeless- only, or dual (both types) tobacco, given a set of socio-demographic characteristics. P TijX ð Þ ¼ expðx0 ibjÞ 1 þ expðx0 ibqÞ ð1Þ ð1Þ Eq (1) shows the logistic regression specification, where, Ti takes the value 1 if household i has positive tobacco expenditure, and 0 otherwise; X is a set of household level characteristics and other relevant control variables, including proportion of male among adult household members, proportion of children, presence of elderly (aged 60+), sex of household head, household size, household income per capita, religion, principal source of household income, 5 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0205120 October 9, 2018 The crowding-out effect of tobacco expenditure on household spending patterns in Bangladesh level of education of household head, and district fixed effects. level of education of household head, and district fixed effects. ehold head, and district fixed effects. P Ti ¼ tjX ð Þ ¼ expðx0 ibjÞ 1 þ P3 q¼1 expðx0 ibqÞ ð2Þ ð2Þ Eq (2) is the multinomial logistic regression specification, where, t represents different cate- gories of tobacco consumption. The base category here is ‘no’ tobacco-user. The results of logistic estimation are usually interpreted using marginal effects, as shown in Eq (3): Eq (2) is the multinomial logistic regression specification, where, t represents different cate- gories of tobacco consumption. The base category here is ‘no’ tobacco-user. The results of logistic estimation are usually interpreted using marginal effects, as shown in Eq (3): dPðTi ¼ tjXÞ dxik ¼ pij bjk X 3 q¼1 bqkpiq ! ð3Þ ð3Þ Eq (3) is the marginal effect of the kth explanatory variable, Xik, on the tth response probabil- ity of household i. Average marginal effects are then obtained by taking arithmetic mean of the marginal effects as following: AMEtk ¼ 1 N X N i¼1 dPðTi ¼ tjXÞ dxik ð4Þ ð4Þ Results from logistic regression show that probability of consuming any tobacco is on aver- age lower for female headed households, and households with larger proportion of children; and higher for households with elderly members (Table 3). However, multinomial logistic regression results indicate that several of these findings could be different for different tobacco use types. Contrary to the lower probability of consuming any tobacco, probability of consum- ing smokeless tobacco-only is higher for female headed households. Likewise, probability of consuming smoking tobacco only is lower for households with elderly members, though prob- ability of consuming any tobacco is higher for these households. These results suggest consid- erable heterogeneity in tobacco use across households, and thus justify our approach of analyzing the crowding out effect for different tobacco use types. PLOS ONE \| https://doi.org/10.1371/journal.pone.0205120 October 9, 2018 The crowding-out effect The crowding out effect of tobacco expenditure entails reduced consumption of goods and ser- vices because of tobacco consumption. Depending on the level of analysis (i.e. individual or household), data availability, and methodological approach, the crowding-out effects have been reported as the differences in the mean expenditure shares of different consumption cate- gories between tobacco users and non-users, and/or the marginal effects of tobacco expendi- ture on other commodities or services. The conceptual framework of this paper followed the most recent generation of empirical studies on the crowding-out impact of tobacco expendi- ture [10, 13, 14, 15, 16, 17]. However, the empirical strategy is most aligned with John, Ross, and Blecher [16] that defined the crowding-out effect as reduced consumption of goods and services as a result of tobacco consumption. Conceptual framework. Households can report zero tobacco expenditures either because none in the household use any form of tobacco, even if they have adequate income; or house- holds cannot afford tobacco products, given their income. The former explanation implies that there is a difference between the spending patterns of smoking and non-smoking house- holds [14]. The crowding-out attribution of tobacco expenditure in displacing expenditure on other commodities comes with the assumption that a household that spends on tobacco decides on the quantity of tobacco to be purchased before deciding on the quantities of the other goods and services. Given this, household’s demand for a particular commodity is condi- tional on the household’s tobacco use status and the remainder of household income after 6 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0205120 October 9, 2018 nthesis. vel of the household head. ‘No’ education is the reference category. The crowding-out effect of tobacco expenditure on household spending patterns in Bangladesh spending on tobacco [10, 15]. Following the recent literature, we estimated and compared a set of Engel curves for tobacco non-user households with conditional Engel curves for tobacco user households for a common set of commodities. If, on average, the quantity demanded of a commodity for the typical tobacco user household is less (more) than the quantity demanded of the same commodity for a typical tobacco non-user household, then the difference can be attributed, ceteris paribus, to tobacco use [16]. Empirical strategy. In the first step of our empirical strategy, we compared the mean expenditure shares for the food and various non-food expenditure categories between house- holds with and without tobacco expenditure using the t-test on the equality of means. Statisti- cally significant differences in the expenditure dedicated to other commodities in the budgets of tobacco user households (i.e. any tobacco, smoking-only, smokeless-only, and dual user households) and non-user households indicate unadjusted crowding-out effect. However, these unadjusted differences in expenditure shares do not take into account households’ socio- economic and demographic characteristics that may have influence on spending pattern. Therefore, we formally tested the crowding-out hypothesis using multivariate regression analysis, which predicted the budget share allocation to each expenditure category according to tobacco use status, controlling for household-specific and other characteristics. To deter- mine differences in spending patterns between tobacco user and non-user households, the regression models estimated conditional Engel curves for 10 expenditure categories using the Quadratic Almost Ideal Demand System (QUAIDS) developed by Banks, Blundell, & Lewbel [23]. Depending on household expenditure level, a particular consumption item could be either necessary or luxury. Use of QUAIDS allows a particular expenditure category to be either a luxury or a necessity by including a quadratic expenditure term in the specification [10, 23]. If household expenditure in one category is correlated with expenditures on other cat- egories, the error terms in the Engle curve estimations are likely to be correlated, potentially leading to increased variance in the estimated coefficients and inefficient coefficient estimates. We addressed this by using Seemingly Unrelated Regression (SUR) model, which estimated all regression equations simultaneously using a Feasible Generalized Least Squares (FGLS) method [16]. The crowding-out effect of tobacco expenditure on household spending patterns in Bangladesh Table 3. Probability of tobacco consumption: average marginal effects of household demographics. Logistic Multinomial Logistic Any Smoking only Smokeless only Dual Proportion of males among adults -0.00006 (-0.00066, 0.00054) 0.00078 (0.00020, 0.00136) -0.00177 (-0.00236, -0.00117) 0.00115 (0.00059, 0.00170) Proportion of children -0.00223 (-0.00270, -0.00176) 0.00080 (0.00034, 0.00126) -0.00190 (-0.00237, -0.00144) -0.00114 (-0.00163, -0.00065) Female headed household -0.20202 (-0.23710, -0.16693) -0.13473 (-0.15446, -0.11500) 0.09133 (0.05658, 0.12608) -0.17513 (-0.19800, -0.15225) Presence of elderly (age 60+) 0.05951 (0.03817, 0.08086) -0.06875 (-0.08861, -0.04890) 0.10122 (0.07949, 0.12294) 0.02156 (0.00236, 0.04076) Education† Primary -0.02540 (-0.04787, -0.00292) -0.00059 (-0.02276, 0.02157) 0.03446 (0.01195, 0.05696) -0.05834 (-0.08108, -0.03560) Secondary -0.10549 (-0.12933, -0.08165) -0.02007 (-0.04057, 0.00042) 0.03133 (0.01032, 0.05233) -0.11530 (-0.13384, -0.09676) Bachelor’s -0.20788 (-0.26908, -0.14669) -0.05217 (-0.09249, -0.01184) 0.08144 (0.03255, 0.13033) -0.23572 (-0.27010, -0.20134) Graduate or Professional -0.31654 (-0.38525, -0.24784) -0.12491 (-0.16116, -0.08866) 0.03419 (-0.02761, 0.09600) -0.22264 (-0.27150, -0.17377) HH per capita income groups 700 to 999 -0.00590 -0.01012 0.02514 -0.01974 (-0.04306, 0.03127) (-0.04127, 0.02103) (-0.00996, 0.06023) (-0.05154, 0.01206) 1000 to 1499 -0.00703 -0.01536 -0.01639 0.02442 (-0.03983, 0.02576) (-0.04236, 0.01163) (-0.04732, 0.01454) (-0.00604, 0.05488) 1500 to 2499 0.00208 -0.00994 -0.00767 0.01993 (-0.03060, 0.03476) (-0.03737, 0.01750) (-0.03603, 0.02069) (-0.00799, 0.04785) 2500 to 4999 -0.00515 -0.01764 0.00688 0.00843 (-0.04127, 0.03097) (-0.04882, 0.01354) (-0.02538, 0.03913) (-0.02449, 0.04136) 5000 & more -0.02302 -0.01397 -0.01569 0.01361 (-0.06550, 0.01946) (-0.05241, 0.02448) (-0.05337, 0.02198) (-0.03010, 0.05732) Household Religion Hinduism -0.02580 0.01496 -0.04982 0.01031 (-0.06029, 0.00868) (-0.01101, 0.04092) (-0.07928, -0.02036) (-0.01580, 0.03643) Other 0.11571 0.07703 -0.06707 0.11115 (0.05612, 0.17530) (-0.02517, 0.17922) (-0.16050, 0.02635) (0.03405, 0.18825) Household Size 3 to 5 0.11429 0.02554 0.02502 0.07579 (0.07668, 0.15189) (-0.00541, 0.05649) (-0.00399, 0.05402) (0.04892, 0.10265) 6 to 9 0.22007 0.00652 0.06289 0.16461 (0.17966, 0.26048) (-0.02867, 0.04171) (0.02808, 0.09769) (0.13239, 0.19682) 10 and more 0.27773 -0.05132 0.05405 0.28242 (0.21793, 0.33753) (-0.11348, 0.01083) (-0.01442, 0.12252) (0.21318, 0.35166) HH major source of income Agri Production 0.02948 0.01490 -0.03243 0.05410 (-0.00059, 0.05955) (-0.00996, 0.03975) (-0.06266, -0.00221) (0.02593, 0.08227) Non-agri Production 0.04984 0.05189 -0.03242 0.03781 (0.01502, 0.08465) (0.02127, 0.08250) (-0.06234, -0.00250) (0.01071, 0.06490) Wage & Salary 0.00390 0.05358 -0.06185 0.02090 (-0.02165, 0.02945) (0.03077, 0.07639) (-0.08623, -0.03748) (-0.00258, 0.04438) Note ability of tobacco consumption: average marginal effects of household demographics. PLOS ONE \| https://doi.org/10.1371/journal.pone.0205120 October 9, 2018 PLOS ONE \| https://doi.org/10.1371/journal.pone.0205120 October 9, 2018 7 / 15 7 / 15 The crowding-out effect of tobacco expenditure on household spending patterns in Bangladesh presence of children aged under five, presence of children aged 5 to 14, presence of elderly (aged 60+), proportion of male among adult household members, household size, sex of household-head, religion, principal source of household income, household head’s highest level of education, occupation, and indicator variable for presence of any chronic diseases of any household member in last 12 months. Districtid controls for regional fixed effects, which takes the value 1 if household i resides in administrative district d, and 0 otherwise. εij is the idiosyncratic error term. We assume that households within a district face similar prices for respective commodities. The system in Eq (5) was estimated using Seemingly Unrelated Regression (SUR) tech- nique. Miscellaneous consumption category was dropped from the system of equations to meet summation restrictions. Following Banks, Blundell, & Lewbel [23] ln M and (ln M)2 were instrumented by natural log of monthly household income, and its squared respectively. wij ¼ b0 þ X 3 t¼1 ltTit þ b2lnMi þ b3ðlnMiÞ 2 þ Xβ4 þ X 63 d¼1 gdDistrictid þ εij ð6Þ ð6Þ The crowding-out effects for the mutually exclusive smoking-only, smokeless-only, and both smoking and smokeless user households are estimated using Eq (6), where Tit denotes t type of tobacco consumption for household i, and it takes the value 1 if tobacco consumption type is t, and 0 otherwise. The coefficients β1 in Eq (5) and λt in Eq (6) estimate the percentage point differences in expenditure shares between households with and without tobacco expen- diture, and therefore captures the crowding out effect. Several studies on the crowding-out effect of smoking emphasized the potential endogene- ity of total expenditure and smoking expenditure, and therefore used the instrumental variable (IV) method to obtain consistent and unbiased estimators [10, 14, 15, 17]. Those studies used income or total value of household assets as instruments for total expenditure, and adult sex ratio or female ratio in the household as instrument for expenditure on tobacco. While in this paper we instrumented household total expenditure by total income, we believe that adult sex ratio (or female ratio) does not satisfy the exclusion restriction requirements due to their potential correlations with other consumption items. More importantly, while adult sex ratio may explain smoking decision in a household, we found that the same is not true for the deci- sion to spend on smokeless tobacco; instead we use proportion of males among adults and other household demographics as control variables in the regression specifications. wij ¼ b0 þ b1Ti þ b2lnMi þ b3ðlnMiÞ 2 þ Xβ4 þ X 63 d¼1 gdDistrictid þ εij ð5Þ ð5Þ Eq (5) shows the regression specification for ‘any’ tobacco consumption, where, wij is household i’s expenditure share of category j. Expenditure shares are calculated after deducting expenditure on tobacco. Ti is a binary variable that takes the value 1 if household i consumes any type of tobacco, and 0 otherwise. Mi is monthly consumption expenditure of household i, excluding tobacco expenditure. X is a vector of household level characteristics; including 8 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0205120 October 9, 2018 The crowding-out effect of tobacco expenditure on household spending patterns in Bangladesh Table 4. Unadjusted differences in consumption share. Household types by tobacco use No Tobacco (n = 3,550) Any tobacco (n = 8,690) Smoking-only (n = 2,063) Smokeless-only (n = 3,281) Dual/both tobacco (n = 3,346) Mean share (%) Difference (% point) Difference (% point) Difference (% point) Difference (% point) Food 55.142 (54.224,56.061) 4.475 (3.601, 5.348) 4.254 (3.291, 5.217) 2.825 (1.780, 3.870) 6.239 (5.167, 7.312) Clothing 6.226 (5.924,6.529) -0.564 (-0.834, -0.294) -0.274 (-0.551, 0.002) -0.639 (-0.932, -0.347) -0.679 (-0.983, -0.375) Housing 10.448 (9.810,11.085) -2.628 (-3.208, -2.049) -1.897 (-2.597, -1.197) -2.033 (-2.716, -1.351) -3.689 (-4.387, -2.992) Education 4.862 (4.411,5.312) -1.165 (-1.580, -0.751) -1.228 (-1.704, -0.752) -0.653 (-1.099, -0.208) -1.628 (-2.095, -1.161) Medical 2.989 (2.768,3.210) 0.772 (0.533, 1.012) 0.524 (0.136, 0.912) 0.888 (0.613, 1.162) 0.821 (0.517, 1.125) Life-style and hygiene 3.159 (3.078,3.240) 0.040 (-0.043, 0.124) 0.093 (-0.011, 0.198) 0.042 (-0.060, 0.144) 0.005 (-0.100, 0.109) Energy 7.321 (7.098,7.544) -0.326 (-0.550, -0.103) -0.127 (-0.426, 0.172) -0.241 (-0.505, 0.022) -0.540 (-0.820, -0.259) Transport and Communication 5.559 (5.295,5.822) -0.715 (-0.988, -0.441) -0.616 (-0.955, -0.276) -0.794 (-1.121, -0.468) -0.701 (-1.046, -0.356) Entertainment 0.588 (0.517,0.659) 0.055 (-0.022, 0.133) 0.153 (0.043, 0.262) 0.032 (-0.058, 0.123) 0.015 (-0.090, 0.120) Miscellaneous 3.707 (3.394,4.019) 0.056 (-0.283, 0.396) -0.882 (-1.279, -0.484) 0.576 (0.151, 1.000) 0.157 (-0.280, 0.594) Note p<0.01 Table 4. Unadjusted differences in consumption share. Previous studies supported crowding-out effect of tobacco expenditure on food expendi- ture, in contrast to what is observed in Table 4 (e.g. [14, 15]). However, for poor households, the expenditure on food and medical expenses may be less discretionary in nature, rather nec- essary expenditure items constituting large budget share. Therefore, the crowding-out effects are manifested in other expenditure items of discretionary nature [13]. In Bangladesh, a closer look at the food and non-food expenditure pattern by household expenditure per capita per- centiles in Fig 1 revealed that expenditure share allocated to food were higher for households that reported any tobacco expenditure than tobacco non-user households. This was observed throughout the expenditure percentile distribution. Accordingly, tobacco user households allocated less budget on non-food items than the tobacco non-user counterparts, throughout the percentile distribution. Unadjusted differences in expenditure share Unadjusted differences in expenditure shares between tobacco user and non-user households, expressed as percentage points are reported in Table 4. Tobacco non-user households being the reference category, a positive percentage point difference implies that households spending on any tobacco (or on a particular type of tobacco), on average, allocated a greater share to that consumption category than non-user households; and a negative percentage point differ- ence implies that tobacco user households allocated a smaller share. Table 4 reveals statistically significant differences expenditure allocations between tobacco user and non-user households. The ‘mean shares’ in second column are the expenditure shares for the tobacco non-user households, and the last four columns show the percentage point differences from the corresponding mean shares of the tobacco non- user households. For instance, a household reporting expenditure on any tobacco, on average allocated 2.63 percentage point less on housing compared to a typical tobacco non-user household that spent 10.4% of its budget on housing. Compared to a tobacco non-user household, tobacco user (any tobacco) households, on average, allocated less on clothing, education, energy, and transport and communication. In contrast, any tobacco user households allocated more of the household total expenditure on food and medical expenses. Albeit difference in magnitudes, a more or less similar pattern is evident for smoking-only, smokeless-only, and dual tobacco user households. In addition to food and medical expenses, the smoking-only and smokeless-only households also allocated 0.153 and 0.032 percentage points more of their household budget on entertainment. The largest differences were observed for the households reporting expenditure on both smoking and smokeless tobacco (i.e. dual tobacco). 9 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0205120 October 9, 2018 https://doi.org/10.1371/journal.pone.0205120.t004 PLOS ONE \| https://doi.org/10.1371/journal.pone.0205120 October 9, 2018 p p<0.1. 95% confidence interval in the parenthesis. Food Clothing Housing Education Medical Hygiene Energy Transport and Communication Entertainment Any Tobacco Rural bottom 1.743 -0.935 -0.731 0.252 0.673 0.0761 -0.115 -0.555 -0.101 Rural top 2.120 -0.22 -1.898 -0.335 0.147 -0.124 -0.0226 -1.328 0.316 Urban bottom 2.327 -0.502 -0.712 -0.557 0.134 -0.0193 0.380 -0.862 -0.128 Urban top 3.435 -0.0534 -1.951 -1.346 0.088 -0.13 -0.0694 0.587 -0.134 Smoking only Rural bottom 1.876 -0.602 -0.45 -0.218 0.404 -0.0287 0.0858 -0.617 -0.0604 Rural top 3.082 0.241 -1.291 -0.542 0.602 0.0567 0.322 -1.265 0.648 Urban bottom 1.929 -0.539 0.26 -0.341 0.054 0.0804 0.00486 -1.045 -0.143 Urban top 2.831 -0.176 -2.803 -2.328 1.22 -0.0596 -0.106 0.321 0.141 Smokeless only Rural Bottom 1.965 -1.044 -0.831 0.262 0.771 0.126 -0.304 -0.689 -0.124 Rural Top 1.286 -0.308 -1.508 0.196 0.0917 -0.205 -0.111 -1.827 0.211 Urban Bottom 1.905 -0.24 -1.330 -0.486 0.129 -0.121 0.924 -0.717 -0.223 Urban Top 3.403 0.0357 -0.207 -0.396 -0.861 -0.136 -0.0595 0.448 -0.298 Dual/Both tobacco Rural Bottom 1.405 -1.092 -0.854 0.623 0.789 0.109 -0.08 -0.366 -0.111 Rural Top 2.983 -0.370 -3.009 -1.154 -0.0587 -0.0981 -0.0936 -0.468 0.282 Urban Bottom 3.152 -0.708 -1.180 -0.856 0.224 -0.0312 0.273 -0.802 -0.024 Urban Top 4.570 -0.0448 -4.501 -1.827 0.304 -0.241 -0.0275 1.378 -0.235 p<0.01 p<0.05 p<0.1. The confidence intervals are not reported in the table. https://doi.org/10.1371/journal.pone.0205120.t006 The crowding-out effect of tobacco expenditure on household spending patterns in Bangladesh PLOS ONE \| https://doi.org/10.1371/journal.pone.0205120 October 9, 2018 12 / 15 Table 6. Adjusted differences (percentage point) in expenditure share by rural and urban and by bottom and top expenditure quintiles. Food Clothing Housing Education Medical Hygiene Energy Transport and Communication Entertainment Any Tobacco Rural bottom 1.743 -0.935 -0.731 0.252 0.673 0.0761 -0.115 -0.555 -0.101 Rural top 2.120 -0.22 -1.898 -0.335 0.147 -0.124 -0.0226 -1.328 0.316 Urban bottom 2.327 -0.502 -0.712 -0.557 0.134 -0.0193 0.380 -0.862 -0.128 Urban top 3.435 -0.0534 -1.951 -1.346 0.088 -0.13 -0.0694 0.587 -0.134 Smoking only Rural bottom 1.876 -0.602 -0.45 -0.218 0.404 -0.0287 0.0858 -0.617 -0.0604 Rural top 3.082 0.241 -1.291 -0.542 0.602 0.0567 0.322 -1.265 0.648 Urban bottom 1.929 -0.539 0.26 -0.341 0.054 0.0804 0.00486 -1.045 -0.143 Urban top 2.831 -0.176 -2.803 -2.328 1.22 -0.0596 -0.106 0.321 0.141 Smokeless only Rural Bottom 1.965 -1.044 -0.831 0.262 0.771 0.126 -0.304 -0.689 -0.124 Rural Top 1.286 -0.308 -1.508 0.196 0.0917 -0.205 -0.111 -1.827 0.211 Urban Bottom 1.905 -0.24 -1.330 -0.486 0.129 -0.121 0.924 -0.717 -0.223 Urban Top 3.403 0.0357 -0.207 -0.396 -0.861 -0.136 -0.0595 0.448 -0.298 Dual/Both tobacco Rural Bottom 1.405 -1.092 -0.854 0.623 0.789 0.109 -0.08 -0.366 -0.111 Rural Top 2.983 -0.370 -3.009 -1.154 -0.0587 -0.0981 -0.0936 -0.468 0.282 Urban Bottom 3.152 -0.708 -1.180 -0.856 0.224 -0.0312 0.273 -0.802 -0.024 Urban Top 4.570 -0.0448 -4.501 -1.827 0.304 -0.241 -0.0275 1.378 -0.235 p<0.01 p<0.05 p<0 1 The confidence intervals are not reported in the table The adjusted differences in expenditure shares for ‘any’ tobacco user households confirm that household tobacco use crowds-out budget allocations in clothing, housing, education, energy, and transport and communication. The budget allocations in a tobacco user household are higher for food and medical expenses, although the coefficients of medical expenses when generated separately for smoking-only, smokeless-only, and dual tobacco use households were not statistically significant. The adjusted differences for food, clothing, housing, and education are relatively larger (in absolute terms) for dual tobacco user households, than the adjusted dif- ferences of those for any-, smoking only-, or smokeless only- tobacco user households. Spending patterns and tobacco consumption could vary across rural and urban areas, and across different level of consumption expenditure. Table 6 presents the adjusted differences in expenditure shares by rural and urban households and for bottom and top expenditure quin- tiles. We observe crowding-out effects of tobacco consumption at different sub groups as well, however, the magnitudes become different. For example, all smokeless-only households on average allocated 0.33 percentage points less in clothing than all non-tobacco households, while smokeless-only households in rural bottom quintile allocated 1.04 percentage points less than non-tobacco households in rural bottom quintile. These results provide a more compre- hensive perspective of the crowding-out effect. Adjusted differences in expenditure share The unadjusted differences in expenditure shares presented in the previous section did not control for other household socio-economic characteristics that may affect budget allocation. We implemented Eqs (5) and (6) to control for these characteristics. Table 5 reports the coeffi- cients of the dichotomous tobacco consumption variable for each budget share equation in the system. The coefficient values are the average percentage point differences in the budget shares for the corresponding expenditure categories; a negative coefficient indicates a lower budget allocation among tobacco-user households than non-user households. 10 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0205120 October 9, 2018 The crowding-out effect of tobacco expenditure on household spending patterns in Bangladesh Fig 1. Differences in food and non-food expenditure shares between tobacco user and non-user households. https://doi.org/10.1371/journal.pone.0205120.g001 Fig 1. Differences in food and non-food expenditure shares between tobacco user and non-user households. Fig 1. Differences in food and non-food expenditure shares between tobacco user and non-user households. Table 5. Adjusted differences in expenditure share. Table 5. Adjusted differences in expenditure share. Household types by tobacco use No Tobacco (n = 3,545) Any tobacco (n = 8,644) Smoking-only (n = 2,050) Smokeless-only (n = 3,264) Dual/both tobacco (n = 3,330) Mean share (%) Difference (% point) Difference (% point) Difference (% point) Difference (% point) Food 54.42 (53.94,54.90) 2.383 (1.916,2.850) 2.118 (1.520,2.716) 1.993 (1.442,2.545) 3.264 (2.669,3.859) Clothing 6.44 (6.33,6.56) -0.410 (-0.524,-0.296) -0.332 (-0.478,-0.186) -0.404 (-0.538,-0.269) -0.510 (-0.655,-0.365) Housing 10.21 (9.88,10.54) -0.788 (-1.144,-0.431) -0.420 (-0.877,0.037) -0.779 (-1.200,-0.358) -1.230 (-1.684,-0.775) Education 4.94 (4.69,5.19) -0.543 (-0.795,-0.291) -0.680 (-1.003,-0.357) -0.167 (-0.465,0.131) -0.934 (-1.256,-0.613) Medical 3.19 (3.02,3.37) 0.187 (-0.029,0.402) 0.168 (-0.110,0.445) 0.176 (-0.08,0.431) 0.225 (-0.051,0.500) Life-style and hygiene 3.24 (3.19,3.29) -0.0319 (-0.095,0.031) 0.024 (-0.057,0.105) -0.082 (-0.157,-0.007) -0.023 (-0.104,0.057) Energy 7.42 (7.29,7.54) -0.170 (-0.297,-0.042) -0.139 (-0.302,0.025) -0.212 (-0.363,-0.062) -0.143 (-0.306,0.019) Transport and Communication 5.54 (5.36,5.71) -0.402 (-0.619,-0.184) -0.330 (-0.610,-0.051) -0.470 (-0.728,-0.213) -0.385 (-0.663,-0.107) Entertainment 0.64 (0.58,0.70) 0.0223 (-0.056,0.100) 0.063 (-0.037,0.163) 0.01 (-0.083,0.102) -0.007 (-0.106,0.093) <0 01 PLOS ONE \| https://doi.org/10.1371/journal.pone.0205120 October 9, 2018 11 / 15 The crowding-out effect of tobacco expenditure on household spending patterns in Bangladesh The adjusted differences in expenditure shares for ‘any’ tobacco user households confirm that household tobacco use crowds-out budget allocations in clothing, housing, education, energy, and transport and communication. The budget allocations in a tobacco user household are higher for food and medical expenses, although the coefficients of medical expenses when generated separately for smoking-only, smokeless-only, and dual tobacco use households were not statistically significant. The adjusted differences for food, clothing, housing, and education are relatively larger (in absolute terms) for dual tobacco user households, than the adjusted dif- ferences of those for any-, smoking only-, or smokeless only- tobacco user households. Spending patterns and tobacco consumption could vary across rural and urban areas, and across different level of consumption expenditure. Table 6 presents the adjusted differences in expenditure shares by rural and urban households and for bottom and top expenditure quin- tiles. We observe crowding-out effects of tobacco consumption at different sub groups as well, however, the magnitudes become different. For example, all smokeless-only households on average allocated 0.33 percentage points less in clothing than all non-tobacco households, while smokeless-only households in rural bottom quintile allocated 1.04 percentage points less than non-tobacco households in rural bottom quintile. These results provide a more compre- hensive perspective of the crowding-out effect. Discussion The deleterious effects of tobacco consumption, including smoking tobacco, smokeless tobacco, and passive smoking, on health is well-documented [1, 2, 3]. The tobacco-attributable Table 6. Adjusted differences (percentage point) in expenditure share by rural and urban and by bottom and top expenditure quintiles. The crowding-out effect of tobacco expenditure on household spending patterns in Bangladesh deaths, disabilities, and diseases cause enormous economic tolls on individuals, households, and society. These economic costs are described as direct medical costs for the individuals and families, and consequent strain on the national level healthcare finances, as well as indirect costs due to absenteeism and presenteeism for the earners and informal caregivers, con- strained education attainments school attendance and learning, and consequent lost produc- tivity at the macro level. Another mechanism through which tobacco use can negatively impact well-being of tobacco users and family members is due to displacement of consump- tion of other basic necessities due to expenditure on tobacco, generally described in the litera- ture as the ‘crowding-out effect’ [13]. Given fixed household budget, the displacements often occur for items that constitute human capital investments (e.g. food and nutrition, education, health and hygiene, housing etc.) rendering life-course implications on household well-being. The existing tobacco expenditure crowding-out literature mainly highlighted the consump- tion displacement effects for smoking tobacco only. Apparently, the expenditure on smokeless tobacco in those literature was included into one of the consumption categories other than tobacco, most probably as part of the food consumption item. This paper examined how the household expenditure patterns for smoking-, smokeless-, and dual- (both smoking and smokeless) user households differ from tobacco non-user households, using household level data from Bangladesh, and provided additional policy insights for much needed different tobacco-use types. The analyses on smokeless and dual tobacco use constitute a major contri- bution of this paper in the tobacco crowding-out literature. The econometric analysis showed that tobacco user households on average allocate less in clothing, housing, education, energy, and transportation and communication compared to tobacco non-user households. Mean expenditure share of food and medical expenditure of tobacco user households, however, are greater than those of tobacco non-user households. Albeit similar patterns were observed for different types of tobacco user households, there are differences in magnitudes depending on the type of tobacco-use, rural-urban locations and economic status. The greater share of medical expenditure of tobacco user households could be linked to tobacco-attributable diseases and related health costs. Tobacco use is a major risk factor for various noncommunicable diseases and the direct cost (healthcare expenditures) of smoking in low-income and lower-middle-income countries was estimated around 4% of the total health expenditure in 2012 [24]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0205120 October 9, 2018 Discussion The deleterious effects of tobacco consumption, including smoking tobacco, smokeless tobacco, and passive smoking, on health is well-documented [1, 2, 3]. The tobacco-attributable 12 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0205120 October 9, 2018 The crowding-out effect of tobacco expenditure on household spending patterns in Bangladesh the macro-nutrient level food consumption pattern may reveal important differences between tobacco user and non-user households. Our findings reaffirm the negative impact of tobacco expenditure on human capital invest- ments, impeding short- and long-term economic well-being potentials at the household level, and in effect, for the society. Policy measures that reduce tobacco use could reduce displace- ment of commodities by households with tobacco users, including those commodities that can contribute to human capital investments. Author Contributions Conceptualization: Muhammad Jami Husain, Biplab Kumar Datta, Mandeep K. Virk-Baker, Mark Parascandola, Bazlul Haque Khondker. Data curation: Muhammad Jami Husain, Biplab Kumar Datta, Bazlul Haque Khondker. Formal analysis: Muhammad Jami Husain, Biplab Kumar Datta. Methodology: Muhammad Jami Husain, Biplab Kumar Datta. Software: Muhammad Jami Husain, Biplab Kumar Datta. Writing – original draft: Muhammad Jami Husain, Biplab Kumar Datta. Writing – review & editing: Muhammad Jami Husain, Biplab Kumar Datta, Mandeep K. Virk-Baker, Mark Parascandola, Bazlul Haque Khondker. Writing – review & editing: Muhammad Jami Husain, Biplab Kumar Datta, Mandeep K. Writing – review & editing: Muhammad Jami Husain, Biplab Kumar Datta, Mandeep K. Virk-Baker, Mark Parascandola, Bazlul Haque Khondker. Acknowledgments The authors are thankful to Deliana Kostova, Samira Asma, Juliette Morgan, and Krishna Pali- pudi for their review of the paper. The findings and conclusions in this study are those of the authors and do not necessarily represent the official position of the Centers for Disease Con- trol and Prevention or National Cancer Institute. Hence, tobacco user households might suffer from greater dis- ease burden, resulting in larger budget share of medical expenses. Findings from the crowding-out literature demonstrate the importance of the economic effects of higher spending on tobacco on household standards of living and expenditure pat- terns and these effects may be different in different countries [8,10,11,20]. On the association between smoking tobacco and food expenditure, the crowding-out effect literature suggests mixed results. For instance, Chewla and Walbeek [15], Wang et al. [11], San and Chaloupka [14], and John [10] found crowding-out of food expenditure for Zambia, China, Turkey, and India, respectively. On the other hand, the studies by Do and Bautista [13] on low and middle income countries and John et al. [16] on Cambodia did not find crowding-out effect for food consumption. Other literature suggest that tobacco user households may crowd-out expendi- ture on quality or nutrition-rich food [25, 26, 27, 28, 29, 30]. In our study, we found that food expenditure shares, in general, were higher for tobacco user households than tobacco non-user households throughout the consumption expenditure distribution. In Bangladesh, after deducting the tobacco expenditure from the total expenditure, a household on average spends between 55% to 61% of their budget on food, depending on household tobacco use status. Therefore, the expenditure on food may be less discretionary in nature. Further research on 13 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0205120 October 9, 2018 References Making the Tobacco and Poverty Link: Results from Research for Advocacy Projects in Africa, Asia, and Latin America. Ottawa, Canada: HealthBridge Foundation of Canada. 2011. 13. Do YK, Bautista MA. Tobacco use and household expenditures on food, education, and healthcare in low-and middle-income countries: a multilevel analysis. BMC public health. 2015; 15(1), 1098. 14. San S, Chaloupka FJ. The impact of tobacco expenditures on spending within Turkish households. Tobacco control, 2015; Aug 20: tobaccocontrol-2014. 15. Chelwa G, Van Walbeek C. Assessing the Causal Impact of Tobacco Expenditure on Household Spending Patterns in Zambia. Economic Research Southern Africa. 2014; Working paper 453:1–40. 16. John RM, Ross H, Blecher E. Tobacco expenditures and its implications for household resource alloca- tion in Cambodia. Tob Control. 2012 May; 21(3):341–6. https://doi.org/10.1136/tc.2010.042598 PMID: 21828230 17. Pu CY, Lan V, Chou YJ, Lan CF. The crowding-out effects of tobacco and alcohol where expenditure shares are low: analyzing expenditure data for Taiwan. Social Science & Medicine. 2008 May; 66 (9):1979–89. 18. Nonnemaker J, Sur M. Tobacco expenditures and child health and nutritional outcomes in rural Bangla- desh. Social Science & Medicine. 2007 Dec; 65(12):2517–26. 19. World Health Organization. Global Adult Tobacco Survey: Bangladesh Report 2009. Dhaka, Bangla- desh: Country Office for Bangladesh. 2009. 20. Husain MJ, Virk-Baker M, Parascandola M, Khondker BH, Ahluwalia IB. Money Gone Up in Smoke: The Tobacco Use and Malnutrition Nexus in Bangladesh. Annals of Global Health. 2016 Sep 1; 82(5), 749–759. https://doi.org/10.1016/j.aogh.2016.07.005 PMID: 28283125 21. Bangladesh Bureau of Statistics. Report of the Household Income & Expenditure Survey 2010. Bangla- desh Bureau of Statistics. 2011. 22. World Bank. Bangladesh—Household income and expenditure survey: key findings and results 2010. Washington, DC: World Bank; 2011. Available from: http://documents.worldbank.org/curated/en/2011/ 06/16494498/bangladesh-household-income-expenditure-survey-key-findings-results-2010 (Cited September 21, 2018) 23. Banks J, Blundell R, Lewbel A. Quadratic Engel curves and consumer demand. Review of Economics and Statistics. 1997 Nov; 79(4): 527–539. 24. Goodchild M, Nargis N, d’Espaignet ET. Global economic cost of smoking-attributable diseases. Tobacco Control. 2017 Jan: tobaccocontrol-2016-053305. 25. de Castro JM, Taylor T. Smoking status relationships with the food and fluid intakes of free-living humans. Nutrition. 2008 Feb 1, 24(2): 109–119. 26. Morabia A, Wynder EL. Dietary habits of smokers, people who never smoked, and exsmokers. The American Journal of Clinical Nutrition. 1990 Nov 1, 52(5): 933–937. 27. Palaniappan U, Jacobs SL, O’Loughlin J, Gray-Donald K. References 1. US Department of Health and Human Services. The health consequences of smoking—50 years of progress: a report of the Surgeon General. Atlanta, GA: US Department of Health and Human Ser- vices, Centers for Disease Control and Prevention, National Center for Chronic Disease Prevention and Health Promotion, Office on Smoking and Health; 2014. 2. World Health Organization. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. Vol- ume 89: Smokeless Tobacco and Some Tobacco-Specific N-Nitrosamines. Lyon (France): World Health Organization, International Agency for Research on Cancer, 2007. 3. Piano MR, Benowitz NL, Fitzgerald GA, Corbridge S, Heath J, Hahn E, et al. Impact of Smokeless Tobacco Products on Cardiovascular Disease: Implications for Policy, Prevention, and Treatment: A Policy Statement from the American Heart Association. Circulation 2010; 122(15):1520–44. https://doi. org/10.1161/CIR.0b013e3181f432c3 PMID: 20837898 4. World health Organization. Tobacco. Fact sheet. Updated May 2017. Media Center. Available from: http://www.who.int/mediacentre/factsheets/fs339/en/. Cited 24 September, 2018. 5. U.S. National Cancer Institute and World Health Organization. The Economics of Tobacco and Tobacco Control. National Cancer Institute Tobacco Control Monograph 21. NIH Publication No. 16-CA-8029A. Bethesda, MD: U.S. Department of Health and Human Services, National Institutes of Health, National Cancer Institute; and Geneva, CH: World Health Organization; 2016. 6. WHO Tobacco Free Initiative. (2004). Tobacco and poverty: a vicious circle. WHO/NMH/TFI/04.01. Available from: http://apps.who.int/iris/handle/10665/68704. Cited 24 September 2018. 7. Chaloupka F, Warner KE, Cuyler A, Newhouse J. The economics of smoking. In: Handbook of Health Economics, 1; 2000. pp 1539–1627. PLOS ONE \| https://doi.org/10.1371/journal.pone.0205120 October 9, 2018 14 / 15 The crowding-out effect of tobacco expenditure on household spending patterns in Bangladesh 8. Efroymson D, Ahmed S, Townsend J, Alam SM, Dey AR, Saha R, et al. Hungry for tobacco: an analysis of the economic impact of tobacco consumption on the poor in Bangladesh. Tob Control. 2001 Sep; 10 (3):212–7. https://doi.org/10.1136/tc.10.3.212 PMID: 11544383 9. John RM. Tobacco consumption patterns and its health implications in India. Health Policy. 2005 Feb; 71(2):213–22. https://doi.org/10.1016/j.healthpol.2004.08.008 PMID: 15607383 10. John RM. Crowding out effect of tobacco expenditure and its implications on household resource alloca- tion in India. Social Science & Medicine. 2008 Mar; 66(6):1356–67. 11. Wang H, Sindelar JL, Busch SH. The impact of tobacco expenditure on household consumption pat- terns in rural China. Social Science & Medicine. 2006 Mar; 62(6):1414–26. 12. Jones L, Efroymson D. References Fruit and vegetable consumption is lower and saturated fat intake is higher among Canadians reporting smoking. The Journal of Nutrition. 2001 Jul 1; 131: 1952–1958. https://doi.org/10.1093/jn/131.7.1952 PMID: 11435513 28. Phillips EL, Arnett DK, Himes JH. Mcgovern PG, Blackburn H, Luepker RV. Differences and trends in antioxidant dietary intake in smokers and non-smokers, 1980–1992: The Minnesota Heart Survey. Annals of Epidemiology. 2000 Oct 1, 10(7): 417–423. PMID: 11018344 29. Subar AF, Harlan LC. Nutrient and food group intake by tobacco use status: the 1987 National Health Interview Survey. Annals of the New York Academy of Sciences. 1993 May; 686(1):310–21. 30. Ma J, Hampl JS, Betts NM. Antioxidant intakes and smoking status: data from the continuing survey of food intakes by individuals 1994–1996. The American journal of clinical nutrition. 2000 Mar 1; 71 (3):774–80. https://doi.org/10.1093/ajcn/71.3.774 PMID: 10702172 15 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0205120 October 9, 2018
https://openalex.org/W2122189759	https://portalrevistas.uct.cl/index.php/cuhso/article/download/899/pdf_17	es		Corrupción y Desarrollo Humano: un estudio de correlación entre fenómenos político-sociales	Cultura-hombre-sociedad	2,015	cc-by	9,294	CUHSO. CULTURA-HOMBRE-SOCIEDAD JULIO 2015 • ISSN 0716-1557 • e -ISSN 0719-2789 • VOL. 25 • NÚM. 1 • PÁGS. 113-138 DOI 10.7770/CUHSO-V25N1-ART899 Corrupción y desarrollo humano: un estudio de correlación entre fenómenos político-sociales Corruption and human development: a correlation study between political-social phenomena. Corrupção e desenvolvimento humano: um estudo de correlação entre fenômenos político-sociais juni o r sa r m i e n t o Universidad Federal de Alagoas, Brasil recepción: 07/04/2015 • aceptación: 30/05/2015 RESUMEN La corrupción es un fenómeno es relevante, prácticamente, para todas las sociedades, en mayor o menor escala. Los actos de corrupción están vinculados a la sobreposición de intereses públicos por los privados con una serie de acciones ilegales concretas que afectan directamente las políticas públicas y la efectividad de la administración pública. A través del índice de correlación de Pearson se constató que la corrupción y el desarrollo humano poseen una fuerte correlación, percibiéndose una tendencia lineal de factores inversamente proporcionales. Pero, esos fenómenos son potenciados por la efectividad o ausencia de accountability, la corrupción, claramente, se vuelve más fuerte cuando hay una precariedad en el intrumento de accountability, ya que cuando ese instrumento se muestra efectivo la promoción de la justicia social y la democracia son fortalecidas y, consecuentemente, hay desarrollo humano. PALABRAS CLAVE Corrupción, desarrollo humano, accountability, corrección. 113 cusho-25-1.indb 113 08-10-15 12:19 p.m. SARMIENTO CORRUPCIÓN Y DESARROLLO HUMANO: UN ESTUDIO DE CORRELACIÓN ENTRE FENÓMENOS POLÍTICO-SOCIALES ABSTRACT Corruption, on a greater or lesser scale, is an important pheno- menon for practically all societies. Acts of corruption occur when private individuals place their own interests above those of society in a series of concrete illegal acts which directly affect public policies and the effectiveness of public administration. Using a Pearson correlation index we found that there is a strong correlation between corruption and human development, in a linear trend of inversely proportional factors. However these phenomena are affected by the effectiveness or absence of accountability. Corruption, clearly, becomes stronger when the instrument for accountability is weak, since when this instrument is seen to be effective the promotion of social justice and democracy is strengthened, and consequently there is human development. KEYWORDS Corruption, human development, accountability, correction. RESUMO A corrupção é um fenômeno que atinge praticamente todas as so- ciedades, em menor ou maior escala. Os atos corruptos estão vinculados a sobreposição do interesse público pelo privado com a concretização de ações revestidas de ilegalidade afetando diretamente as políticas públicas e a efetividade da administração pública. Através do índice de correlação de Pearson constatou-se que a corrupção e o desenvolvimento humano possuem uma forte correlação, percebendo uma tendência linear de fatores inversamente proporcionais. Mas, esses fenômenos são potencializados pela efetividade ou ausência da accountability; a corrupção, claramente, torna-se mais forte quando há uma precariedade no instrumento de accountability, já quando esse instrumento mostra-se efetivo a promoção da justiça social e a democracia são fortalecidas e, consequentemente, há o desenvolvimento humano. PALAVRAS CHAVE Corrupção, Desenvolvimento Humano, Accountability, Correlação. Introdução Quando se fala de corrupção, o cidadão comum associa o fenômeno como culpa de outrem que se aproveita dos seus status econômicos e sociais em benefício próprio em detrimento do bem comum. Há uma delimitação natural na avaliação da corrupção, pois os meios ilícitos geralmente estão camuflados 114 cusho-25-1.indb 114 08-10-15 12:19 p.m. CUHSO. CULTURA-HOMBRE-SOCIEDAD JULIO 2015 • ISSN 0716-1557 • e -ISSN 0719-2789 • VOL. 25 • NÚM. 1 • PÁGS. 113-138 e apenas quando descobertos, a população tem conhecimento dos fatos. E, como há dificuldades em descobrir todos os atos corruptos, os órgãos de controle (accountability horizontal) utilizam-se de ferramentas e métodos para levantamento de evidências que possam configurar as práticas ilícitas, passivas de punição. Segundo Avritzer et al (2008), a corrupção acontece quando alguém, na busca de seus interesses pessoais, age de forma pérfida, burlando as normas estabelecidas. O foco não é a repressão dos interesses pessoais, pois em algum grau sempre haverá a existência dos interesses privado, mas a utilização de mecanismos que prejudiquem o interesse coletivo e o bem comum por meios escusos. Ao longo dos anos, tenta-se minimizar o fenômeno através de marcos regulatórios e criação de organismos de accountability. Avritzer et al (2008) complementa que a transparência das informações é uma ferramenta indispensável para o controle social em uma democracia representativa, pois auxilia o combate à corrupção e aproxima a população dos atos e fatos políticos. Percebe-se, que a delimitação do poder através de normas e leis, mecanismos e órgãos paralelos é um procedimento adotado historicamente no «controle» dos poderes concedidos pelo meio econômico e político e, não obstante, o cidadão precisa atuar ativamente como agente fiscalizador e transformador na tentativa de garantir a execução de políticas públicas efetivas que visam o bem-comum. E, quando se fala em bem-comum, as políticas públicas são importantes para a inclusão da população mais pobre e garante por meio de «liberdades instrumentais» o desenvolvimento humano com uma maior justiça social (Rawls, 2000). Porém, apesar de um tema recorrente entre a mídia e a população, falta literatura científica atualizada que trate sobre o fenômeno corrupção, suas reais implicações, variáveis, medidas preventivas eficientes entre outros. As formas de identificação da corrupção ainda dependem muito da visibilidade dos escândalos e isso torna o processo de avaliação de corrupção um tanto quanto subjetivo. Além disso, é preciso realizar o cruzamento de dados com demais dimensões e variáveis para identificar suas causas e os efeitos; especificamente na relação com a renda, saúde educação —base de cálculo para o índice de desenvolvimento humano. Observa-se que o conhecimento produzido sobre o tema, muitas vezes é tratado de forma pontual, com um curto espaço temporal e com uma amostra estratificada que pode gerar alguns vieses. Tais escolhas podem gerar uma pesquisa «míope» e os resultados encontrados podem não retratar a realidade. 115 cusho-25-1.indb 115 08-10-15 12:19 p.m. SARMIENTO CORRUPCIÓN Y DESARROLLO HUMANO: UN ESTUDIO DE CORRELACIÓN ENTRE FENÓMENOS POLÍTICO-SOCIALES Portanto, a presente pesquisa utiliza o lapso temporal de um (1) quadriênio com uma amostragem de 171 países. E, tendo em vista que nenhum outro estudo contempla essa amplitude de informações, é aplicado o método quantitativo a fim de obter dados objetivos correlacionando as afirmações teóricas com os resultados estatísticos obtidos. Com isso, a pesquisa tem como objetivo principal analisar a seguinte hipótese: «Existe uma forte correlação entre corrupção e o desenvolvimento humano». O estudo utiliza métodos estatísticos para obtenção dos resultados, com ênfase no valor obtido através do Coeficiente de Correlação de Pearson e a Regressão Linear. Como objetivos específicos, tem-se a análise de perspectiva histórica da corrupção nas macrorregiões, evidenciar o grau de corrupção institucionalizada e análise das demais variáveis qualitativas com impacto na corrupção. Devido ao grau de importância do tema que abrange o interesse de toda a população, o presente trabalho demonstra-se viável para apreciação de pesquisadores, acadêmicos, gestores públicos, empresários e toda a sociedade civil. Revisão de literatura Conceito e aspectos históricos da corrupção Primeiramente, é importante esclarecer que definir corrupção é um desafio, pois delimitar a palavra é considerar que tal fenômeno é imutável e estático ao longo do tempo e, ao consider os aspectos históricos, a própria palavra corrupção possui vários significados e conotações, não se mostra viável delimitar através de um único conceito o que seria corrupção (Heidenheimer e Johnston, 2002). Weber (1962) afirma que não se deve começar com uma definição, mas obter aspectos e exemplos específicos para um conceito que, porém, pode ser alterada ao decorrer do tempo. A partir dessa afirmação, serão explanadas as ideias dos principais estudiosos do assunto e evolução da corrupção. Os estudos sobre corrupção vêm sendo, a cada dia mais relevantes e, apesar de não ser o centro dos trabalhos dos cientistas sociais, a questão sobre as implicações da corrupção no desenvolvimento político-sócio-econômico vendo sendo cada vez mais abordadas (Heffernan e Kleinig, 2004). Mesmo com tal relevância, a tentativa de delimitação da palavra através de um conceito mostra-se um desafio por ser tratar de um fenômeno que varia entre as sociedades e momentos históricos. O filósofo Aristóteles (2012) Retrata corrupção 116 cusho-25-1.indb 116 08-10-15 12:19 p.m. CUHSO. CULTURA-HOMBRE-SOCIEDAD JULIO 2015 • ISSN 0716-1557 • e -ISSN 0719-2789 • VOL. 25 • NÚM. 1 • PÁGS. 113-138 através da tipologia de governo e seus respectivos desvios de finalidade do bem comum e sobreposição dos interesses privados (de um pequeno grupo) aos interesses públicos. O pensador Maquiavel (2009) trata corrupção como fenômeno latente na política diante do poder concebido pela mesma, sendo possível apenas medidas retardatárias do acontecimento do fenômeno. Os grandes pensadores de outrora relacionavam a corrupção com os atos advindos dos governantes ou a cúpula que exercia poder sobre assuntos públicos. Weber (apud Motta, 2004), no campo organizacional —administração burocrática—, trata da estrutura como meio de manter a legalidade e cumprimento das regras, separando os interesses públicos (organizacionais) do privado, ou seja, uma evolução do patrimonialismo —fragilmente corruptível— para um sistema regrado de maior controle. Já a partir da segunda metade do século XX, os casos de corrupção de grande repercussão em vários países e diversas áreas foram surgindo e, com isso, a necessidade de estudos mais aprofundados, com uma nova visão e conceito e consequência. Como marco dos estudos consagrados, podemos citar um dos grandes estudiosos do tema, Huntington (1973) explica sobre a necessidade da distinção entre o interesse público e privado, pois quando não há essa distinção é impossível determinar o que ou não corrupção. O autor retomou a ideia entre público e privado, deixando clara a obrigação do Estado e dos particulares. Outra grande contribuição acerca do tema pode ser atribuída também a Huntington (1973), que reforça a corrupção como fenômeno latente e que surge mais fortemente em períodos de crescimento e modernização acelerados, devido ao aumento da riqueza e poder. O estudioso aborda uma visão condicionante para o surgimento da corrupção com foco no contexto econômico. Outra brilhante estudiosa sobre o tema foi Rose-Ackerman (1978, p. 6-7): An act is commercially corrupt if a member of an organization uses his/ her position, his/her rights to make decisions, his/her access to information, or other resources of the organization, to the advantage of a third party and thereby receives money or other economically valuable goods or services where either the payment itself or the services provided are illegal and/or against the organization’s own aims or rules. A autora retrata da relação de interesse entre os envolvidos no fenômeno da corrupção, onde um lado utiliza, geralmente, a vantagem política ou técnica e 117 cusho-25-1.indb 117 08-10-15 12:19 p.m. SARMIENTO CORRUPCIÓN Y DESARROLLO HUMANO: UN ESTUDIO DE CORRELACIÓN ENTRE FENÓMENOS POLÍTICO-SOCIALES o outro lado o poder financeiro. A abordagem de Rose-Ackerman é comportamental correlacionada aos agentes que se beneficiam da corrupção, denominados de rent-seeking. Rose Ackerman (1978) atribui, também, a corrupção aos incentivos do «sistema» que funcionam como catalisadores do fenômeno, permitindo aos agentes políticos maximizarem o suborno ou propina. Cockcroft (2012) cita ainda que o agente político não é o rent-seeking, empresários e burocratas estão constantemente ligados ao fenômeno agindo de forma passiva ou ativa, dependendo da posição ocupada, recursos disponíveis e objetivos. Em sua obra, Klitgaard (1994, p.38) cita que corrupção é: «a indução por meio de considerações impróprias a cometer uma violação do dever. Heidenheimer e Johnston (2002) definem corrupção como termo geral que abrange abuso de autoridade na espera de ganhos pessoais, monetários ou não. Porém, aprofundando-se no tema, Heidenheimer (1970) agrupa em três os tipos básicos de definição mais utilizadas por estudiosos no assunto: 1) uma definição centrada no ofício público, essa definição parte da corrupção do desvio dos deveres formais do cargo de um funcionário público, devido à busca de recompensas para si ou para terceiros; 2) uma definição centrada no mercado, que tem como fator preponderante para a corrupção a maximização da renda, ou seja, o fator da renda pessoal; 3) uma definição centrada na ideia de bem público, segundo essa definição, uma prática é considerada corrupta quando o interesse comum é violado em função da preocupação com ganhos particulares. Tradicionalmente, os escândalos de corrupção que envolve agentes políticos têm suas consequências suprimidas com uma tendência protecionista aos corruptores do alto escalão político, como lembra Gingerich (2006), que todos os atores da corrupção têm interesse em ocultar ou revelar escândalos, o fator determinante para a escolha geralmente é a avaliação dos ganhos x perdas. Habermas (2002) observa que a neutralidade do direito —e do procedimento democrático de privação do direito— é ocasionalmente entendida como se questões políticas de natureza ética tivessem de ser afastadas da agenda política por meio de «gag rules» e suprimidas das discussões por serem inacessíveis a uma regulamentação jurídica imparcial. A partir dos casos concretos de corrupção e envolvimento de agentes políticos, tornam-se necessários mecanismos de prevenção ou remediação do problema. Porém, quando o «sistema» é apático quanto ao combate de práticas dessa natureza, cria-se um ambiente favorável para práticas antiéticas. Até então o foco para entendimento da corrupção foi a relação humana 118 cusho-25-1.indb 118 08-10-15 12:19 p.m. CUHSO. CULTURA-HOMBRE-SOCIEDAD JULIO 2015 • ISSN 0716-1557 • e -ISSN 0719-2789 • VOL. 25 • NÚM. 1 • PÁGS. 113-138 através do prisma político-econômico. Porém, há autores que tratam esse fenômeno, também, através do prisma cultural, reforçando as questões dos valores éticos e morais. Avritzer et al (2008) relata que as diferenças culturais, do ponto de vista de pesquisas comparativas, mostram que a corrupção é variável conforme o plano de valores. Pareto (1984) afirma que a diferença entre os países será identificada, substancialmente, no sentimento do povo (valores), ou seja, um povo honesto gera um governo honesto. A conjuntura de todos os fatores explanados é decisiva para o acontecimento do fenômeno, o poder político, econômico e social. Nesse sentido, partir-se do pressuposto de que esses sistemas interagem de forma permanente, e que nenhum deles funciona de forma independente (Alves, 2009). Dessa forma, não se pode também analisar a corrupção apenas por um enfoque, tem-se que levar em consideração todo o contexto e sistema para entendimento do fenômeno. Transferindo todos os ensinamentos para a atualidade, tem-se a responsabilidade direta do poder público —técnico ou político— no cumprimento legal de servir ao interesse público, evitando sempre a sobreposição do interesse privado sobre o público (Nogueira, 2005). E, sendo a corrupção «regulável», é importante levantar os meios utilizados para esse controle. Accountability como instrumento de combate à corrupção Nesse tópico, apesar da importância do tema, não será abordado diretamente o conceito, mas sim as características e implicações, pois há grande dificuldade em conceituar a palavra, como relata Behn (2001, p.3): «accountability is an important yet elusive concept whose meaning and characteristic differ depending upon of context.» O dicionário da língua inglesa conceitua accountability como qualidade ou estado de ser responsabilizado, ou seja, a imputação da obrigação em responder por algo. Contudo, o mais desejável é obter a noção, ao invés da definição, já que o assunto faz parte das ciências social, que se transforma ao longo do tempo. Na mesma obra Behn (2001) cita que accountability pode ser utilizada de formas distintas quando ligadas a finanças, equidade, performance e entre outros. Além disso, os objetivos e meios específicos são diferentes entre si. É preciso frisar que para alguém tenha a obrigação de responder ou prestar conta, deve haver alguém que responsável pela solicitação e verificação. Behn (2001) 119 cusho-25-1.indb 119 08-10-15 12:19 p.m. SARMIENTO CORRUPCIÓN Y DESARROLLO HUMANO: UN ESTUDIO DE CORRELACIÓN ENTRE FENÓMENOS POLÍTICO-SOCIALES destaca que existe dois grupos: • Accountability Holders: pessoas, órgãos, organizações que fiscalizam, verificam as ações dos holdees; e • Accountability Holdees: tem o dever de «prestar contas» aos holders. Para que haja o processo de Accountability é preciso definir de quem é a responsabilidade das informações e a quem será informado. Geralmente, a responsabilização recai sobre aquele (pessoa física ou jurídica) que recebe algum benefício pecuniário ou não, enquanto o direito e a percepção da necessidade de receber o report fica a cargo dos stakeholders. Behn (2001) cita «everyone wants —other people— to be held accountable». Essa afirmação é visível em todas as áreas, política, organizacional e social, uma vez que todo direito traz consigo a imputabilidade, ou seja, a punição pelo mau uso desse direito. É importante ressaltar que a accountability, assim como a punição, depende do tipo de accountable. Como explica O’Donnel (1998), existem dois tipos de Accountability: 1) accountability horizontal, que diz respeito ao controle de órgãos governamentais e 2) accountability vertical, quando cidadãos ou um grupo da sociedade civil, de forma organizada, exercem essa ação de controle ou «punição». Para facilitar a compreensão, tem-se como exemplo nacional de accountability horizontal, o Tribunal de Contas da União e dos Estados exercem essa função de verificação da aplicação de recursos por parte de órgãos da administração direta e indireta e, quando necessário, há a punição dos agentes públicos pela má utilização desses recursos. Já a accountability vertical, geralmente é exercida pelos cidadãos de forma avulsa e, quando não organizada, se mostra frágil tornando um dos seus poderes de punição ineficaz - o voto. Mas, um fator é primordial para o alcance da accountability: a informação. E, além da organização, é necessária a percepção da população nesse processo para efetivação da democracia que, segundo Dahl (2001) corresponde a participação, igualdade, conhecimento, controle e inclusão de forma indissociável e permanente. No que diz respeito ao governo — decisões políticas—, sua contribuição pode acontecer em duas frentes, segundo Avritzer et al (2008): 1) Reforçar o princípio da virtude cívica, por meio da promoção de incentivos à participação política e de ferramentas que facultem a interação entre arenas participativas e representativas, diminuindo a assimetria informacional entre representantes e representados; 2) podem ser dissua120 cusho-25-1.indb 120 08-10-15 12:19 p.m. CUHSO. CULTURA-HOMBRE-SOCIEDAD JULIO 2015 • ISSN 0716-1557 • e -ISSN 0719-2789 • VOL. 25 • NÚM. 1 • PÁGS. 113-138 sórias relativamente à prática da corrupção, através da organização de mecanismos que aumentem a publicidade dos atos e das omissões dos governantes ou através da efetivação de instrumentos de punição que tornem proibitivos os custos da corrupção e incertos os seus benefícios. Como foi explanado acima, a informação sistematizada e atualizada é um forte aliado, pois funcionam como uma ponte entre os accountability holders e accountability holdees. A informação é considerada um meio de «transparência» dos atos e fatos pertinentes, sendo um método de prestação de contas e medida de combate a corrupção. Segundo Bobbio (1986), «escândalo é a corrupção que vem a público». Portanto dar publicidade à corrupção é uma medida de combate a essas práticas. Dessa forma, a imprensa torna-se peça fundamental para a formação da percepção do nível de corrupção. É imprescindível que todo o trabalho da imprensa, quanto a veiculação da informação, seja imparcial e retrate de forma fidedigna todos os fatos. Avritzer et al (2008) relata que os escândalos midiáticos representam a ação de controle social mais saliente em termos de luta contra a corrupção, pois permitem dar conhecimento à opinião pública. Toda essa discussão sobre accountability e combate à corrupção tem um fim de promover a justiça social, onde em uma sociedade democrática, todos os serem humanos são dignos e têm a mesma importância. Por isso, possuem direitos e deveres iguais não apenas em aspectos econômicos, mas também relativos à saúde, educação, trabalho, direito à justiça e manifestação cultural. Justiça social, liberdade e desenvolvimento humano Através da justiça social, é possível diminuir drasticamente as diferenças de oportunidade, aumentando a potencialidade do ser, já a inexistência dessa igualdade vai na contramão da liberdade e, consequentemente, do desenvolvimento do indivíduo. Como esclarece Sen (2010), toda forma de privação de capacidade é considerada como uma perda da amplitude da liberdade pessoal. Tem-se como exemplo: fome, analfabetismo, doenças físicas e mentais. Segundo Sen, essas capacidades representam a «liberdade substantiva», que permitem um maior leque de escolhas para cada indivíduo. Sen (2010) reforça que a expansão das «capacidades» pode ser aumentada pela política pública, mas também, pode ser influenciada pelo uso efetivo das capacidades participativas do povo. O autor também cita que as «liberdades 121 cusho-25-1.indb 121 08-10-15 12:19 p.m. SARMIENTO CORRUPCIÓN Y DESARROLLO HUMANO: UN ESTUDIO DE CORRELACIÓN ENTRE FENÓMENOS POLÍTICO-SOCIALES instrumentais» devem ser consideradas no processo social de desenvolvimento pois estão relacionadas às liberdades políticas, às facilidades de mercado e às oportunidades sociais. Ou seja, A justiça social só é alcançada através das «liberdades instrumentais» que condicionam a «liberdade substantiva» e, assim, potencializar o desenvolvimento humano. Essas implicações levantas por Sen, de certa forma, são complementares às ideias de Rawls (2000), que parte de uma concepção geral de justiça baseada na ideia de que todos os bens primários —liberdades, oportunidades, riqueza e entre outros—, devem ser distribuídos de maneira igual ou compatível com o sistema semelhante. Rawls (2000) argumenta que os «princípios de justiça» emergem na posição original através de um acordo unânime: • Cada pessoa tem um direito igual a um esquema plenamente adequado de liberdades básicas iguais que seja compatível com um esquema similar de liberdade para todos; • As desigualdades sociais e econômicas devem satisfazer duas condições. Primeira, elas devem estar associadas a cargos e posições abertos a todos em condições de igualdade equitativa de oportunidades. Segunda, elas devem ser para o maior benefício dos membros menos favorecidos da sociedade Fica claro que a visão da liberdade adotada envolve tanto os processos que permitem a liberdade de ações e decisões como as oportunidades reais que as pessoas têm, dadas as suas circunstâncias pessoais e sociais. E, assim como há a expansão da capacidade, também pode haver a sua diminuição através da privação de «liberdade» que pode surgir em razão de processos inadequados —como a violação do direito ao voto ou de outros direitos políticos ou civis—, ou da falta de oportunidade para realizar o que gostariam —também podem ser denominadas de condições, como a fome, analfabetismo—. O conceito de desenvolvimento humano tem suas origens no pensamento clássico e , em particular, nas ideias de Aristóteles, que acreditava que alcançar a plenitude do florescimento das capacidades humanas é o sentido e fim de todo desenvolvimento. Ford e Lerner (1992, p.49) apresentam um conceito complementar: «o desenvolvimento humano envolve processos de incremento e transformação que através do fluxo de interações entre características atuais da pessoa e os contextos em que está inserida, produz uma sucessão de mudanças relativamente duradouras [...]». 122 cusho-25-1.indb 122 08-10-15 12:19 p.m. CUHSO. CULTURA-HOMBRE-SOCIEDAD JULIO 2015 • ISSN 0716-1557 • e -ISSN 0719-2789 • VOL. 25 • NÚM. 1 • PÁGS. 113-138 Wolfensohn (1999) defende uma «estrutura de desenvolvimento ampla» com abordagem integrada e multifacetada, visando a um progresso simultâneo em diferentes frentes. Sen (2010) defende que a plenitude da liberdade requer mais do que suprir com as necessidades básicas, mas requer a inserção do cidadão no meio político interagindo e influenciando nas próprias diretrizes das políticas públicas. Método da Pesquisa A pesquisa caracteriza-se como uma pesquisa de análise documental já que os objetos de análise são os relatórios do Transparency Internacional (TI): «Corruption Perceptions Index», CPI e pela United Nations Development Programme (UNDP): «Human Development Index» (HDI). Ambos os relatórios podem ser obtidos de forma impressa ou digital em seus respectivos endereços eletrônicos. Primeiramente, foram obtidos os dados do CPI referente a um Quadriênio (2010 – 2013). Transparency Internacional, Technical Methodology Note (2012): «Corruption Perceptions Index» segue 4 (quatro) etapas: 1) seleção de dados, 2) redimensionamento dos dados, 3) agregação dos dados redimensionados e 4) análise da medida de incerteza». A pesquisa mede a percepção do grau de corrupção aferido por empresários, analistas de riscos e o público em geral. O Transparency Internacional é uma organização não-governamental que monitora a corrupção no mundo. O IDH, criado pela ONU no início da década de noventa, tem sido calculado anualmente para diversos países. Foi idealizado para servir como a base empírica dos Relatórios de desenvolvimento humano, responsáveis por monitorar o processo de desenvolvimento mundial. O «Human Development Index» é constituído por 3 pilares: Saúde, Educação e Renda. Esses três fatores são calculados, obtendo-se uma média e, partir de então, é atribuído um valor ao país de acordo com a média obtida. Também se caracteriza como uma pesquisa quantitativa, uma vez que, a pesquisa tem como proposta a interpretação dos dados dos dois índices citados no período de 1 (um) Quadriênio, 2010 a 2013. O presente estudo buscou analisar, através do coeficiente de correlação de Pearson (r), a seguinte hipótese: «existe uma forte correlação entre corrupção e o desenvolvimento humano». Garson (2009) afirma que correlação é uma medida de associação bivariada (força) do grau de relacionamento entre duas variáveis. Para Moore 123 cusho-25-1.indb 123 08-10-15 12:19 p.m. SARMIENTO CORRUPCIÓN Y DESARROLLO HUMANO: UN ESTUDIO DE CORRELACIÓN ENTRE FENÓMENOS POLÍTICO-SOCIALES (2007: 100-101), «A correlação mensura a direção e o grau da relação linear entre duas variáveis quantitativas». O estudo também contempla a estatística descritiva, com o cálculo da média, desvio-padrão, amplitude dos índices e histograma. Como ferramenta, foi utilizado o programa computacional Statistical Package for Social Science (SPSS). Em paralelo foi realizado ainda o teste de regressão linear devido a sua capacidade preditiva. A pesquisa tem como característica a estatística amostral, já que realizou a análise com 171 países, onde o universo contém 193 países reconhecidos, segundo a ONU, correspondendo a uma amostra de 87%. Os 171 países foram divididos em 6 macrorregiões, a saber: África Subsaariana (AF), Américas (AM), Ásia-Pacífico (AP), Europa Oriental/Ásia Central (EOr/AC), União Europeia/Europa Ocidental (UE/EOc) e Médio Oriente e Norte Africano (ME/ NA). Análise dos dados Índice de Percepção da Corrupção e Desenvolvimento Humano Atualmente, o índice de corrupção mais utilizado em pesquisas sobre corrupção é formulado pelo Transparency Internacional (TI), uma organização nãogovernamental de coalização contra a corrupção com sede em Berlim (ALE). A organização chega a esse índice através de survey, aplicados por especialistas no tema e direcionado a pessoas de vários setores de cada país com objetivo de obter a «percepção da corrupção» em cada um dos países. A amostra do presente estudo contém 171 países, divididos em 6 macrorregiões. África Subsaariana (AF) —Angola, Benin, Botswana, Burkina Faso, Burundi, Camarões, Cabo Verde, República Centro-Africano, Chade, Comores, República do Congo, Costa do Marfim, República Democrática do Congo, Djibouti, Guiné Equatorial, Eritreia, Etiópia, Gabão, Gâmbia, Gana, Guiné, Guiné-Bissau, Quênia, Lesoto, Libéria, Madagáscar, Malaui, Mali, Mauritânia, Maurício, Moçambique, Namíbia, Níger, Nigéria, Ruanda, São Tomé e Príncipe, Senegal, Seicheles, Serra Leoa, África do Sul, Suazilândia, Tanzânia, Togo, Uganda, Zâmbia e Zimbábue; Américas (AM) —Argentina, Bahamas, Barbados, Bolívia, Brasil, Canadá, Chile, Colômbia, Costa Rica, Cuba, Dominica, República Dominicana, Equador, El Salvador, Guatemala, Guiana, Haiti, Honduras, Jamaica, México, Nicarágua, Panamá, Paraguai, Peru, Costa Rica, Santa Lúcia, São Vicente e Granadinas, Suriname, Trinidade e Tobago, Esta- 124 cusho-25-1.indb 124 08-10-15 12:19 p.m. CUHSO. CULTURA-HOMBRE-SOCIEDAD JULIO 2015 • ISSN 0716-1557 • e -ISSN 0719-2789 • VOL. 25 • NÚM. 1 • PÁGS. 113-138 dos Unidos, Uruguai e Venezuela; Ásia-Pacífico (AP) —Afeganistão, Austrália, Bangladesh, Butão, Brunei, Camboja, China, Hong Kong, Índia, Indonésia, Japão, Coreia do Sul, Laos, Malásia, Mongólia, Mianmar, Nepal, Nova Zelândia, Paquistão, Papua Nova Guiné, Filipinas, Cingapura, Sri Lanka, Tailândia, Timor-Leste e Vietnã; Europa Oriental/Ásia Central (EOr/AC) —Albânia, Armênia, Azerbaijão, Bielo-Rússia, Bósnia e Herzegovina, Geórgia, Cazaquistão, Quirguistão, Macedônia, Moldávia, Montenegro, Rússia, Sérvia, Tajiquistão, Turquia, Turcomenistão, Ucrânia e Uzbequistão; União Europeia/Europa Ocidental (UE/EOc) —Áustria, Bélgica, Bulgária, Croácia, Chipre, República Tcheca, Dinamarca, Estônia, Finlândia, França, Alemanha, Grécia, Hungria, Islândia, Irlanda, Itália, Letônia, Lituânia, Luxemburgo, Malta, Holanda, Noruega, Polônia, Portugal, Romênia, Eslováquia, Eslovênia, Espanha, Suécia, Suíça e Reino Unido; Médio Oriente e Norte Africano (ME/NA) —Argélia, Bahrain, Egito, Iémen, Emirados Árabe, Irã, Iraque, Israel, Kuwait, Líbano, Líbia, Marrocos, Omã, Catar, Arábia Saudita, Sudão, Síria e Tunísia. Considerando os dados da ONU, que reconhece 193 territórios como países, a amostra corresponde a 87% do universo. Os países do estudo foram selecionados a partir do Transparency Internacional (TI) e o United Nations Development Programme (UNPD), que publicam os Índice de Percepção da Corrupção e o Índice de Desenvolvimento Humano, respectivamente. O Índice de Percepção da Corrupção IPCorr é demonstrado através de uma pontuação atribuída aos países. Essa pontuação varia em uma escala de 0 a 100, onde 0 corresponde a um país extremamente corrupto (Highly Corrupt) e 100 ao país totalmente livre de corrupção (Very Clean). A fim de facilitar a visualização, os países foram substituídos pelas macrorregiões e a pontuação Tabela 1. Pontuação no Índice de Percepção da Corrupção Região 2013 2012 2011 2010 Média África Subsaariana 33,50 34,35 29,92 29,28 31,76 Américas 44,88 45,59 42,39 42,91 43,94 Ásia-Pacífico 43,69 43,27 42,37 41,19 42,63 Europa Oriental e Ásia Central 32,72 32,72 28,42 28,33 30,55 Médio Oriente e Norte da África 36,56 38,06 37,59 37,72 37,48 União Europeia e Europa Ocidental 65,19 65,1 64,79 64,52 64,90 Fonte: Adaptado do Transparency Internacional – TI. Disponível em: https://www.transparency.org 125 cusho-25-1.indb 125 08-10-15 12:19 p.m. SARMIENTO CORRUPCIÓN Y DESARROLLO HUMANO: UN ESTUDIO DE CORRELACIÓN ENTRE FENÓMENOS POLÍTICO-SOCIALES de cada país constituiu a média das suas respectivas regiões. No estudo foi analisado o período de um quadriênio 2010/2013, possibilitando uma análise de tendência histórica e correlação entre as variáveis. A tabela demonstra a média agrupada dos quatro anos analisados e, percebe-se que, dentro da escala, cinco das seis macrorregiões estão abaixo de 50 pontos (ponto médio), ou seja, em situação de média-alta corrupção. Apenas a macrorregião UE/EOc apresenta uma situação «favorável», acima do ponto médio. Porém, deve-se considerar que os países de cada macrorregião possuem pontuações divergentes dentro da escala, portanto, além da média, também foi analisado os respectivos desvios-padrão. Os resultados demonstraram como maior desvio-padrão a região AP 22,59, representando uma alta variação entre as pontuações dos países dessa região, já a região EOr/AC apresenta a menor variação entre as macrorregiões -10,17, ou seja, uma menor heterogeneidade entre as pontuações dos países correspondentes. Destaca-se ainda a variação negativa da macrorregião Médio Oriente/Norte da África, que a partir da perspectiva histórica no período do quadriênio analisado (2010-2013), é a única macrorregião com a média menor do que o valor do primeiro ano (2010) com 0,24 negativos de divergência. Já as demais macrorregiões apresentam uma variação positiva entre 0,38 e 2,48. Gráfico 1. Histograma do Índice de Corrupção (Fonte: Adaptado do Transparency Internacional – TI. Disponível em: https://www.transparency.org). 126 cusho-25-1.indb 126 08-10-15 12:19 p.m. CUHSO. CULTURA-HOMBRE-SOCIEDAD JULIO 2015 • ISSN 0716-1557 • e -ISSN 0719-2789 • VOL. 25 • NÚM. 1 • PÁGS. 113-138 O gráfico acima demonstra, graficamente, o resultado dos dados quanto as posições dos países na escala de corrupção proposta pela Transparency Internacional. Identifica-se que a média (mean) do Índice de Corrupção dos países observados (N) é preocupante, tendo em vista que a escala produzida pela Transparency Internacional, onde 0 representa um um país extremamente corrupto e 100 um país totalmente livre da corrupção. Verifica-se uma amplitude de 82,5 no IPCorr, isso demonstra o abismo entre alguns países, que se torna ainda mais vísivel entre países da região da África Subsaariana e região da União Europeia e Europa Ocidental. Essas extremidades regionalizadas podem ser vistas como uma dicotomia do fenômeno corrupção. Apesar das demais regiões estudas apresentarem pontuação próximas as duas extremidades, as mesmas não possuem uniformidade, ou seja, nas demais regiões encontram-se países muito corruptos e pouco corruptos. A região AF é caracterizada pela extrema pobreza, elevados índices de analfabetismo e a não ou precária oferta dos serviços públicos básicos (PNUD). Cockcroft (2012) ressalta ainda como problemas existentes, os conflitos etnicos com ligações diretas no tráfico de drogas e fraudes financeiras e a instabilidade politica. Todos esses fatores implicam diretamente no desenvolvimento do índividuo pois a privação das capacidades limita a «liberdade substantiva» (Sen, 2010). Quanto mais a população for desprovida das condições básicas, menor será o interesse por questões mais complexas, porém de grande significância. Rawls (2000) defende a ideia da distribuição dos bens primários para que haja a evolução de estágio da sociedade. Essa concepção justiça social é mais facilmente observado na região EU/EOc, possuindo uma distribuição de renda mais igualitária, um alto indice educacional e a melhor oferta de serviços públicos básicos do mundo. Através do Índice de Gini é observado uma diferença considerável entre a duas regiões em destaque, enquanto a EU/EOc tem uma média de 30,49, já a região AF tem uma média de 41,77, com a análise de uma regressão linear verifica-se a o relacionamento entre a variável ‘IPCorr’ (dependente) e a variável ‘GINI’ (independente). Diferentemente da escala utilizada no IPCorr e IDH, o índice de Gini tem como como uma condição ideal de total distribuição quando o valor é igual a 0 e de total desigualdade (concentração total) quando o valor é igual a 100. Primeiramente, é obtido como resultado do grau de significância (p-value) 0,00 ( = 0,05). O grau de correlação ‘r’ de 0,62 apresenta uma força moderada, segundo Dancey e Reidy (2006). Esse valor demonstra a força, linearidade 127 cusho-25-1.indb 127 08-10-15 12:19 p.m. SARMIENTO CORRUPCIÓN Y DESARROLLO HUMANO: UN ESTUDIO DE CORRELACIÓN ENTRE FENÓMENOS POLÍTICO-SOCIALES Gráfico 2. Regressão Linear: Corrupção x Desigualdade Econômica (Fonte: Elaboração própria). e direção das variáveis. Já na análise de regressão linear, tem-se como poder preditivo o valor de 0,385 (r-squared). Percebe-se então a relevância da desigualdade na distribuição de renda para o aumento da corrupção, onde 1 ponto no aumento no índice de GINI (maior desigualdade) corresponde a diminuição de 0,385 na escala IPCorr (maior corrupção). Esse resultado corrobora estatisticamente a teoria de Heidenheimer e Johnston (2002), que relaciona abuso de autoridade e corrupção, e nos países onde há uma grande concentração da renda os mais ricos detém naturalmente uma autoridade com pouco ou nenhum limite. Esse cenário torna-se ainda mais crítico em países com várias outras deficiências estruturais e sociais, ou seja, esses demais fatores agravam e aumentam exponencialmente a corrupção. Fica então evidente a necessidade de políticas públicas que viabilizem uma melhor igualdade da renda, contudo, somente ações voltadas a esse propósito não são suficientes para a promoção da justiça social. Huntington (1973), afirma que quanto mais rico economicamente for um país, mais propenso à corrupção o mesmo estará. Contudo, essa regra não se aplica a todos os países, pois dados do PIB demonstram crescimento econômico de países extremamente corruptos assim como de países com um nível de corrupção baixa, logo o fator riqueza não tem influencia direta nos atos corruptos. Já a variável de distribuição de renda demonstra ser uma variável muito mais consistente para a construção de uma escala ou uma análise pre128 cusho-25-1.indb 128 08-10-15 12:19 p.m. CUHSO. CULTURA-HOMBRE-SOCIEDAD JULIO 2015 • ISSN 0716-1557 • e -ISSN 0719-2789 • VOL. 25 • NÚM. 1 • PÁGS. 113-138 ditiva. A cultura é um dos pontos cruciais para entendimento e transformação de valores. Inglehart e Baker (2000) comentam que a cultura existente em uma região (povo) pode dificultar ou facilitar o enraizamento natural de valores ou normas que orientem a boa conduta e o cumprimento das normas. Rothestein (2011) afirma que a transformação da cultura é frágil, contínua e dependente. O autor lembra que a cultura de privilégios destinada a alguns poucos que ocupam cargos políticos (decision makers) impede o avanço moral e ético de uma sociedade. Outro ponto relevante levantado pelo cientista político, diz respeito à necessidade de uma reforma política organizacional do governo induzindo uma mudança comportamental na população, como exemplo a Suécia – país que passou por um «Big Bang Institucional»-. Com isso, observa-se que a corrupção possui características culturais, mas não é culturalmente determinada. Ou seja, através de uma decisão top-down do governo e uma rígida estrutura administrativa, os rent-seekers perceberam que não havia mais um campo fértil para práticas corruptas. Isso demonstra que o sistema organizacional político-administrativo de um país é de suma importância para a transformação, sendo um catalisador que possui duas forças de direções distintas a depender da governança e accountability. A partir dessa observação podemos então redefinir alguns pontos importantes na composição das variáveis independentes. Esta segunda tabela demonstra a percepção dos cidadãos comuns quanto a conduta de setores e órgãos tendo em vista o grau de corrupção nos mesmos. Esse índice pode ser utilizado como insight pelos gestores públicos e agente políticos como fonte de informações para tomada de decisão no combate à Tabela 2. Percepção da Corrupção por Instituição (2013) Região Part. políticos Policia Judiciário Func. Públicos Parlam./legis. AF 2 14 3 - - AM 9 4 1 - 2 AP 7 9 2 1 3 EOr/AC 5 3 3 5 - MO/NA 4 2 1 1 - UE/EOc 16 - 4 - 1 Total % 42% 31% 14% 7% 6% Fonte: Adaptado do Transparency Internacional – TI. Disponível em: https://www.transparency.org 129 cusho-25-1.indb 129 08-10-15 12:19 p.m. SARMIENTO CORRUPCIÓN Y DESARROLLO HUMANO: UN ESTUDIO DE CORRELACIÓN ENTRE FENÓMENOS POLÍTICO-SOCIALES corrupção de forma direcionada. O resultado da pesquisa mostra-se válido pois demonstra a percepção da população que está cotidianamente interagindo com as instituições pública e/ou privadas. A organização Transparency Internacional - TI realiza um survey utilizando 12 (doze) setores e órgãos no questionário e, partir do questionário, o entrevistado atribui notas em uma escala de 0 a 100, onde 0 seria um setor ou órgão isento de corrupção e 100 corresponde a um estágio extremo de corrupção. O questionário elenca os seguintes setores e órgãos: partidos políticos, parlamento/legislativo, militares (exército), ONG’s, mídia, organizações religiosas, organizações privadas, sistema educacional, sistema judiciário, serviços de saúde, polícia e funcionalismo público (administração). Porém, a tabela acima não apresenta as pontuações individuais, mas aponta quantas vezes cada uma das instituições apareceu como o principal «ambiente» de corrupção do país. Mais uma vez, foram utilizadas as macrorregiões devido à grande quantidade da amostra. A fim de uma melhor análise, foram escolhidos apenas os 5 setores/órgãos com maiores índices de corrupção: partidos políticos, parlamento/legislativo, funcionalismo público (administração), polícia e sistema judiciário. Dentre esses, dá-se o destaque negativo aos partidos políticos – podendo inserir os próprios componentes do partido – que obteve o maior percentual dentre os demais (42%), sendo 37% (16 apontamentos) correspondente a região da União Europeia e Europa Ocidental – UE/EOc. Outra instituição com um percentual substancial de apontamentos foi a Polícia (14 apontamentos), correspondendo a 31% do geral. Os países da África Subsaariana foram os países que mais vezes apontaram a instituição policial como a mais corrupta (44%). Observa-se que os partidos políticos e judiciário foram apontados, pelo menos uma vez, em todas as macrorregiões como instituições mais corruptas, isso mostra um certo grau de homogeneidade quanto a institucionalização da corrupção nos países. A tabela demonstra a fragilidade de vários organismos públicos que deveriam trabalhar em prol da população. Assim, a accountability horizontal e vertical mostram-se ainda mais necessárias nesses contextos, pois apenas com transparência, controle e punibilidade é possível iniciar o processo de mudanças e tornar a administração pública sem disfunções e com uma maior efetividade. Apesar dos apontamentos dos resultados quanto ao nível de corrupção, Cockcroft (2012) ressalta que a corrupção também está presente no setor privado, nas mais diversas áreas: indústria, comércio e serviços. Contudo, devido 130 cusho-25-1.indb 130 08-10-15 12:19 p.m. CUHSO. CULTURA-HOMBRE-SOCIEDAD JULIO 2015 • ISSN 0716-1557 • e -ISSN 0719-2789 • VOL. 25 • NÚM. 1 • PÁGS. 113-138 Tabela 3. Índice de Corrupção x Índice de Desenvolvimento Humano 2013 2012 2011 2010 Correlação Regiões IPCorr IDH IPCorr IDH IPCorr IDH IPCorr IDH R - Pearson AF 33,50 0,5 34,35 0,45 29,92 0,46 29,28 0,42 0,68 AM 44,88 0,73 45,59 0,73 42,39 0,72 42,91 0,70 0,70 AP 43,69 0,69 43,27 0,67 42,37 0,65 41,19 0,64 0,89 EOr/AC 32,72 0,72 32,72 0,72 28,42 0,71 28,33 0,67 0,51 MO/NA 36,56 0,73 38,06 0,71 37,59 0,69 37,72 0,68 0,76 UE/EOc 65,19 0,86 65,1 0,87 64,79 0,86 64,52 0,84 0,78 Fonte: Adaptado do Transparency Internacional – TI e United Nations Development Programme – UNDP. Dis poníveis em https://www.transparecy.org e https://www.unpd.org a menor visibilidade dada pela accountability social (mídia) e a dificuldade no acesso de informações dessas empresas, dificultam a mensuração da participação dessas empresas em atos corruptos. A tabela 3 demonstra o confronto entre o Índice de Percepção da Corrupção – IPCorr e o índice do Desenvolvimento Humano – IDH. A análise foi realizada com uma perspectiva correlação entre os países ano a ano a um método de estatística descritiva e correlação: Desvio Padrão e Coeficiente de Correlação Linear de Pearson (r). O método consiste em analisar a correlação entre duas variáveis (x, y) que, no presente estudo refere-se aos índices já citados. O objetivo da análise consiste em detectar qual o grau de correlação entre a corrupção e o desenvolvimento humano. Utilizando o cálculo de Pearson, obtivemos r de correlação de cada macrorregião, com objetivo de demonstrar que dependendo da quantidade e tipo da amostra o resultado pode apresentar uma variação substancial, prejudicando a análise do fenômeno, resposta de hipóteses e a construção inferências. Por isso, é importante levantar o maior número possível de elementos para que a composição da análise retrate o quadro real. «the sampling distribution of Pearson’s r does not approximate normality for small samples. When n increases, distributions will aproximate normal more slowly when 𝜌≠0 then when 𝜌=0» (Chen e Popovic, 2002, p.15). Por exemplo, caso fossem adotados apenas os países da região Ásia-Pacífico, o resultado seria uma forte correlação (0,88), mas se fossem adotados apenas os países pertencentes à região Europa Oriental e Ásia Central, o resultado seria uma correlação média (0,51), ou seja, a 131 cusho-25-1.indb 131 08-10-15 12:19 p.m. SARMIENTO CORRUPCIÓN Y DESARROLLO HUMANO: UN ESTUDIO DE CORRELACIÓN ENTRE FENÓMENOS POLÍTICO-SOCIALES escolha de pequenas amostras dentro de um universo pode tornar a pesquisa tendenciosa e falha. Primeiramente, demonstraremos os resultados da média da correlação de Pearson (r) em uma análise entre países no quadriênio 2010-2013. Também foram evidenciados os resultados das correlações de cada macrorregião propositalmente pelos motivos já citados no parágrafo anterior. Os dados dos países em questão foram levantados e compilados para obtenção do coeficiente de correlação linear de Pearson (r). A correlação obtida foi r = 0,72. Dancey e Reidy (2005) apontam a seguinte classificação: r = 0,10 até 0,30 (fraco); r = 0,40 até 0,6 (moderado); r = 0,70 até 1 (forte). Também foi obtido o f de significação = 0,00. Menor do que 0,05, ou seja, é significativa. Com esses dados, mostra-se que há uma associação e linearidade forte e positiva entre os índices (corrupção e desenvolvimento humano). Vale lembrar que a escala de Pearson varia entre 1 e -1. Além disso, os resultados apresentaram um r² = 0,525. Ou seja, isto significa que 52,5% da variável dependente consegue ser explicado pelo regressor presente no modelo. Através do gráfico pode-se observar a tendência positiva da correlação, como já demonstrada. Além disso, o gráfico facilita a compreensão do valor de r (0,72), pois quanto mais próximos estiverem os pontos da reta traçada, maior será o grau de correlação e quanto mais distante, menor será a. Ou seja, apresenta uma correlação forte, mas não perfeita quanto a associação e linearidade. Como exemplo, pode-se adotar as extremidades de uma das variáveis para análise, por exemplo: no eixo (y) – IDH, observa-se que a pontuação da Gráfico 3. Diagrama: Coeficiente de Correlação Linear de Pearson (Fonte: Elaboração própria). 132 cusho-25-1.indb 132 08-10-15 12:19 p.m. CUHSO. CULTURA-HOMBRE-SOCIEDAD JULIO 2015 • ISSN 0716-1557 • e -ISSN 0719-2789 • VOL. 25 • NÚM. 1 • PÁGS. 113-138 última amostra (República Democrática do Congo) equivale a 0,32 e localizase entre os últimos no IPCorr, representando alto nível de corrupção, mas para que se torna uma correlação perfeita (1), o IPCorr também deveria ser o último, assim como o IDH. Isso também acontece no outro extremo que equivale a 0,95 no IDH (Noruega) e localiza-se entre os primeiros no IPCorr, baixo nível de corrupção, mas a correlação perfeita somente aconteceria com o IPCorr em 90,5 – 1º lugar -. Esses são exemplos que demonstram o porquê da correlação forte, mas não perfeita. Foi observado ainda a amplitude dos dois (2) índices em questão. O IDH apresentou como último e primeiro valor 0,32 a 0,95, respectivamente. Amplitude de 0,63. Ou seja, os países elencados perpassam do desenvolvimento humano baixo ao desenvolvimento humano muito alto, segundo a ONU. Já o IPCorr apresenta como último e primeiro valor 8 e 90,5, respectivamente. Amplitude de 82,5. Ou seja, os países elencados partem de um baixo nível de corrupção ao nível muito alto de corrupção, segundo a escala do TI. O resultado da amplitude demonstra o contraste existente entre os vários países do mundo. No presente estudo foi preciso eliminar um (1) país – Jordânia, pois apresentou uma variação acentuada no eixo y, fora dos padrões das demais amostras, comprometendo a análise do fenômeno. Esse tipo de amostra configurase com um outlier, um ponto destoante dos demais. Tanto a direção quanto a magnitude do coeficiente são afetadas pela presença de observações destoantes. Outliers podem gerar correlações espúrias e/ou esconder associações reais (Figueiredo Filho & Silva Junior, 2009). Os dados revelaram que apenas 53 países alcançam o desenvolvimento humano muito alto (condição ideal), sendo 30 desses países localizadas na União Europeia e Europa Ocidental – UE/EOc e, negativamente, existem 40 países na zona do desenvolvimento humano baixo, sendo 33 deles localizados na África Subsaariana – AF. Já no IPCorr, como baixo nível de corrupção foi adotado a pontuação a partir de 70 pontos dentro da escala da TI, o resultado foi que apenas 25 países possuem um baixo nível de corrupção, sendo 13 deles localizados na UE/EOc e, negativamente, existem 53 países com alto nível de corrupção, sendo 22 deles localizados na AF. Considerações Finais O presente estudo buscou analisar a seguinte hipótese: «há uma forte correlação entre a corrupção e o desenvolvimento humano». Como objeto de análi133 cusho-25-1.indb 133 08-10-15 12:19 p.m. SARMIENTO CORRUPCIÓN Y DESARROLLO HUMANO: UN ESTUDIO DE CORRELACIÓN ENTRE FENÓMENOS POLÍTICO-SOCIALES se foram escolhidos 171 países localizadas nos seis (6) continentes. A amostra, que abrange 87% do universo, apresentou um alto nível de aproximação da realidade e que o resultado obtido pelo coeficiente de correlação de Pearson (r), possivelmente, apresentará um resultado similar em uma análise dos 193 países (100%). Além disso, é importante ressaltar que a amostra não deve ser tendenciosa e, por isso, a estratificação pode auxiliar ou comprometer a pesquisa. Porém, quanto maior o número de países, maior será o número de variáveis, elementos, indicadores que pode influenciar no resultado: cultura, governo, sistema econômico, educação dentre outros. Devido à complexidade e extensão do assunto, não foi levada em consideração maiores particularidades dos países, por isso foi definido que os índices de Percepção da Corrupção do TI e Desenvolvimento Humano do PNUD seriam a base de dados para a análise proposta. A padronização da obtenção desses dados e a possibilidade de verificação da metodologia aplicada dá credibilidade aos dados fornecidos por essas organizações. Para chegar aos resultados apresentados foi preciso aplicar o Coeficiente de Correlação Linear de Pearson. Contudo, outros cálculos e análises foram realizados, demonstrandose pertinentes, como: Média, Desvio Padrão, Amplitude, Grau de Significância e Regressão Linear. Todos esses métodos foram imprescindíveis para a validação, conclusão e melhor compreensão do fenômeno. Ressalta-se que ambos os índices – IDH e IPCorr, são índices compostos por várias outras variáveis e que uma análise bivariada entre ambas consiste em uma análise multivariada «sintetizada», com isso, os dados analisados permitem, com maior credibilidade, o pesquisador constatar resultados válidos de inferência. Como resultados, que auxiliam na corroboração da hipótese, observa-se: • A maioria dos países de desenvolvimento humano ‘muito alto’ está localizada na macrorregião União Europeia/Europa Ocidental que, por sua vez, possui os menores índices de corrupção no mundo, a partir de uma perspectiva de proporcionalidade. • A maioria dos países de desenvolvimento humano ‘baixo’ está localizada na macrorregião África Subsaariana que, por sua vez, possui os maiores índices de corrupção no mundo, a partir de uma perspectiva de proporcionalidade. • O Coeficiente de Correlação Linear de Pearson, variou entre médio e forte grau de correlação mesmo em menores amostras estratificadas, 134 cusho-25-1.indb 134 08-10-15 12:19 p.m. CUHSO. CULTURA-HOMBRE-SOCIEDAD JULIO 2015 • ISSN 0716-1557 • e -ISSN 0719-2789 • VOL. 25 • NÚM. 1 • PÁGS. 113-138 0,51 a 0,88. Já a correlação dos 171 países foi de 0,72, uma correlação forte e positiva, segundo Dancey e Reidy (2005). • Com a eliminação de um (1) país – Jordânia -, que se encontrava como ponto de distorção no eixo y (outlier), o grau de correlação aumentou drasticamente. • Quanto as instituições e setores, constatou-se que a população tem uma grande desconfiança sobre a legalidade dos atos dessas instituições. E, um dos reflexos disso pode ser visto no «termômetro» de confiança publicado pelo Transparency Internacional. O resultado apresenta um cenário desfavorável com desafios de mudança organizacional e comportamental. Essa análise quantitativa dos dados levanta ainda mais questões sobre o tema e, sendo a correlação considerada forte – mas não perfeita (1) – é necessário que se observe quais são as demais variáveis que influenciam negativamente no desenvolvimento humano, de forma à auxiliar a construção de um instrumento de análise. Isso também é válido para a corrupção, já que a metodologia apresenta alguns «gaps» a serem preenchidos. Como meio de validação, a abordagem qualitativa do impacto da cultura sobre a corrupção demonstrou o povo, apesar da sua direta influência sobre os fenômenos sociais, a mesma não possui força para modificar drasticamente uma cultura enraizada de corrupção; apenas com apoio ou iniciativa do poder público (Estado) seria possível a mudança de cenário. Ao analisar as instituições, percebe-se claramente a associação de algumas dessas instituições com a corrupção, sendo necessário um estudo de «reforma» política e cultural. Apesar da limitação do estudo, os dados verificados foram suficientes para análise da hipótese e validar, de forma quantitativa, as teorias e afirmações de muitos estudiosos que defendem o combate à corrupção por ser um dos fatores de geração de injustiça social, privação das liberdades e retardamento no desenvolvimento humano. É visto que as duas variáveis são inversamente proporcionais, ou seja, na medida em que a corrupção aumenta o desenvolvimento humano diminui e vice-versa, porém, a relação apesar de forte, não é perfeita. Todo o processo de construção de entendimento do fenômeno deve ser analisado contextualmente levando em consideração todos os aspectos pertinentes que existem em cada região. A participação social torna-se um catalisador para o processo de mudança e combate à corrupção pois a accountabililty sustenta-se em um tripé: accou135 cusho-25-1.indb 135 08-10-15 12:19 p.m. SARMIENTO CORRUPCIÓN Y DESARROLLO HUMANO: UN ESTUDIO DE CORRELACIÓN ENTRE FENÓMENOS POLÍTICO-SOCIALES ntability vertical (cidadãos comuns), horizontal (governo) e social (grupos organizados). A pressão exercida pelo povo faz com que os gestores públicos e agentes políticos busquem ferramentas e métodos fundamentais para o auxílio na busca de uma sociedade mais justa com liberdade, igualdade, democracia e desenvolvimento. Os instrumentos de accountability mostram-se ainda mais importante na tentativa de combate e repressão à corrupção. Percebe-se quanto menos instrumentos de accountability, maior a possibilidade de atos corruptos. De certa forma os tipos de accountability mantêm uma interdependência de efetividade; geralmente os atos corruptos surgem a partir de denúncias divulgadas pela mídia, posteriormente, os órgãos públicos competentes utilizam as informações veiculadas começam o processo de accountability horizontal e através dos relatórios de constatações a população pode exercer com maior eficácia o direito garantido por lei de accountability vertical. É válido lembrar que accontability vertical não é apenas exercida através do voto, mas por demais mecanismos legais que conferem à população o poder de influenciar decisões que afetam diretamente a sociedade. 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https://openalex.org/W3102232514	https://centaur.reading.ac.uk/78674/1/17m1119056.pdf	English	null	Long-Time Stability and Accuracy of the Ensemble Kalman--Bucy Filter for Fully Observed Processes and Small Measurement Noise	SIAM journal on applied dynamical systems	2,018	cc-by	16,219	Article Published Version Creative Commons: Attribution 4.0 (CC-BY) Open Access Open Access de Wiljes, J., Reich, S. and Stannat, W. (2018) Long-time stability and accuracy of the ensemble Kalman--Bucy filter for fully observed processes and small measurement noise. SIAM Journal on Applied Dynamical Systems, 17 (2). pp. 1152-1181. ISSN 1536-0040 doi: https://doi.org/10.1137/17M1119056 Available at https://centaur.reading.ac.uk/78674/ It is advisable to refer to the publisher’s version if you intend to cite from the work. See Guidance on citing . Published version at: http://dx.doi.org/10.1137/17M1119056 To link to this article DOI: http://dx.doi.org/10.1137/17M1119056 Publisher: Society for Industrial and Applied Mathematics outputs in CentAUR are protected by Intellectual Property Rights law, All outputs in CentAUR are protected by Intellectual Property Rights law, including copyright law. Copyright and IPR is retained by the creators or other copyright holders. Terms and conditions for use of this material are defined in the End User Agreement . www.reading.ac.uk/centaur www.reading.ac.uk/centaur Central Archive at the University of Reading Reading’s research outputs online SIAM J. APPLIED DYNAMICAL SYSTEMS Vol. 17, No. 2, pp. 1152–1181 c⃝2018 SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license c⃝2018 SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license ⃝2018 SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license §TU Berlin, Institut f¨ur Mathematik, Str. des 17. Juni 136, D-10623 Berlin, Germany (stannat@math.tu-berlin. de). Long-Time Stability and Accuracy of the Ensemble Kalman–Bucy Filter for Fully Observed Processes and Small Measurement Noise∗ Jana de Wiljes† , Sebastian Reich‡ , and Wilhelm Stannat§ Abstract. The ensemble Kalman ﬁlter has become a popular data assimilation technique in the geosciences. However, little is known theoretically about its long term stability and accuracy. In this paper, we investigate the behavior of an ensemble Kalman–Bucy ﬁlter applied to continuous-time ﬁltering problems. We derive mean ﬁeld limiting equations as the ensemble size goes to inﬁnity as well as uniform-in-time accuracy and stability results for ﬁnite ensemble sizes. The later results require that the process is fully observed and that the measurement noise is small. We also demonstrate that our ensemble Kalman–Bucy ﬁlter is consistent with the classic Kalman–Bucy ﬁlter for linear systems and Gaussian processes. We ﬁnally verify our theoretical ﬁndings for the Lorenz-63 system. Key words. data assimilation, Kalman–Bucy ﬁlter, ensemble Kalman ﬁlter, stability, accuracy, asymptotic behavior AMS subject classiﬁcations. 65C05, 62M20, 93E11, 62F15, 86A22 AMS subject classiﬁcations. 65C05, 62M20, 93E11, 62F15, 86A22 ‡Universit¨at Potsdam, Institut f¨ur Mathematik, Karl-Liebknecht-Str. 24/25, D-14476 Potsdam, Germany, and University of Reading, Department of Mathematics and Statistics, Whiteknights, PO Box 220, Reading RG6 6AX, UK (sreich@math.uni-potsdam.de). ∗Received by the editors March 1, 2017; accepted for publication (in revised form) by E. Kostelich February 20, 2018; published electronically April 17, 2018. http://www.siam.org/journals/siads/17-2/M111905.html Funding: This research has been partially funded by Deutsche Forschungsgemeinschaft (DFG) through grant CRC 1294 “Data Assimilation”, Project (A02) “Long-time stability and accuracy of ensemble transform ﬁlter algo- rithms”. †Universit¨at Potsdam, Institut f¨ur Mathematik, Karl-Liebknecht-Str. 24/25, D-14476 Potsdam, Germany (wiljes@ uni-potsdam.de). ‡Universit¨at Potsdam, Institut f¨ur Mathematik, Karl-Liebknecht-Str. 24/25, D-14476 Potsdam, Germany, and University of Reading, Department of Mathematics and Statistics, Whiteknights, PO Box 220, Reading RG6 6AX, UK (sreich@math.uni-potsdam.de). §TU Berlin, Institut f¨ur Mathematik, Str. des 17. Juni 136, D-10623 Berlin, Germany (stannat@math.tu-berlin. de). 1152 p y p , http://www.siam.org/journals/siads/17-2/M111905.htm †Universit¨at Potsdam, Institut f¨ur Mathematik, Karl-Liebknecht-Str. 24/25, D-14476 Potsdam, Germany (wiljes@ uni-potsdam.de). Funding: This research has been partially funded by Deutsche Forschungsgemeinschaft (DFG) through grant CRC 1294 “Data Assimilation”, Project (A02) “Long-time stability and accuracy of ensemble transform ﬁlter algo- rithms”. LONG-TIME BEHAVIOR OF THE ENSEMBLE KALMAN FILTER Brownian motion of dimensions Nw and Ny, respectively. It is well known that the ﬁltering distribution πt, i.e., the conditional distribution in Xt for given observations Ys, s ∈[0, t], satisﬁes the Kushner–Zakai equation [Jaz70, BC08], which we write as an evolution equation in the expectation values (3) πt[g] = Z RNx g(x)πt(x)dx πt[g] = Z RNx g(x)πt(x)dx (3) of smooth and bounded functions g : RNx →R, i.e., of smooth and bounded functions g : RNx →R, i.e., (4) dπt[g] = πt[f · ∇g]dt + πt[∇· D∇g]dt + (πt[gh] −πt[g]πt[h])T R−1 (dYt −πt[h]d (4) dπt[g] = πt[f · ∇g]dt + πt[∇· D∇g]dt + (πt[gh] −πt[g]πt[h])T R−1 (dYt −πt[h]dt) . (4) In order to have a properly formulated ﬁltering problem, we also have to specify the distribu- tion at initial time t = 0. wnloaded 08/17/18 to 134.225.109.120. Redistribution subject to CCBY license Popular numerical methods for approximating solutions to (4) include direct ﬁnite-diﬀerence or ﬁnite-element discretizations of (4) and sequential Monte Carlo methods, also called parti- cle ﬁlters [BC08, DdFe01]. These methods lead to consistent approximations but are typically restricted to low-dimensional problems. In recent years, particle ﬁlter methods have become popular, which are applicable to higher-dimensional problems but are no longer consistent. These include the ensemble Kalman ﬁlter (EnKF) [Eve06, LSZ15, RC15], which is now widely used in the geosciences. Abstractly spoken, particle ﬁlters are deﬁned as follows. First, one deﬁnes M weighted random variables Xi t, called particles, which are i.i.d. at initial time t = 0 with distribution π0, and weights wi t ≥0 with wi 0 = 1/M at initial time. A particle ﬁlter is then characterized by appropriate evolution laws for the particles and the weights. Most known particle ﬁlters lead to particles which remain identically distributed while no longer being independent, so called interacting particle systems [Mor13]. If the weights are furthermore kept uniform either through resampling or appropriately deﬁned evolution equations, then expectation can be taken with respect to the law πM t of the Mth particle and consistency of a particle ﬁlter means that limM→∞πM t [g] →πt[g]. t [ ] [ ] The classic bootstrap ﬁlter [AMGC02] uses (1) for the evolution of the particles and (2) for the update of the weights in combination with an appropriate resampling strategy in order to avoid the weights to degenerate. DOI. 10.1137/17M1119056 1. Introduction. In this paper, we consider the continuous-time ﬁltering problem [Jaz70, BC08] for diﬀusion processes of type (1) dXt = f(Xt)dt + √ 2CdWt dXt = f(Xt)dt + √ 2CdWt (1) and observations, Yt, given by and observations, Yt, given by (2) dYt = h(Xt)dt + R1/2dBt. dYt = h(Xt)dt + R1/2dBt. (2) Here Xt denotes the state variable of the Nx-dimensional diﬀusion process with Lipschitz- continuous drift f : RNx →RNx and constant diﬀusion tensors D = CCT and C ∈RNx×Nw. Th b i Y N di i l i h f d h RN RN d Here Xt denotes the state variable of the Nx-dimensional diﬀusion process with Lipschitz- continuous drift f : RNx →RNx and constant diﬀusion tensors D = CCT and C ∈RNx×Nw. The observations Yt are Ny-dimensional with forward map h : RNx →RNy and measurement error covariance matrix R ∈RNy×Ny. Finally, Wt ∈RNw and Bt ∈RNy denote independent continuous drift f : RNx →RNx and constant diﬀusion tensors D = CCT and C ∈RNx×Nw. The observations Yt are Ny-dimensional with forward map h : RNx →RNy and measurement error covariance matrix R ∈RNy×Ny. Finally, Wt ∈RNw and Bt ∈RNy denote independent The observations Yt are Ny-dimensional with forward map h : RNx →RNy and measurement error covariance matrix R ∈RNy×Ny. Finally, Wt ∈RNw and Bt ∈RNy denote independent p // g/j / / / Funding: This research has been partially funded by Deutsche Forschungsgemeinschaft (DFG) through grant CRC 1294 “Data Assimilation”, Project (A02) “Long-time stability and accuracy of ensemble transform ﬁlter algo- rithms”. p // g/j / / / Funding: This research has been partially funded by Deutsche Forschungsgemeinschaft (DFG) through grant CRC 1294 “Data Assimilation”, Project (A02) “Long-time stability and accuracy of ensemble transform ﬁlter algo- rithms”. †Universit¨at Potsdam, Institut f¨ur Mathematik, Karl-Liebknecht-Str. 24/25, D-14476 Potsdam, Germany (wiljes@ uni-potsdam.de). ‡ ‡Universit¨at Potsdam, Institut f¨ur Mathematik, Karl-Liebknecht-Str. 24/25, D-14476 Potsdam, Germany, and University of Reading, Department of Mathematics and Statistics, Whiteknights, PO Box 220, Reading RG6 6AX, UK (sreich@math.uni-potsdam.de). ‡Universit¨at Potsdam, Institut f¨ur Mathematik, Karl-Liebknecht-Str. 24/25, D-14476 Potsdam, Germany, and University of Reading, Department of Mathematics and Statistics, Whiteknights, PO Box 220, Reading RG6 6AX, UK (sreich@math.uni-potsdam.de). de). 1152 1153 ⃝2018 SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license JANA DE WILJES, SEBASTIAN REICH, AND WILHELM STANNAT 1154 appropriate Lyapunov function can be guaranteed. While such ergodicity results of [MH12] are important, they do not imply the accuracy of a ﬁlter. In fact, it is well known that EnKF can diverge and techniques, such as ensemble inﬂation [Eve06], have been developed in order to stabilize a ﬁlter. Furthermore, it has been rigorously demonstrated, for example, in [KLS14] that a judicious choice of inﬂation can lead to uniform-in-time accurate state estimates. At the same time, [KMT15] provides an example of catastrophic ﬁlter divergence, i.e., an expo- nential blow-up of the ensemble systems, for a linear forward map h(x) = Hx with strongly nonnormal operator H. In this paper, we investigate a time-continuous EnKF formulation which is consistent with the classic Kalman ﬁlter in the linear case and which is also stable and accurate uniformly in time without additional ensemble inﬂation. In this ﬁrst study, we will assume for simplicity that the system is fully observable, i.e., h(x) = x in (2), and that the measurement errors are small. These assumptions can be relaxed under appropriate assumptions on the stochastic process (1) and the observation process (2), well known from the theory of classic Kalman ﬁlter theory (i.e., observability and controlability) [Jaz70]. We will also investigate in future work whether the proposed ﬁlter formulations can prevent catastrophic ﬁlter divergence for strongly nonlinear and partially observed systems. Downloaded 08/17/18 to 134.225.109.120. Redistribution subject to CCBY licen The speciﬁc ensemble Kalman–Bucy ﬁlter (EnKBF) formulation, which we will investigate in this paper, is given by drawing M independent realizations (called particles or ensemble members) Xi 0 ∼π0, which then follow the system of diﬀerential equations (5) dXi t = f(Xi t)dt + D(P M t )−1(Xi t −¯xM t )dt −1 2QM t R−1 h(Xi t)dt + ¯hM t dt −2dYt (5) for t ≥0. These equations of motion for the particles are closed through the empirical estimates 6) ¯xM t = 1 M M X i=1 Xi t, P M t = 1 M −1 M X i=1 (Xi t −¯xM t )(Xi t −¯xM t )T (6) (7) ¯hM t = 1 M M X i=1 h(Xi t), QM t = 1 M −1 M X i=1 (Xi t −¯xM t )(h(Xi t) −¯hM t )T. LONG-TIME BEHAVIOR OF THE ENSEMBLE KALMAN FILTER The EnKF, on the contrary, introduces modiﬁed evolution equations for the particles which include the observations and keep the weights uniform in- stead. Most available EnKF formulations are stated for discrete-in-time observations [Eve06]. While the robust behavior of EnKFs has been demonstrated for many applications primarily arising from the geosciences, our theoretical understanding of their long-time stability and accuracy is still rather limited. Large sample size limits have been, for example, investigated in [GMT11, KM15] and it has been demonstrated that the EnKF converges to the classic Kalman ﬁlter for linear systems (1), linear observations (2), and Gaussian initial conditions. Using concepts from shadowing, [GTH13] showed that the EnKF is stable and accurate uni- formly in time for hyperbolic dynamical systems provided the ensemble size is larger than the dimension of the chaotic attractor. Stability and ergodicity of EnKFs have also been studied in [TMK16]. The authors demonstrate that the extended system consisting of (1), (2), and the ﬁlter algorithm possesses a unique ergodic invariant measure provided the existence of an SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license c⃝2018 SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license LONG-TIME BEHAVIOR OF THE ENSEMBLE KALMAN FILTER 1155 In case P M t is not invertible, which is surely the case for M ≤Nx, the inverse of P M t is replaced by its generalized inverse (P M t )+. This generalization is unproblematic from a mathematical perspective since (P M t )+ gets multiplied by a vector which is in the range of P M t , and we show that the equations are well-posed in section 2. At the same time it is known that M ≪Nx often requires application of localization [Eve06, RC15] in order to obtain a full rank approximation of the covariance matrix and to prevent ﬁlter divergence. The impact of localization has been studied in [Ton17] from a rigorous mathematical perspective for high-dimensional linear systems. Given the evolution equations (5), one can derive associated evolution equations for the ensemble mean, ¯xM t , and the ensemble covariance matrix, P M t . These are given by (9) d¯xM t = ¯fM t dt −QM t R−1(¯hM t dt −dYt) d¯xM t = ¯fM t dt −QM t R−1(¯hM t dt −dYt) (9) ownloaded 08/17/18 to 134.225.109.120. Redistribution subject to CCBY license with ¯fM t = ˆπM t [f] and with ¯fM t = ˆπM t [f] and (10) d dtP M t = 1 M −1 M X i=1 (f(Xi t) −¯ft)(Xi t −¯xt)T + (Xi t −¯xt)(f(Xi t) −¯ft)T + D(P M t )+P M t + P M t (P M t )+D −QM t R−1(QM t )T. (10) We will study the behavior of the EnKBF for fully observed processes, i.e., h(x) = x and regular measurement error covariance matrix R in sections 2 and 3. More speciﬁcally, it is shown in section 2 that strong solutions of (5) exist for all times and are unique. This result implies that catastrophic ﬁlter divergence [KMT15] cannot arise under the setting considered in this paper. Next, uniform-in-time stability and accuracy of (5) are proven in section 3 under the additional assumption that R = ϵI, ε > 0, suﬃciently small, and that M > Nx, i.e., the empirical covariance matrix P M t is invertible. In sections 4 and 5, we return to the ﬁltering problem for general observation operator, h, and measurement error covariance matrix R. JANA DE WILJES, SEBASTIAN REICH, AND WILHELM STANNAT (7) Finally, given a solution of (5), we deﬁne the empirical expectation values of a function g and the empirical distribution ˆπM t by (8) ¯gM t := ˆπM t [g], ˆπM t (x) := 1 M M X i=1 δ(x −Xi t) , (8) respectively. Here δ(·) denotes the standard Dirac delta function. The formulation (5) has been stated ﬁrst in [BR10, BR12]. Alternative ensemble Kalman–Bucy formulations in- clude stochastically perturbed formulations [Rei11, LSZ15, RC15] and the extended ensemble Kalman–Bucy ﬁlter, whose exponential stability and propagation of chaos properties have been studied in [DMKT16]. ⃝2018 SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license LONG-TIME BEHAVIOR OF THE ENSEMBLE KALMAN FILTER It is demonstrated in section 4 that in the case of linear systems, (9) and (10) are consistent with the classic Kalman–Bucy ﬁltering equations [Jaz70]. Note that this does not imply that the empirical distribution of the extended EnKBF is asymptotically normal. In fact, we will identify in section 5 its asymptotic distribution for M →∞. To this end we will prove in Theorem 16 that the ensemble Xi t, 1 ≤i ≤M, converges as M →∞to independent solutions ˆXi t, i = 1, 2, 3, . . ., of the following McKean– Vlasov equation: (11) d ˆXt = f( ˆXt)dt + D(Pt)−1( ˆXt −¯xt)dt −1 2QtR−1 h( ˆXt)dt + ¯htdt −2dYt , (11) with ¯xt = ˆπt[x], ht = ˆπt[h], with ¯xt = ˆπt[x], ht = ˆπt[h], (12) Pt = Cov ( ˆXt, ˆXt), Qt = Cov ( ˆXt, h( ˆXt)) . (12) Here ˆπt denotes the distribution of ˆXt. c⃝2018 SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license 1156 JANA DE WILJES, SEBASTIAN REICH, AND WILHELM STANNAT Using Itˆo’s formula, it is then easy to derive from (11) the weak formulation of the nonlinear Fokker–Planck equation driving the distribution ˆπt of ˆXt (13) dˆπt[g] = ˆπt ∇g · fdt + DP−1 t (x −ˆπt[x])dt −1 2QtR−1(h(x)dt + ˆπt[h]dt −2dYt) + ˆπt 1 2∇· QtR−1QT t ∇g dt . (13) te the diﬀerence between (13) and the Kushner–Zakai equation (4). Note the diﬀerence between (13) and the Kushner–Zakai equation (4). Some numerical results, supporting our theoretical estimates, will be presented in section 6 using a stochastically perturbed Lorenz-63 system [Lor63, LSZ15]. 2. Well-posedness of the ensemble Kalman–Bucy ﬁlter for fully observed processes. In this section, we specify the problem setting which is investigated in detail in this paper. We will also derive a ﬁrst well-posedness result for the system (5)–(7) implying that the ﬁlter is not subject to catastrophic ﬁlter divergence. More speciﬁcally, we assume that the process is fully observed, i.e., h(x) = x, that the diﬀusion tensor D has full rank, and that the drift function f is globally Lipschitz continuous. Since the ensemble size, M, will be ﬁxed in this section, we also drop the superscript M in (5). LONG-TIME BEHAVIOR OF THE ENSEMBLE KALMAN FILTER Hence (5) is replaced by (14) dXi t = f(Xi t)dt + DP + t (Xi t −¯xt)dt −1 2PtR−1 Xi tdt + ¯xtdt −2dYt , (14) i = 1, . . . , M. The standard inner product in RNx will be denoted by ⟨·, ·⟩and we recall that (15) ⟨a, b⟩= tr (baT). (15) Hence we quickly verify that Hence we quickly verify that (16) 1 M −1 M X i=1 ⟨Xi t −¯xt, DP + t (Xi t −¯xt)⟩= tr (DP + t Pt) (16) (17) 1 M −1 M X i=1 ⟨Xi t −¯xt, PtR−1(Xi t −¯xt)⟩= tr (PtR−1Pt) = ∥R−1 2 Pt∥2 F. (17) Here ∥A∥F denotes the Frobenius norm of a matrix A. We also introduce the notation ⟨A, B⟩ = tr (BAT), i.e., ∥A∥2 F = ⟨A, A⟩. W h l f h bl d f h Here ∥A∥F denotes the Frobenius norm of a matrix A. We also introduce the notation ⟨A, B⟩ = tr (BAT), i.e., ∥A∥2 F = ⟨A, A⟩. We now investigate the l2-norm of the ensemble deviations from the mean, i.e., Here ∥A∥F denotes the Frobenius norm of a matrix A. We also introduce the notation ⟨A, B⟩ = tr (BAT), i.e., ∥A∥2 F = ⟨A, A⟩. We now investigate the l2-norm of the ensemble deviations from the mean, i.e., = tr (BA ), i.e., ∥A∥F = ⟨A, A⟩. We now investigate the l2-norm of the ensemble deviations from the mean, i.e., ( ), , ∥∥F ⟨, ⟩ We now investigate the l2-norm of the ensemble deviations from the mean, i.e., (18) Vt = 1 M −1 M X i=1 ∥Xi t −¯xt∥2, (18) ⃝2018 SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license c⃝2018 SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license LONG-TIME BEHAVIOR OF THE ENSEMBLE KALMAN FILTER LONG-TIME BEHAVIOR OF THE ENSEMBLE KALMAN FILTER 1157 which satisﬁes the evolution equation which satisﬁes the evolution equation which satisﬁes the evolution equation which satisﬁes the evolution equation es the evolution equation (19) 1 2 dVt dt = 1 M −1 M X i=1 ⟨Xi t −¯xt, f(Xi t) −¯ft⟩+ 1 M −1 M X i=1 ⟨Xi t −¯xt, DP + t (Xi t −¯xt)⟩ − 1 2ε 1 M −1 M X i=1 ⟨Xi t −¯xt, PtR−1(Xi t −¯xt)⟩ = 1 M −1 M X i=1 ⟨Xi t −¯xt, f(Xi t) −¯ft⟩+ tr (DP + t Pt) −1 2∥R−1 2 Pt∥2 F = 1 M −1 M X i=1 ⟨Xi t −¯xt, f(Xi t) −f(¯xt)⟩+ tr (DP + t Pt) −1 2∥R−1 2 Pt∥2 F . 1 2 dVt dt = 1 M −1 M X i=1 ⟨Xi t −¯xt, f(Xi t) −¯ft⟩+ 1 M −1 M X i=1 ⟨Xi t −¯xt, DP + t (Xi t −¯xt)⟩ − 1 2ε 1 M −1 M X i=1 ⟨Xi t −¯xt, PtR−1(Xi t −¯xt)⟩ (19) Here we have used Here we have used (20) X i ⟨Xi t −¯xt, f(¯xt) −¯ft⟩= 0 X i ⟨Xi t −¯xt, f(¯xt) −¯ft⟩= 0 (20) and that the evolution equation (9) for the mean, ¯xt, reduces to (21) d¯xt = ¯ftdt −PtR−1(¯xtdt −dYt) and that the evolution equation (9) for the mean, ¯xt, reduces to (21) d¯xt = ¯ftdt −PtR−1(¯xtdt −dYt) (21) in our setting. Lemma 1. The Frobenius norm of Pt satisﬁes Lemma 1. The Frobenius norm of Pt satisﬁes (22) 1 √ M Vt ≤∥Pt∥F ≤Vt . (22) Proof. We ﬁrst note the following identity: Proof. We ﬁrst note the following identity: (23) ∥Pt∥2 F = 1 (M −1)2 X i,j ⟨Xi t −¯xt, Xj t −¯xt⟩2 . (23) For the proof of the upper bound it is now suﬃcient to observe that 1 (M −1)2 X i,j ⟨Xi t −¯xt, Xj t −¯xt⟩2 ≤ 1 (M −1)2 X i,j ∥Xi t −¯xt∥2∥Xj t −¯xt∥2 = 1 M −1 X i ∥Xi t −¯xt∥2 !2 . (24) For the proof of the lower bound observe that For the proof of the lower bound observe that For the proof of the lower bound observe that 1 (M −1)2 X i,j ⟨Xi t −¯xt, Xj t −¯xt⟩2 ≥ 1 (M −1)2 X i ∥Xi t −¯xt∥4 (25) ≥1 M 1 M −1 X i ∥Xi t −¯xt∥2 !2 . LONG-TIME BEHAVIOR OF THE ENSEMBLE KALMAN FILTER (25) This completes the proof. This completes the proof. This completes the proof. This completes the proof. c⃝2018 SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license ⃝2018 SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license 1158 JANA DE WILJES, SEBASTIAN REICH, AND WILHELM STANNAT JANA DE WILJES, SEBASTIAN REICH, AND WILHELM STANNAT Published by SIAM under the terms of the Creative Commons 4.0 license LONG-TIME BEHAVIOR OF THE ENSEMBLE KALMAN FILTER 1159 This implies that Vt ≤max{V0, λmax(R)L+M + q (λmax(R)ML+)2 + 2λmax(R)M tr (D)} uni- formly in t. Similarly, we obtain the lower bound This implies that Vt ≤max{V0, λmax(R)L+M + q (λmax(R)ML+)2 + 2λmax(R)M tr (D)} uni- formly in t. Similarly, we obtain the lower bound (35) 1 2 dVt dt ≥L−Vt + λmin(D) −λmax(R−1) 2 V 2 t , (35) which implies that Vt ≥min{V0, λmin(R)L−+ q (λmin(R)L−)2 + 2λmin(R)λmin(D)} uniformly in t and Vt > 0 provided V0 > 0. which implies that Vt ≥min{V0, λmin(R)L−+ q (λmin(R)L−)2 + 2λmin(R)λmin(D)} uniformly in t and Vt > 0 provided V0 > 0. Theorem 3. Assume that the drift term f in (1) is globally Lipschitz continuous and sat- isﬁes a linear growth condition (36) ∥f(x)∥≤˜c1(1 + ∥x∥) (36) ∥f(x)∥≤˜c1(1 + ∥x∥) ∥f(x)∥≤˜c1(1 + ∥x∥) (36) for an appropriate ˜c1 > 0. Then the system (14) together with (6)–(7) possesses strong solutions for all times t ≥0. for an appropriate ˜c1 > 0. Then the system (14) together with (6)–(7) possesses strong solutions for all times t ≥0. Proof. We can decompose the equations (14) into ordinary diﬀerential equations in Xi t−¯xt, i = 1, . . . , M and (21) for the mean, ¯xt. Since the l2-norm, Vt, remains bounded, the equations in Xi t −¯xt are well-posed. Furthermore, since ∥Pt∥remains bounded as well, the combined drift term in (21), written as (37) d¯xt = f(¯xt)dt + btdt −PtR−1(¯xtdt −dYt), (37) with bt = ¯ft−f(¯xt), is Lipschitz continuous in ¯xt and, hence, satisﬁes a linear growth condition, i.e., t = ¯ft−f(¯xt), is Lipschitz continuous in ¯xt and, hence, satisﬁes a linear growth condition, with bt = ¯ft−f(¯xt), is Lipschitz continuous in ¯xt and, hence, satisﬁes a linear growth condition, i.e., (38) ∥f(¯xt) + bt −PtR−1¯xt∥≤∥f(¯xt) −PtR−1¯xt∥+ ∥¯ft −f(¯xt)∥≤˜c2(1 + ∥¯xt∥) (38) an appropriate ˜c2 > 0, and, hence, strong solutions to (21) exist for all times [Øks00]. Remark 4. For the analysis of the asymptotic behavior of M →∞the upper bound on V not suﬃcient, because it diverges as M →∞. However, since we need a control only locall Remark 4. For the analysis of the asymptotic behavior of M →∞the upper bound on Vt is not suﬃcient, because it diverges as M →∞. JANA DE WILJES, SEBASTIAN REICH, AND WILHELM STANNAT Remark 2. We recall the standard relations between the Frobenius and the spectral norm of a matrix A, i.e., of a matrix A, i.e., ∥A∥≤∥A∥F (26) and ∥A∥F ≤ p Nx∥A∥. (27) We are now ready to obtain uniform-in-time upper and lower bounds on Vt. First, we can estimate the ﬁrst term of (19) from above and from below as follows: 1 M −1 X i ⟨Xi t −¯xt, f(Xi t) −f(¯xt)⟩≤L+ 1 M −1 X i ∥Xi t −¯xt∥2 = L+Vt (28) Downloaded 08/17/18 to 134.225.109.120. Redistribution subject to CCBY licen (29) 1 M −1 X i ⟨Xi t −¯xt, f(Xi t) −f(¯xt)⟩≥L− 1 M −1 X i ∥Xi t −¯xt∥2 = L−Vt, (29) respectively, where respectively, where (30) L+ := sup x̸=y ⟨f(x) −f(y), x −y⟩ ∥x −y∥2 L−:= inf x̸=y ⟨f(x) −f(y), x −y⟩ ∥x −y∥2 (30) are the upper and lower control on the “dissipativity” constant of f. We clearly have L+ ≤ ∥f∥Lip and L−≥−∥f∥Lip for globally Lipschitz continuous f with Lipschitz constant ∥f∥Lip. Provided Vt ̸= 0, we also ﬁnd that (31) λmin(D) ≤tr (DP + t Pt) ≤tr (D) . (31) Here, λmin(A) and λmax(A) denote the smallest and largest singular values of a matrix A, respectively. Here, λmin(A) and λmax(A) denote the smallest and largest singular values of a matrix A, respectively. Finally, the third term in (19) can be estimated from above and from below using (32) λmin(R−1)∥Pt∥2 F = tr(Ptλmin(R−1)Pt) ≤tr(PtR−1Pt) = ∥R−1 2 Pt∥2 F (32) λmin(R−1)∥Pt∥2 F = tr(Ptλmin(R−1)Pt) ≤tr(PtR−1Pt) = ∥R−1 2 Pt∥2 F and (33) ∥R−1 2 P ∥2 tr(P R−1P ) ≤tr(P λmax(R−1)P ) λmax(R−1) tr(P 2) (λmin(R))− (32) ) R−1 2 Pt∥2 F = tr(PtR−1Pt) ≤tr(Ptλmax(R−1)Pt) = λmax(R−1) tr(P 2 t ) = (λmin(R))−1∥Pt∥2 F , which follow from the inequalities Ptλmin(R−1)Pt ≤PtR−1Pt ≤Ptλmax(R−1)Pt, where ≤is meant in the sense of (symmetric) positive (semi-) deﬁnite matrices. which follow from the inequalities Ptλmin(R−1)Pt ≤PtR−1Pt ≤Ptλmax(R−1)Pt, where ≤is meant in the sense of (symmetric) positive (semi-) deﬁnite matrices. Inserting these estimates and the previous two identities into (19), we ﬁrst obtain th per bound (34) 1 2 dVt dt ≤L+Vt + tr (D) −λmin(R−1) 2M V 2 t . (34) c⃝2018 SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license SIAM. ⃝2018 SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license JANA DE WILJES, SEBASTIAN REICH, AND WILHELM STANNAT JANA DE WILJES, SEBASTIAN REICH, AND WILHELM STANNAT 1160 In addition, we assume the error covariance to be of the type R = εI with suﬃciently small ε > 0, implying In addition, we assume the error covariance to be of the type R = εI with suﬃciently small ε > 0, implying (41) dYt = Xref t dt + √εdBt , (41) and that P M t is invertible which necessitates that M > Nx. We drop the superscript M from all relevant quantities throughout this section, as we are interested in the accuracy of the ﬁlter for ﬁxed ensemble size, M. and that P M t is invertible which necessitates that M > Nx. We drop the superscript M from all relevant quantities throughout this section, as we are interested in the accuracy of the ﬁlter for ﬁxed ensemble size, M. We ﬁnd that the estimation error satisﬁes the evolution equation (42) det = f(Xref t )dt + √ 2CdWt −¯ftdt −1 εPt(etdt + ε1/2dBt). (42) We introduce the squared estimation error norm Et = ∥et∥2/2 = ⟨et, et⟩/2. Then Ito’s formula implies that We introduce the squared estimation error norm Et = ∥et∥2/2 = ⟨et, et⟩/2. Then Ito’s formula implies that Downloaded 08/17/18 to 134.225.109.120. Redistribution subject to CCBY lic dEt = ⟨f(Xref t ) −¯ft, Xref t −¯xt⟩dt −1 ε⟨et, Ptet⟩dt + ⟨et, √ 2CdWt⟩−1 √ε⟨et, PtdBt⟩+ tr (D) dt + 1 2ε tr(P 2 t ) dt , (43) (43) which can be rewritten as (44) dEt = Etdt + dMt (44) Et = ⟨Xref t −¯xt, f(Xref t ) −¯ft⟩−1 ε⟨et, Ptet⟩+ tr (D) + 1 2ε∥Pt∥2 F (45) (45) and the martingale and the martingale Mt = Z t 0 ⟨es, −ε−1/2PsdBs + √ 2CdWs⟩, t ≥0 . To make further progress we need bounds for the smallest and largest singular values λmin t = λmin(Pt) and λmax t = λmax(Pt) of Pt, respectively. An upper bound for the largest singular value has already been derived in section 2, since λmax t = ∥Pt∥≤∥Pt∥F ≤Vt. Since Pt is assumed to be invertible, the explicit evolution equation for Pt reduces to d dtPt = 1 M −1 X i (f(Xi t) −¯f(t))(Xi t −¯xt)T + (Xi t −¯xt)(f(Xi t) −¯f(t))T + 2D −1 εP 2 t . JANA DE WILJES, SEBASTIAN REICH, AND WILHELM STANNAT However, since we need a control only locally in time, we can use (34) to estimate 1 2 d dtVt ≤L+Vt + tr (D) which implies the upper bound ﬃ g y y e, we can use (34) to estimate 1 2 d dtVt ≤L+Vt + tr (D) which implies the upper bound (39) Vt ≤e2L+t V0 + tr (D) L+ , (39) which becomes uniform in M (but, of course, not in t) if the particles at time t = 0 are chosen with uniform upper bound on V0 = V M 0 . which becomes uniform in M (but, of course, not in t) if the particles at time t = 0 are chosen with uniform upper bound on V0 = V M 0 . 3. Accuracy of the ensemble Kalman–Bucy ﬁlter for ﬁnite ensemble sizes and small measurement noise. The goal of this section is to derive bounds on the estimation error (40) et = Xref t −¯xt, et = Xref t −¯xt, (40) where Xref t denotes the reference trajectory of (1) which generated the data. We again restrict the discussion to fully observed processes and globally Lipschitz-continuous drift functions f. c⃝2018 SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license c⃝2018 SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license ⃝2018 SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license JANA DE WILJES, SEBASTIAN REICH, AND WILHELM STANNAT ) Next, we make use of the fact that Pt can be diagonalized, i.e., there are orthogonal matrices Qt and diagonal matrices Λt such that Next, we make use of the fact that Pt can be diagonalized, i.e., there are orthogonal matrices Qt and diagonal matrices Λt such that (47) Pt = QT t ΛtQt. Pt = QT t ΛtQt. (47) c⃝2018 SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license LONG-TIME BEHAVIOR OF THE ENSEMBLE KALMAN FILTER 1161 While the orthogonal matrices Qt are, in general, only continuous in t, the diagonal matrix of singular values can be chosen to be diﬀerentiable in t [Rel69]. As shown in [DE99], the evolution equation for diagonal matrix of eigenvalues, Λt, is of the form dΛt dt = diag (QtUtQT t ) + 2diag (QtDQT t ) −1 εΛ2 t (48) (48) with (49) Ut := 1 M −1 X i f(Xi t) −f(¯xt) Xi t −¯xt T + Xi t −¯xt f(Xi t) −f(¯xt) T . (49) Here diag (A) denotes a diagonal matrix with diagonal entries equal to the diagonal of A. More speciﬁcally, the diagonal entries of diag (QtUtQT t ) are given by Here diag (A) denotes a diagonal matrix with diagonal entries equal to the diagonal of A. More speciﬁcally, the diagonal entries of diag (QtUtQT t ) are given by diag (QtUtQT t ) ii = eT i QtUtQT t ei, 0) diag (QtUtQT t ) ii = eT i QtUtQT t ei, (50) where ei ∈RNx denotes the ith basis vector in RNx. where ei ∈RNx denotes the ith basis vector in RNx. Next, we derive the following estimate using the fact that f is globally Lipschitz continuous. Then, given any unit vector v, it holds that \| 1 M −1 X i ⟨f(Xi t) −f(¯xt), v⟩⟨Xi t −¯xt, v⟩\| (51) ≤ 1 M −1 X i ⟨f(Xi t) −f(¯xt), v⟩2 ! 1 2 1 M −1 X i ⟨Xi t −¯xt, v⟩2 ! 1 2 ≤∥f∥LipVt ≤∥f∥Lip p NxM∥Pt∥, ≤ 1 M −1 X i ⟨f(Xi t) −f(¯xt), v⟩2 ! 1 2 1 M −1 X i ⟨Xi t −¯xt, v⟩2 ! 1 2 p (51) ≤∥f∥LipVt ≤∥f∥Lip p NxM∥Pt∥, ≤∥f∥LipVt ≤∥f∥Lip p NxM∥Pt∥, where we have used Vt ≤√NxM∥Pt∥. Hence setting v = QT t ei, we obtain where we have used Vt ≤√NxM∥Pt∥. Hence setting v = QT t ei, we obtain (52) \| diag (QtUtQT t ) ii \| ≤2∥f∥Lip p NxM∥Pt∥= 2∥f∥Lip p NxMλmax t . (52) \| diag (QtUtQT t ) ii \| ≤2∥f∥Lip p NxM∥Pt∥= 2∥f∥Lip p NxMλmax t . c⃝2018 SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license ⃝2018 SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license LONG-TIME BEHAVIOR OF THE ENSEMBLE KALMAN FILTER Since λmax t = (Λt)ii for some index i, we hence deduce that (52) Since λmax t = (Λt)ii for some index i, we hence deduce that Since λmax t = (Λt)ii for some index i, we hence deduce that dλmax t dt ≤2∥f∥Lip p NxMλmax t + 2λmax(D) −(λmax t )2 ε . (53) This implies that (53) This implies that (54) λmax t ≤max n λmax 0 , ε∥f∥Lip p NxM + q ε2∥f∥2 LipNxM + 2ελmax(D) o . (54) Hence we have shown the following lemma. Hence we have shown the following lemma. Lemma 5 (upper bound on spectral radius of Pt). There is a constant (55) C1 = C1(∥f∥Lip, M, Nx, D, ε0) (55) such that λmax 0 ≤C1ε1/2 at initial time t = 0 implies λmax t ≤C1ε1/2 for all times and all ε ≤ε0. such that λmax 0 ≤C1ε1/2 at initial time t = 0 implies λmax t ≤C1ε1/2 for all times and all ε ≤ε0. c⃝2018 SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license c⃝2018 SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license JANA DE WILJES, SEBASTIAN REICH, AND WILHELM STANNAT SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license JANA DE WILJES, SEBASTIAN REICH, AND WILHELM STANNAT 1162 We now use our upper bound on λmax t = ∥Pt∥2 from Lemma 5 in order to get the estimate (56) \| diag (QtUtQT t )ii\| ≤2L p NxMC1ε1/2. We now use our upper bound on λmax t = ∥Pt∥2 from Lemma 5 in order to get the estimat (56) \| diag (QtUtQT t )ii\| ≤2L p NxMC1ε1/2. (56) Hence, we deduce that (57) dλmin t dt ≥−2∥f∥Lip p NxMC1ε1/2 + 2λmin(D) −(λmin t )2 ε and (57) (58) λmin t ≥min{λmin 0 , q 2ελmin(D) −2ε 3 2 ∥f∥Lip p NxMC1} , (58) which implies the desired lower bound on λmin t . Here λmin(D) denotes the smallest eigenvalue of D. We now ﬁx ε0 > 0 such that which implies the desired lower bound on λmin t . Here λmin(D) denotes the smallest eigenvalue of D. We now ﬁx ε0 > 0 such that (59) λmin(D) −√ε∥f∥Lip p NxMC1 > 0 . (59) Lemma 6 (lower bound on smallest singular value of Pt). There is a constant Lemma 6 (lower bound on smallest singular value of Pt). There is a constant (60) C2 = C2(∥f∥Lip, M, Nx, D, ε0) C2 = C2(∥f∥Lip, M, Nx, D, ε0) (60) such that λmin 0 ≥C2ε1/2 at initial time t = 0 implies λmin t ≥C2ε1/2 for all t > 0 and all ε ≤ε0. such that λmin 0 ≥C2ε1/2 at initial time t = 0 implies λmin t ≥C2ε1/2 for all t > 0 and all ε ≤ε0. Remark 7. The upper and lower bounds for the largest and smallest, respectively, eigenvalue of Pt depend on the ensemble size, M. This dependence can be eliminated for the price of the estimates no longer being valid uniformly in time. We now derive such M-independent upper and lower bounds. Let us assume that (61) ∥f∥LipVs ≤λmax(D) ∥f∥LipVs ≤λmax(D) (61) for all s ∈[0, t]. Such a bound can be found because of (39) and for ε suﬃciently small, i.e., ε ≤εt. Then (53) implies that for all s ∈[0, t]. Such a bound can be found because of (39) and for ε suﬃciently small, i.e., ε ≤εt. Then (53) implies that (62) λmax s ≤2 (λmax(D)ε)1/2 λmax s ≤2 (λmax(D)ε)1/2 (62) (62) λmax s ≤2 (λmax(D)ε)1/2 for all s ∈[0, t] and all ε ≤εt. Similarly, (57) implies that (63) λmin s ≥ 2λmin(D)ε 1/2 . JANA DE WILJES, SEBASTIAN REICH, AND WILHELM STANNAT (63) λmin s ≥ 2λmin(D)ε 1/2 . λmin s ≥ 2λmin(D)ε 1/2 . (63) Hence we have traded the M-dependent constants C1 and C2 in the previous two lemmas by M-independent constants ˜C1 = 2λmax(D)1/2 and ˜C2 = λmin(D)1/2, respectively. However, the estimates hold for ε ≤εt only, where the upper bound εt = εt(∥f∥Lip, D) decreases in time. Hence we have traded the M-dependent constants C1 and C2 in the previous two lemmas by M-independent constants ˜C1 = 2λmax(D)1/2 and ˜C2 = λmin(D)1/2, respectively. However, the estimates hold for ε ≤εt only, where the upper bound εt = εt(∥f∥Lip, D) decreases in time. The upper and lower bounds of the eigenvalues of Pt obtained in the previous two lemmas hold with constants C1 and C2 independent of the driving Wiener processes. They only depend on the initial conditions (which might be random), but we can impose deterministic LONG-TIME BEHAVIOR OF THE ENSEMBLE KALMAN FILTER 1163 bounds on the spectral radius of the covariance matrix. Hence we can take expectations on both sides of (44) in order to obtain the following integral inequality: (64) ( ) E [Et] ≤E [E0] + Z t 0 E [Es] ds ≤E [E0] + Z t 0 E " tr (D) + NxC2 1 + 2ε1/4∥f∥LipC1/2 1 (NxM)1/4E1/2 s −2C2 −ε1/2∥f∥Lip ε1/2 Es # ds , where we used where we used (65) ⟨f(Xref t ) −¯ft, Xref t −¯xt⟩= ⟨f(Xref t ) −f(¯xt), Xref t −¯xt⟩+ ⟨f(¯xt) −¯ft, Xref t −¯xt⟩ ≤2L+Et + √ 2∥f∥LipV 1/2 t E1/2 t ≤2∥f∥Lip Et + ε1/4C1/2 1 (MNx)1/4E1/2 t . (65) The next step is to close the right-hand side in E[Es]. To this end, we ﬁrst derive the following ω-wise estimate: (66) Es ≤ tr (D) + NxC2 1 + 2ε1/4∥f∥LipC1/2 1 (NxM)1/4E1/2 s −2C2 −ε1/2∥f∥Lip ε1/2 Es ! ≤C3 + ε1/4C4E1/2 s −2C2 −ε1/2∥f∥Lip ε1/2 Es ≤ C3 + εC2 4 C2 −C2 −2ε1/2∥f∥Lip ε1/2 Es =: Φ (Es) (66) for C3 = tr (D) + NxC2 1, C4 = 2∥f∥LipC1/2 1 (NxM)1/4, and a linear function Φ(Es). Taking expectations and using E [Φ(Es)] = Φ (E[Es]) we arrive at the integral inequality for C3 = tr (D) + NxC2 1, C4 = 2∥f∥LipC1/2 1 (NxM)1/4, and a linear function Φ(Es). Taking expectations and using E [Φ(Es)] = Φ (E[Es]) we arrive at the integral inequality for C3 = tr (D) + NxC2 1, C4 = 2∥f∥LipC1/2 1 (NxM)1/4, and a linear function Φ(Es). Taking expectations and using E [Φ(Es)] = Φ (E[Es]) we arrive at the integral inequality (67) E [Et] ≤E [E0] + Z t 0 Φ (E [Es]) ds, (67) and we can now apply the Gronwall lemma or comparison techniques for integral inequalities. More precisely, let α = ε−1/2(C2 −2ε1/2∥f∥Lip) > 0, then the time-dependent Ito’s-formula implies that and we can now apply the Gronwall lemma or comparison techniques for integral inequalities. More precisely, let α = ε−1/2(C2 −2ε1/2∥f∥Lip) > 0, then the time-dependent Ito’s-formula implies that (68) eαtE [Et] ≤E [E0] + Z t 0 eαs C3 + εC2 4 C2 ds (68) and, hence, and, hence, (69) E [Et] ≤e−αtE [E0] + α−1K E [Et] ≤e−αtE [E0] + α−1K (69) with K := C3 + ε C2 4 C2 . LONG-TIME BEHAVIOR OF THE ENSEMBLE KALMAN FILTER Note that α−1 = O(ε 1 2 ). Hence we have shown the following the with K := C3 + ε C2 4 C2 . Note that α−1 = O(ε 1 2 ). Hence we have shown the following theorem. with K := C3 + ε C2 4 C2 . Note that α−1 = O(ε 1 2 ). Hence we have shown the following theorem. c⃝2018 SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license 1164 JANA DE WILJES, SEBASTIAN REICH, AND WILHELM STANNAT JANA DE WILJES, SEBASTIAN REICH, AND WILHELM STANNAT Theorem 8 (estimation error). If the measurement error variance ε is chosen suﬃciently small, the initial ensemble is chosen such that P0 is invertible and the bounds of Lemmas 5 and 6 are satisﬁed at initial time, then the mean squared estimation error is of order ε1/2 asymptotically in time. Using Markov’s inequality the above estimate on the measurement error now yields for ﬁxed t the following estimate: (70) P [Et ≥cεq] ≤1 cεq E [Et] = O ε1/2−q . P [Et ≥cεq] ≤1 cεq E [Et] = O ε1/2−q . (70) In particular, for any q ∈(0, 1/2) the estimation error Et = ∥et∥2/2 is of order O (εq) with probability close to one. Note that this does not imply that for a given realization of the EnKBF, the estimation error Et will be small all the time, i.e., that supt≥0 Et (or maxt∈[0,T] Et) is of order O (εq) with probability close to one. This latter statement requires a pathwise control, i.e., a (locally) uniform in time control of Et, which we will derive in the next step. To this end note that (44) together with the inequality (66) imply the pathwise estimate Et ≤e−αtE0 + K α (1 −e−αt) + Z t 0 e−α(t−s) dMs = e−αtE0 + K α (1 −e−αt) + e−αtMt + α Z t 0 e−α(t−s)(Mt −Ms) ds , Et ≤e−αtE0 + K α (1 −e−αt) + Z t 0 e−α(t−s) dMs = e−αtE0 + K α (1 −e−αt) + e−αtMt + α Z t 0 e− (71) (72) sup t≤T Et ≤ E0 + K α + sup t≤T e−αt\|Mt\| + α Z t 0 e−α(t−s)\|Mt −Ms\| ds . (72) In order to control the third term, ﬁrst note that the quadratic variation of the martingale is given as In order to control the third term, ﬁrst note that the quadratic variation of the martingale is given as (73) ⟨M⟩t = Z t 0 ε−1∥Pses∥2 + 2∥Ces∥2 dr , (73) so that so that (74) ⟨M⟩t −⟨M⟩s = Z t s ε−1∥Prer∥2 + 2∥Cer∥2 dr ≤(C1 + 2) Z t s Er dr . (74) In the following let LT,δ := sup0≤s<t≤T \|Mt −Ms\|/ (⟨M⟩t −⟨M⟩s) 1 2 −δ for δ ∈(0, 1 2). LONG-TIME BEHAVIOR OF THE ENSEMBLE KALMAN FILTER LONG-TIME BEHAVIOR OF THE ENSEMBLE KALMAN FILTER 1165 for some constant C, depending on γ, δ, T, C1 and on the bound on the mean squared error obtained in Theorem 8. We can therefore estimate (77) E " sup t≤T e−αt\|Mt\| + α Z t 0 e−α(t−s)\|Mt −Ms\| ds # ≤E " sup t≤T e−αt⟨M⟩ 1 2 −δ t + α Z t 0 e−α(t−s) (⟨M⟩t −⟨M⟩s) 1 2 −δ ds LT,δ # ≤(C1 + 2)E " sup t≤T e−αtt 1 2 −δ + α Z t 0 e−α(t−s)(t −s) 1 2 −δ ds sup t≤T E 1 2 −δ t LT,δ # ≤(C1 + 2)Γ 3 2 −δ α 1 2 −δ E " sup t≤T E 1 2 −δ t LT,δ # . (77) Applying Young’s inequality with p = 1 1 2 −δ and q = 1 1 2 +δ we can further estimate the right-hand side from above by (78) (C1 + 2)Γ 3 2 −δ α 1 2 −δ E " sup t≤T E 1 2 −δ t LT,δ # ≤ 1 2 −δ E " sup t≤T Et # + C α 1−2δ 1+2δ E " L 1 1 2 +δ T,δ # , (78) for some ﬁnite constant C depending on C2 and δ. Taking expectation in (72) and using (76) to estimate the third term gives (79) E " sup t≤T Et # ≤ E [E0] + K α + 1 2 −δ E " sup t≤T Et # + C α 1−2δ 1+2δ E " L 1 1 2 +δ T,δ # ≤ E [E0] + K α + 1 2 −δ E " sup t≤T Et # + C α 1−2δ 1+2δ ε δ 1+2δ (79) with some diﬀerent constant C. Under the assumptions of Theorem 8, in particular E [E0] = O ε 1 2 , and thus α−1 = O ε 1 2 for ε ≤ε0, ε0 suﬃciently small, we can now ﬁnd for any η ∈ 0, 1 4 a ﬁnite constant C such that with some diﬀerent constant C. JANA DE WILJES, SEBASTIAN REICH, AND WILHELM STANNAT Theorem 5.1 in [BY82] now implies for any γ ≥1 that there exists a ﬁnite constant Cδ,γ such that In the following let LT,δ := sup0≤s<t≤T \|Mt −Ms\|/ (⟨M⟩t −⟨M⟩s) 1 2 −δ for δ ∈(0, 1 2). Theorem 5.1 in [BY82] now implies for any γ ≥1 that there exists a ﬁnite constant Cδ,γ such that (75) E [(LT,δ)γ] 1 γ ≤Cδ,γE h ⟨M⟩δγ T i 1 γ . (75) Combining the last estimate with Theorem 8 we obtain for γδ ≤1 that Combining the last estimate with Theorem 8 we obtain for γδ ≤1 that (76) E [(LT,δ)γ] 1 γ ≤Cδ,γE h ⟨M⟩δγ T i 1 γ ≤Cδ,γE [⟨M⟩T ]δ ≤Cδ,γ(C1 + 2)δE Z T 0 Et dt δ ≤Cε δ 2 (76) c⃝2018 SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license ⃝2018 SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license LONG-TIME BEHAVIOR OF THE ENSEMBLE KALMAN FILTER Under the assumptions of Theorem 8, in particular E [E0] = O ε 1 2 , and thus α−1 = O ε 1 2 for ε ≤ε0, ε0 suﬃciently small, we can now ﬁnd for any η ∈ 0, 1 4 a ﬁnite constant C such that (80) E " sup t≤T Et # ≤Cε 1 2 −η . (80) E " sup t≤T Et # ≤Cε 1 2 −η . In particular, (80) In particular, (81) P " sup t≤T Et ≥cεq # ≤1 cεq E " sup t≤T Et # = O ε1/2−η−q , (81) which implies that for any q ∈(0, 1/2) the estimation error Et = ∥et∥2/2 is of order O (εq) uniformly on [0, T] with probability close to one. which implies that for any q ∈(0, 1/2) the estimation error Et = ∥et∥2/2 is of order O (εq) uniformly on [0, T] with probability close to one. c⃝2018 SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license JANA DE WILJES, SEBASTIAN REICH, AND WILHELM STANNAT 1166 4. Consistency of the ensemble Kalman–Bucy ﬁlter for linear systems. In this section, we provide a detailed analysis of the EnKBF in the case of linear model dynamics, i.e., f(x) = Ax+b, linear forward map, i.e. h(x) = Hx, full rank diﬀusion tensor, D, and initial ensemble, Xi 0, chosen such that P M 0 is invertible. Then the EnKBF (5) reduces to (82) dXi t = (AXi t +b)dt+D(P M t )−1(Xi t −¯xM t )dt−1 2P M t HTR−1 HXi tdt + H¯xM t dt −2dYt , i = 1, . . . , M, from which we can extract the equation for the empirical mean, ¯xt, i = 1, . . . , M, from which we can extract the equation for the empirical mean, ¯xt, (83) d¯xM t = A¯xM t dt + bdt −P M t HTR−1(H¯xM t dt −dYt) (83) and the equation for the empirical covariance matrix, as deﬁned in (6), (84) d dtP M t = AP M t + P M t AT + 2D −P M t HTR−1HP M t (84) provided P M t has full rank. These equations correspond exactly to the classic Kalman–Bucy ﬁlter formulas for the mean and the covariance matrix [Jaz70]. LONG-TIME BEHAVIOR OF THE ENSEMBLE KALMAN FILTER However, while one would set P M 0 and ¯xM 0 equal to the mean and the covariance matrix, respectively, of the given initial Gaussian distribution N(¯x0, P0) in the classic Kalman–Bucy ﬁlter formulation, the P M t and ¯xM t arise in our context from sampling from the initial distribution, i.e., Xi 0 ∼N(¯x0, P0). Remark 9. It is well known that solutions to (84) have full rank for all t > 0 even if the initial P M 0 is singular. However, note that (84) holds true only if P M 0 is nonsingular and that the diﬀusion induced contribution in (84) needs to be replaced by D(P M t )+P M t otherwise. This discrepancy between the Riccati equation for the classic Kalman–Bucy ﬁlter and the EnKBF is caused by our interacting particle approximation to the diﬀusion term in (1). We will now investigate the asymptotic behavior of the EnKBF in the large ensemble size limit. More speciﬁcally, we will show that the empirical distribution of the EnKBF converges under appropriate conditions towards a distribution with mean and covariance determined by the Kalman–Bucy ﬁltering equations. Note that this does not imply that the empirical distribution of the EnKBF converges to the conditional distribution πt given by the solution of the Kushner–Zakai equation (4), but by the nonlinear Fokker–Planck equation (13) instead as we will show in section 5. Let us ﬁrst state the following a.s. result on the asymptotic behavior of P M t . Proposition 10. Let π0 be the initial distribution on RNx with ﬁnite second moments and invertible covariance matrix with entries (85) ¯P0(k, l) = π0[xkxl] −π0[xk]π0[xl] , (85) 1 ≤k, l ≤Nx. Let Xi 0, i = 1, 2, . . ., be i.i.d. (π0), and let ¯Pt be the solution of the Kalman– Bucy ﬁltering equation (99) with initial condition ¯P0. Then there exists a constant 1 ≤k, l ≤Nx. Let Xi 0, i = 1, 2, . . ., be i.i.d. (π0), and let ¯Pt be the solution of the Kalman– Bucy ﬁltering equation (99) with initial condition ¯P0. Then there exists a constant (86) ˜C = ˜C t, A, D, HT R−1H, max 0≤s≤t ∥¯Ps∥F, sup M≥2 V M 0 (86) c⃝2018 SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license ⃝2018 SIAM. LONG-TIME BEHAVIOR OF THE ENSEMBLE KALMAN FILTER Published by SIAM under the terms of the Creative Commons 4.0 license 1167 LONG-TIME BEHAVIOR OF THE ENSEMBLE KALMAN FILTER such that (87) ∥P M t −¯Pt∥2 F ≤et ˜C∥P M 0 −¯P0∥2 F , (87) ∥P M t −¯Pt∥2 F ≤et ˜C∥P M 0 −¯P0∥2 F , ∥P M t −¯Pt∥2 F ≤et ˜C∥P M 0 −¯P0∥2 F , (87) where V M 0 is deﬁned by (18) with t = 0. where V M 0 is deﬁned by (18) with t = 0. where V M 0 is deﬁned by (18) with t = 0. Note that the strong law of large numbers implies that supM≥2 V M 0 < ∞π0-a.s. Proof. Using the dynamical equations (84) for P M t and (99) for ¯Pt (which, of course, coincides with (84)), we immediately obtain that (88) 1 2 d dt∥P M t −¯Pt∥2 F ≤⟨A(P M t −¯Pt), P M t −¯Pt⟩+ ⟨ P M t −¯Pt AT, P M t −¯Pt⟩ −⟨P M t HTR−1HP M t −¯PtHTR−1H ¯Pt, P M t −¯Pt⟩. (88) Using Using Using (89) ⟨P M t HTR−1HP M t −¯PtHTR−1H ¯Pt, P M t −¯Pt⟩ = ⟨P M t HTR−1H P M t −¯Pt , P M t −¯Pt⟩+ ⟨ P M t −¯Pt HTR−1H ¯Pt, P M t −¯Pt⟩ ≤∥HT R−1H∥F ∥P M t ∥F + ∥¯Pt∥F ∥P M t −¯Pt∥2 F, (89) we arrive at the following diﬀerential inequality: we arrive at the following diﬀerential inequality: (90) 1 2 d dt∥P M t −¯Pt∥2 F ≤ 2∥A∥F + ∥HT R−1H∥F ∥P M t ∥F + ∥¯Pt∥F ∥P M t −¯Pt∥2 F . (90) Integrating up the last inequality w.r.t. time t yields (91) ) ∥P M t −¯Pt∥2 F ≤exp 4t∥A∥F + ∥HT R−1H∥F Z t 0 ∥P M s ∥F + ∥¯Ps∥F ds ∥P M 0 −¯P0∥2 F . In the next step we will need a uniform in M upper bound on ∥P M t ∥F that holds (locally) uniform w.r.t. time t. To this end ﬁrst note that (39) implies (92) ∥P M t ∥F ≤V M t ≤et∥A∥F V M 0 + tr (D) ∥A∥F , (92) thereby using L+ ≤∥A∥F. LONG-TIME BEHAVIOR OF THE ENSEMBLE KALMAN FILTER Since the solution ¯Pt of (99) is continuous, hence, also locally bounded, we can estimate the exponential in (91) from above by 2t 2∥A∥F + ∥R−1∥F∥H∥2 F et ∥A∥F V M 0 + tr (D) ∥A∥F + max 0≤s≤t ∥¯Ps∥F which implies the assertion. which implies the assertion. which implies the assertion. We can now state our main result on the asymptotic consistency of the ensemble Kalman er We can now state our main result on the asymptotic consistency of the ensemble Kalman ﬁlter. c⃝2018 SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license c⃝2018 SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license c⃝2018 SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license ⃝2018 SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license 1168 JANA DE WILJES, SEBASTIAN REICH, AND WILHELM STANNAT 1168 JANA DE WILJES, SEBASTIAN REICH, AND WILHELM STANNAT JANA DE WILJES, SEBASTIAN REICH, AND WILHELM STANNAT Theorem 11. Suppose that Xi 0, i = 1, 2, 3, . . ., are i.i.d. (π0) where the initial distribution π0 has ﬁnite second-order moments and invertible covariance matrix (85). Let ¯Pt be the solution of the Kalman–Bucy ﬁltering equation (99) with initial condition ¯P0, and let ¯xt be the unique solution of (93) d¯xt = A¯xt dt + b dt −¯PtHT R−1 (H¯xt dt −dYt) d¯xt = A¯xt dt + b dt −¯PtHT R−1 (H¯xt dt −dYt) (93) with initial condition ¯x0 := π0[x]. Then limM→∞¯xM t = ¯xt in L2, in particular in probability, for all t ≥0. Proof. Since Xi 0 are i.i.d., the strong law of large numbers implies that limM→∞P M 0 = ¯P0 π0-a.s. and in L2, since π0 has ﬁnite second moments, thus limM→∞P M t = ¯Pt a.s. and in L2 for t ≥0 due to Proposition 10. ownloaded 08/17/18 to 134.225.109.120. Redistribution subject to CCBY license To see that ¯xM t converges towards the unique solution ¯xt of (93) note that d ¯xM t −¯xt = A ¯xM t −¯xt dt − P M t HTR−1H¯xM t −¯PtHTR−1H¯xt dt + P M t −¯Pt HTR−1 dYt (94) and, consequently, (95) and, consequently, ( ) (95) ∥¯xM t −¯xt∥≤∥¯xM 0 −¯x0∥+ Z t 0 ∥A∥F + ∥HT R−1H∥F∥¯Ps∥F ∥¯xM s −¯xs∥ds + Z t 0 ∥HT R−1H∥F∥P M s −¯Ps∥F∥¯xM s ∥ds + Z t 0 P M s −¯Ps HTR−1 dYs . Taking expectations we arrive at Taking expectations we arrive at (96) E ∥¯xM t −¯xt∥ ≤E ∥¯xM 0 −¯x0∥ + Z t 0 ∥A∥F + ∥H∥2 F∥R−1∥F∥¯Ps∥F E ∥¯xM s −¯xs∥ ds + Z t 0 ∥H∥2 F∥R−1∥FE ∥P M s −¯Ps∥F ∥¯xM s ∥ds + E Z t 0 P M s −¯Ps HTR−1 dYs . E ∥¯xM t −¯xt∥ ≤E ∥¯xM 0 −¯x0∥ + Z t 0 ∥A∥F + ∥H∥2 F∥R−1∥F∥¯Ps∥F + Z t 0 ∥H∥2 F∥R−1∥FE ∥P M s −¯Ps∥F ∥¯xM s ∥ds + E Z t 0 P M s −¯Ps HTR−1 dYs . c⃝2018 SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license ⃝2018 SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license (100) (See [KS72, Theorem 4.11], and [OP96, Lemma 2.2].) (See [KS72, Theorem 4.11], and [OP96, Lemma 2.2].) Now recall that we have assumed in sections 2 and 3 that h(x) = x, i.e., H = I, and that D = CCT has full rank. In other words, we have assumed a restricted case of (nonlinear) controllability and observability. It would be of interest to explore, in as far the conditions of sections 2 and 3 can be relaxed while maintaining the well-posedness, stability, and accuracy of the associated EnKBF. 5. Asymptotic limiting equations for the extended EnKBF. In this section, we will derive the non-Markovian stochastic diﬀerential equation (11) with (12) of McKean–Vlasov type. We ﬁrst have to show now that (11) is well-posed. To this end we assume that f, h are globally Lipschitz continuous and that the initial condition ˆX0 has ﬁnite second moments with invertible covariance matrix P0. Recall that—given Xt = Xref t —the observation process Yt can be interpreted as Brownian motion with covariance operator R and drift term h(Xref t ), so that we can solve (11) uniquely up to the ﬁrst time τ where Pτ becomes singular. Clearly, τ > 0 a.s. (w.r.t. the distribution of {Ys}). Using Itˆo’s formula, it is then straightforward to see that the distribution ˆπt of ˆXt indeed satisﬁes the nonlinear Fokker–Planck equation (13) (up to time τ). 5.1. Lower bounds on λmin(Pt) and well-posedness of (11). We will prove in Lemma 15 a strictly positive lower bound on the smallest eigenvalue λmin(Pt) of Pt locally uniformly w.r.t. t, a.s. w.r.t. the distribution of {Ys}, under appropriate assumptions on the coeﬃcients f, h, D, and R. This implies, in particular, that Pt will stay invertible for all t, a.s. and yields existence and uniqueness of a strong solution of (11) for all times t (for typical observation {Ys}). On the other hand, using the algebraic identity (101) (P M s )−1 −P−1 s = (P M s )−1 Ps −P M s P−1 s , (101) we also obtain the following control: (102) ∥(P M s )−1 −P−1 s ∥2 ≤C(t)2∥Ps −P M s ∥2 , s ≤t , (102) for the distance between the inverse covariance matrix of the EnKBF and Pt. Here, C(t) is a joint upper bound of ∥P−1 s ∥2 and ∥(P M s )−1∥2 (uniform in M) for s ≤t. LONG-TIME BEHAVIOR OF THE ENSEMBLE KALMAN FILTER LONG-TIME BEHAVIOR OF THE ENSEMBLE KALMAN FILTER and the solution Pt of the matrix Riccati equation (99) d dtPt = APt + PtAT + 2D −PtHTR−1HPt , (99) converges for any initial condition P0 towards P∞as t →∞with exponential rate λ < λ∗, where converges for any initial condition P0 towards P∞as t →∞with exponential rate λ < λ∗, where (100) λ∗:= inf{−Re(λ) \| λ eigenvalue of A −P∞HTR−1H} . SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license JANA DE WILJES, SEBASTIAN REICH, AND WILHELM STANNAT (96) Using limM→∞E ∥P M t −¯Pt∥2 F = 0 it follows that Using limM→∞E ∥P M t −¯Pt∥2 F = 0 it follows that Using limM→∞E ∥P M t −¯Pt∥2 F = 0 it follows that (97) lim M→∞E Z t 0 P M s −¯Ps HTR−1 dYs = 0 (97) by dominated convergence, and then Gronwall’s lemma implies that limM→∞E ∥¯xM t −¯xt∥ = 0. by dominated convergence, and then Gronwall’s lemma implies that limM→∞E ∥¯xM t −¯xt∥ = 0. Remark 12. It is well known that if (A, H) is observable, i.e., rank HT, (HA)T, . . . , (HANx−1)T = Nx, and (A, C) is controllable, i.e., rank C, AC, . . . , ANx−1C = Nx, then there exists a unique positive deﬁnite solution P∞of the matrix Riccati equation Remark 12. It is well known that if (A, H) is observable, i.e., rank HT, (HA)T, . . . , (HANx−1)T = Nx, and (A, C) is controllable, i.e., rank C, AC, . . . , ANx−1C = Nx, then there exists a unique positive deﬁnite solution P∞of the matrix Riccati equation 0 = AP∞+ P∞AT + 2D −P∞HTR−1HP∞, 98) 0 = AP∞+ P∞AT + 2D −P∞HTR−1HP∞, (98) c⃝2018 SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license 1169 (100) for the distance between the inverse covariance matrix of the EnKBF and Pt. Here, C(t) is a joint upper bound of ∥P−1 s ∥2 and ∥(P M s )−1∥2 (uniform in M) for s ≤t. s s To this end let us ﬁrst state the dynamical equations for the mean ¯xt and the covariance matrix Pt (analogous to (9) and (10) for the EnKBF): (103) d¯xt = ¯ft dt −QtR−1 ¯ht dt −dYt , t < τ , (103) c⃝2018 SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license c⃝2018 SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license JANA DE WILJES, SEBASTIAN REICH, AND WILHELM STANNAT 1170 with ¯ft = E f( ˆXt) and ( 4) with ¯ft = E f( ˆXt) and with ¯ft = E f( ˆXt) and (104) d with ¯ft = E f( ˆXt) and (104) d dtPt = E h (f( ˆXt) −¯ft)( ˆXt −¯xt)T + ( ˆXt −¯xt)(f( ˆXt) −¯ft)Ti + 2D −QtR−1QT t , t < τ . (104) d dtPt = E h (f( ˆXt) −¯ft)( ˆXt −¯xt)T + ( ˆXt −¯xt)(f( ˆXt) −¯ft)Ti + 2D −QtR−1QT t , t < τ . Lemma 13. We have that Lemma 13. We have that (105) 1 √Nx E h ∥ˆXt −¯xt∥2i ≤∥Pt∥F ≤E h ∥ˆXt −¯xt∥2i , t ≤τ . (105) Proof. Similar to the proof of Lemma 1, we have the following. Upper bound: Proof. Similar to the proof of Lemma 1, we have the following. Upper bound: Upper bound: ∥Pt∥2 F = X k,l E h ˆXt −¯xt (k) ˆXt −¯xt (l) i2 ≤ X k,l E ˆXt −¯xt 2 (k) E ˆXt −¯xt 2 (l) = E h ∥ˆXt −¯xt∥2i2 . (106) Lower bound: (107) ∥Pt∥2 F = X k,l E ˆXt −¯xt (k) ˆXt −¯xt (l) 2 ≥ X k E ˆXt −¯xt 2 (k) 2 . (107) Lemma 14. For all t < τ there exists some ﬁnite constant C4(t)—independent of {Ys}— such that Lemma 14. For all t < τ there exists some ﬁnite constant C4(t)—independent of {Ys}— such that such that (108) sup 0≤s≤t E h ∥ˆXs −¯xs∥2i ≤C4(t) . (108) Proof. The diﬀerence ˆXt −¯xt satisﬁes the ordinary diﬀerential equation Proof. SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license (100) The diﬀerence ˆXt −¯xt satisﬁes the ordinary diﬀerential equation (109) d dt ˆXt −¯xt = f( ˆXt) −¯ft + DP−1 t ˆXt −¯xt −1 2QtR−1 h( ˆXt) −¯ht (109) up to time τ so that for t < τ up to time τ so that for t < τ (110) d dtE h ∥ˆXt −¯xt∥2i = 2E h ⟨f( ˆXt) −¯xt, ˆXt −¯xt⟩ i + 2E h ⟨DP−1 t ˆXt −¯xt , ˆXt −¯xt⟩ i −E h ⟨QtR−1 h( ˆXt) −¯ht , ˆXt −¯xt⟩ i ≤2L+E h ∥ˆXt −¯xt∥2i + 2 tr (D), (110) ≤2L+E h ∥ˆXt −¯xt∥2i + 2 tr (D), thereby using thereby using (111) E h ⟨QtR−1h( ˆXt) −¯ht , ˆXt −¯xt⟩ i = ∥R−1/2QT t ∥2 F ≥0 . (111) c⃝2018 SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license LONG-TIME BEHAVIOR OF THE ENSEMBLE KALMAN FILTER 1171 This implies the same bound This implies the same bound (112) Var ( ˆXt) := E h ∥ˆXt −¯xt∥2i ≤e2L+t E h ∥ˆX0 −¯x0∥2i + tr (D) L+ (112) as stated in Remark 4 for the EnKBF for h(x) = x; therefore, as stated in Remark 4 for the EnKBF for h(x) = x; therefore, as stated in Remark 4 for the EnKBF for h(x) = x; therefore, (113) sup 0≤s≤t Var ( ˆXs) = sup 0≤s≤t E h ∥ˆXs −¯xs∥2i ≤C4(t) (113) for some ﬁnite constant C4(t) depending on t. Note that C4(t) clearly is independent of {Ys}. for some ﬁnite constant C4(t) depending on t. Note that C4(t) clearly is independent of {Ys}. Lemma 15. Let ∥f∥2 Lip < 2λmin(D)∥R−1∥F∥h∥2 Lip. If (114) λmin(P0) ≥κ−:= 2λmin(D)∥R−1∥F∥h∥2 Lip −∥f∥2 Lip 2∥R−1∥2 F∥h∥4 LipC4(t) , (114) where C4(t) is the upper bound (108) obtained in Lemma 14, then λmin(Ps) ≥κ−for all s < τ ∧t. In particular, τ > t. where C4(t) is the upper bound (108) obtained in Lemma 14, then λmin(Ps) ≥κ−for all s < τ ∧t. In particular, τ > t. Proof. We will use the representation λmin(Pt) = inf∥v∥=1⟨Ptv, v⟩. So ﬁx v with ∥v∥= 1. Then Proof. We will use the representation λmin(Pt) = inf∥v∥=1⟨Ptv, v⟩. So ﬁx v with ∥v∥= 1 Then (115) d dt⟨Ptv, v⟩= 2E h ⟨f( ˆXt) −¯ft, v⟩⟨ˆXt −¯xt, v⟩ i + 2⟨Dv, v⟩−⟨R−1QT t v, QT t v⟩. c⃝2018 SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license (100) Then (119) lim M→∞E " 1 M M X i=1 ∥Xi t −ˆXi t∥2 # = 0 . In particular, (120) lim M→∞ 1 M M X i=1 g(Xi t) −ˆπt[g] = 0 lim M→∞E " 1 M M X i=1 ∥Xi t −ˆXi t∥2 # = 0 . (119) In particular, ownloaded 08/17/18 to 134.225.109.120. Redistribution subject to CCBY license Downloaded 08/17/18 to 134.225.109.120. Redistribution subject to CCBY license (120) lim M→∞ 1 M M X i=1 g(Xi t) −ˆπt[g] = 0 (120) in L2(P), hence in probability, for any Lipschitz continuous function g. Here, the expectation is taken also w.r.t. the distribution of {Ys}. in L2(P), hence in probability, for any Lipschitz continuous function g. Here, the expectation is taken also w.r.t. the distribution of {Ys}. Remark 17. The last theorem implies by general theory that the empirical distribution ˆπM t , deﬁned in (8), of the extended EnKBF with M ensemble members converges weakly towards the distribution ˆπt of the mean ﬁeld process (11) in probability w.r.t. the distribution of {Ys}. Remark 18. The conditions of Theorem 16 are satisﬁed for fully observed processes h(x) = x, measurement error covariance matrix R = εI, ε > 0 suﬃciently small, and full rank diﬀusion tensor D, i.e., for the ﬁltering setting considered in sections 2 and 3. Proof of Theorem 16. Itˆo’s formula implies that ( ) Proof of Theorem 16. Itˆo’s formula implies that (121) d 1 M M X i=1 ∥∆Xi t∥2 ! = 2 M M X i=1 ⟨f(Xi t) −f( ˆXi t), ∆Xi t⟩dt + 2 M M X i=1 ⟨D (P M t )−1 Xi t −¯xM t −P−1 t ˆXi t −¯xt , ∆Xi t⟩dt −1 M M X i=1 ⟨QM t R−1 h(Xi t) + ¯hM t −QtR−1h( ˆXi t) + ¯ht , ∆Xi t⟩dt + 2 M M X i=1 ⟨ QM t −Qt R−1 dYt, ∆Xi t, ⟩ + 1 M tr QM t −Qt R−1 QM t −Qt T dt = I + · · · + V , with the abbreviation ∆Xi t = Xi t −ˆXi t. Our aim is to estimate the right-hand side of (121) in terms of 1 M PM i=1 ∥Xi t −ˆXi t∥2 and then to apply the Gronwall inequality. This requires, c⃝2018 SIAM. (100) Using d dt⟨Ptv, v⟩= 2E h ⟨f( ˆXt) −¯ft, v⟩⟨ˆXt −¯xt, v⟩ i + 2⟨Dv, v⟩−⟨R−1QT t v, QT t v⟩. ⟨Ptv, v⟩= E h ⟨ˆXt −¯xt, v⟩2i (116) ⟨Ptv, v⟩= E h ⟨ˆXt −¯xt, v⟩2i (116) and (117) ⟨R−1QT t v, QT t v⟩= ⟨R−1E (h( ˆXt) −¯ht)⟨ˆXt −¯xt, v⟩ , E (h( ˆXt) −¯ht)⟨ˆXt −¯xt, v⟩ ⟩ ≤∥R−1∥FE h ∥h( ˆXt) −¯ht∥2i E h ⟨ˆX −¯xt, v⟩2i ≤∥R−1∥F∥h∥2 Lip Var ˆXt ⟨Ptv, v⟩, (117) we can estimate we can estimate (118) d ) d dt⟨Ptv, v⟩≥−2∥f∥Lip Var( ˆXt) 1 2 ∥v∥⟨Ptv, v⟩ 1 2 + 2⟨Dv, v⟩−∥h∥2 Lip∥R−1∥FVar( ˆXt)⟨Ptv, v⟩ ≥−2∥f∥LipC4(t)1/2⟨Ptv, v⟩ 1 2 + 2⟨Dv, v⟩−∥h∥2 Lip∥R−1∥FC4(t)⟨Ptv, v⟩ ≥2λmin(D) − ∥f∥2 Lip ∥R−1∥F∥h∥2 Lip −2∥h∥2 Lip∥R−1∥FC4(t)⟨Ptv, v⟩. Now λmin(P0) ≥κ−implies that ⟨P0v, v⟩≥κ−and thus ⟨Psv, v⟩≥κ−for all s < τ ∧t. Hence λmin(Ps) ≥κ−> 0 for all s < τ ∧t so that τ > t, since otherwise lims↑τ λmin(Ps) = 0. Now λmin(P0) ≥κ−implies that ⟨P0v, v⟩≥κ−and thus ⟨Psv, v⟩≥κ−for all s < τ ∧t. Hence λmin(Ps) ≥κ−> 0 for all s < τ ∧t so that τ > t, since otherwise lims↑τ λmin(Ps) = 0. The lower bound on λmin(Pt), locally uniformly w.r.t. t, implies that the coeﬃcients of (11) are globally Lipschitz on bounded time-intervals, which gives existence and uniqueness of strong solutions by standard results for all t, a.s. (w.r.t. the distribution of {Ys}). c⃝2018 SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license JANA DE WILJES, SEBASTIAN REICH, AND WILHELM STANNAT 1172 5.2. Convergence of the extended EnKBF to the solution of (11). We are now ready to state our main result on the asymptotic behavior of the extended EnKBF. 5.2. Convergence of the extended EnKBF to the solution of (11). We are now ready to state our main result on the asymptotic behavior of the extended EnKBF. Theorem 16. Assume that ∥f∥2 Lip < 2λmin(D)∥R−1∥F∥h∥2 Lip. Let π0 be a distribution on RNx with ﬁnite support and invertible covariance matrix P0 satisfying λmin(P0) ≥κ−, where κ−is as in Lemma 15. Let ˆXi t be solutions of the mean-ﬁeld process (11) with initial conditions ˆXi 0 = Xi 0 and Xi 0 are i.i.d. (π0), so that the solutions ˆXi t to the mean ﬁeld processes are i.i.d., too. ⃝2018 SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license (100) Published by SIAM under the terms of the Creative Commons 4.0 license 1173 LONG-TIME BEHAVIOR OF THE ENSEMBLE KALMAN FILTER in particular, controlling the stochastic integral IV w.r.t. the observation {Ys}. Using the decomposition dYt = h(Xref t ) dt + R1/2dBt we can split up the stochastic integral IV into (122) 2 M M X i=1 ⟨ QM t −Qt R−1dYt, ∆Xi t⟩= 2 M M X i=1 ⟨ QM t −Qt R−1h Xref t , ∆Xi t⟩dt + 2 M M X i=1 ⟨ QM t −Qt R−1/2dBt, ∆Xi t⟩ (122) = IV a + IV b . = IV a + IV b . We can now estimate the right-hand side of the above equation for t ≤T from above as follows: (123) d 1 M M X i=1 ∥Xi t −ˆXi t∥2 ! ≤UM(t) 1 M M X i=1 ∥Xi t −ˆXi t∥2 + RM(t) ! dt + 2 M M X i=1 ⟨ QM t −Qt R−1/2dBt, Xi t −ˆXi t⟩, Downloaded 08/17/18 to 134.225.109.120. Redistribution subject to CCBY lic (123) thereby keeping the stochastic integral IV b. Here, thereby keeping the stochastic integral IV b. Here, (124) UM(t) = CT 1 + h Xref 0:T 2 ∞+ 1 M M X i=1 ∥ˆXi t∥2 ! × 1 + 1 M M X i=1 ∥Xi t −¯xM t ∥2 + 1 M M X i=1 ∥ˆXi t −¯xt∥2 ! , (124) with some ﬁnite constant CT , and a remainder RM(t) that converges to zero in Lp(P) as M →∞for all ﬁnite p. Indeed, this is obvious for term I, using that f is globally Lipschitz, for terms III, IVa, and V using (133) in Lemma 19 in the appendix and for term II it follows from (131) in Lemma 19 in the appendix in combination with (102). Applying Itˆo’s product formula to the process e− R t 0 UM(s)ds 1 M PM i=1 ∥Xi t −ˆXi t∥2 and taking expectations w.r.t. the distribution of {Ys}, we arrive at the following estimate: (125) E " e− R t 0 UM(s)ds 1 M M X i=1 ∥Xi t −ˆXi t∥2 # ≤CT E Z t 0 e− R s 0 UM(r)drUM(s)RM(s)ds (125) for t ≤T. ⃝2018 SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license ⃝2018 SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license (100) Since UMRM is bounded by some ﬁnite constant plus some power of 1 M PM i=1 ∥ˆXi t∥2 and the latter one has some ﬁnite exponential moment by Lemma 21, it follows that for t ≤T. Since UMRM is bounded by some ﬁnite constant plus some power of 1 M PM i=1 ∥ˆXi t∥2 and the latter one has some ﬁnite exponential moment by Lemma 21, it follows that (126) lim M→∞E " e−αT R t 0 1 M PM i=1 ∥ˆ Xi s∥2ds 1 M M X i=1 ∥Xi t −ˆXi t∥2 # = 0 , t ≤T , (126) c⃝2018 SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license Downloaded 08/17/18 to 134.225.109.120. Redistribution subject to CCBY license 25.109.120. Redistribution subject to CCBY license (128) f(x) =   10(x2 −x1) (28 −x3)x1 −x2 x1x2 −8 3x3  , (128) where x = (x1, x2, x3)T. Solutions of the Lorenz-63 system diverge exponentially fast and ﬁltering is required in order to track a reference solution. Although (128) is only locally Lipschitz continuous, the results from this paper are likely to be applicable to the Lorenz-63 system due to the existence of a Lyapunov function. where x = (x1, x2, x3)T. Solutions of the Lorenz-63 system diverge exponentially fast and ﬁltering is required in order to track a reference solution. Although (128) is only locally Lipschitz continuous, the results from this paper are likely to be applicable to the Lorenz-63 system due to the existence of a Lyapunov function. We apply the EnKBF with ensemble size M = 4 for values of the measurement error variances ε ∈{10−1, . . . , 10−4, 10−5}. The stochastic evolution equations of the EnKBF are solved by the following modiﬁed Euler-Maruyama scheme (129) ( ) Xi n+1 = Xi n + ∆tf(Xi n) + ∆t(P M n )−1(Xi n −¯xM n ) −1 2P M n P M n + ε ∆tI3 −1 Xi n + ¯xi n −2∆Yn ∆t with step-size ∆t = 0.00005 over a total of 107 time-steps. Note that P M n + ε ∆tI3 −1 ≈∆t ϵ I3 (130) P M n + ε ∆tI3 −1 ≈∆t ϵ I3 JANA DE WILJES, SEBASTIAN REICH, AND WILHELM STANNAT JANA DE WILJES, SEBASTIAN REICH, AND WILHELM STANNAT 1174 for some αT > 0. Now, using Lemma 21 again, we also may now conclude that (127) for some αT > 0. Now, using Lemma 21 again, we also may now conclude that (127) ) m →∞E " 1 M M X i=1 ∥Xi t −ˆXi t∥ #2 ≤sup M≥2 E h eαT R t 0 1 M PM i=1 ∥ˆ Xi s∥2dsi × lim M→∞E " e−αT R t 0 1 M PM i=1 ∥ˆ Xi s∥2ds 1 M M X i=1 ∥Xi t −ˆXi t∥2 # = 0 for all t ≤T. for all t ≤T. for all t ≤T. 6. Numerical example. We consider the stochastically perturbed Lorenz-63 system [Lor63, LSZ15], which leads to Nx = 3, D = C = I3, and drift term given by 6. Numerical example. We consider the stochastically perturbed Lorenz-63 system [Lor63, LSZ15], which leads to Nx = 3, D = C = I3, and drift term given by 6. Numerical example. We consider the stochastically perturbed Lorenz-63 system [Lor63, LSZ15], which leads to Nx = 3, D = C = I3, and drift term given by LONG-TIME BEHAVIOR OF THE ENSEMBLE KALMAN FILTER LONG-TIME BEHAVIOR OF THE ENSEMBLE KALMAN FILTER 1175 0 50 10 20 20 x3-coordinate 30 reference trajectory 10 x2-coordinate 40 0 x1-coordinate 50 0 -10 -50 -20 10-5 10-4 10-3 10-2 10-1 measurement noise variance 10-3 10-2 10-1 100 101 time-averaged mean squared error time-averaged mean squared error reference line with slope p =1/2 Figure 1. Reference trajectory (left panel) and time-averaged mean squared error as a function of the measurement error variance ε (right panel). time-averaged mean squared error Downloaded 08/17/18 to 134.225.109.120. Redistribution subject to CCBY license nloaded 08/17/18 to 134.225.109.120. Redistribution subject to CCBY license Figure 1. Reference trajectory (left panel) and time-averaged mean squared error as a function of the measurement error variance ε (right panel). 10-5 10-4 10-3 10-2 10-1 measurement noise variance 10-3 10-2 10-1 100 101 time-averaged largest eigenvalue of P t time-averaged largest eigenvalue reference line with slope p =1/2 10-5 10-4 10-3 10-2 10-1 measurement noise variance 10-3 10-2 10-1 100 time-averaged smallest eigenvalue of P t time-averaged smallest eigenvalue reference line with slope p =1/2 Figure 2. Time-averaged largest (left panel) and smallest (right panel) eigenvalues of Pt as a function of the measurement error variance ε 10-5 10-4 10-3 10-2 10-1 measurement noise variance 10-3 10-2 10-1 100 time-averaged smallest eigenvalue of P t time-averaged smallest eigenvalue reference line with slope p =1/2 10-5 10-4 10-3 10-2 10-1 measurement noise variance 10-3 10-2 10-1 100 101 time-averaged largest eigenvalue of P t time-averaged largest eigenvalue reference line with slope p =1/2 time-averaged largest eigenvalue of P t time-averaged largest eigenvalue of P t time-averaged smallest eigenvalue of P t Figure 2. Time-averaged largest (left panel) and smallest (right panel) eigenvalues of Pt as a function of the measurement error variance ε 7. Conclusions. In this paper, we have taken the ﬁrst steps towards an understanding of the long-time behavior of the EnKBF and have derived limiting mean-ﬁeld equations. Natural extensions include partially observed processes and conﬁgurations which lead to singular em- pirical covariance matrices P M t . We also plan to extend our analysis to other ensemble ﬁlter algorithms, such as the stochastically perturbed EnKBF and the ensemble transform particle ﬁlter. See, for example, [RC15] for more details. Appendix: Supplement to the proof of Theorem 16. (130) (130) for ∆t suﬃciently small and the modiﬁcation is introduced for numerical stability reasons. See [AKIR14] for more details. The results can be found in Figures 1 and 2. The numerical results are in agreement with our theoretical ﬁndings, which predicted an O(ε1/2) behavior of these quantities. While this scaling holds for the time-averaged mean squared error and the time-averaged largest eigenvalue of P M t for the whole range of considered values of ε, the time-averaged smallest eigenvalue truncates slightly oﬀfor the larger values of ε. We can also see that there is a gap between the smallest and largest eigenvalues of P M t on average. We repeated the experiment for ensemble sizes of M = 2 and M = 3, in which case P M t is singular. We still ﬁnd that the time-averaged mean squared error is roughly of O(ε1/2). See Figure 3. The results are in line with those obtained in [GTH13] for hyperbolic dynamical systems. We will further investigate the theoretical properties of the EnKBF under singular P M t in a separate paper. ⃝2018 SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license c⃝2018 SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license LONG-TIME BEHAVIOR OF THE ENSEMBLE KALMAN FILTER The purpose of this appendix is to provide two lemmata on the control of ∥P M t −Pt∥F and on the existence of exponential moments of R t 0 1 M PM i=1 ∥ˆXi s∥2ds used in the proof of Theorem 16. ⃝2018 SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license JANA DE WILJES, SEBASTIAN REICH, AND WILHELM STANNAT JANA DE WILJES, SEBASTIAN REICH, AND WILHELM STANNAT 1176 10-5 10-4 10-3 10-2 10-1 measurement noise variance 10-3 10-2 10-1 100 101 time-averaged mean squared error time-averaged mean squared error reference line with slope p =1/2 10-5 10-4 10-3 10-2 10-1 measurement noise variance 10-3 10-2 10-1 100 101 time-averaged mean squared error time-averaged mean squared error reference line with slope p =1/2 Figure 3. Time-averaged mean squared error as a function of the measurement error variance ε for ensemble sizes M = 2 (left panel) and M = 3 (right panel). 10-5 10-4 10-3 10-2 10-1 measurement noise variance 10-3 10-2 10-1 100 101 time-averaged mean squared error time-averaged mean squared error reference line with slope p =1/2 10-5 10-4 10-3 10-2 10-1 measurement noise variance 10-3 10-2 10-1 100 101 time-averaged mean squared error time-averaged mean squared error reference line with slope p =1/2 time-averaged mean squared error time-averaged mean squared error time-averaged mean squared error reference line with slope p =1/2 10-2 Figure 3. Time-averaged mean squared error as a function of the measurement error variance ε for ensemble sizes M = 2 (left panel) and M = 3 (right panel). Lemma 19. We have that Lemma 19. We have that (131) ∥P M t −Pt∥F ≤2Σ(t) 1 M −1 X i ∥Xi t −ˆXi t∥2 ! 1 2 + RM(t) (131) with limM→∞RM(t) = 0 a.s. and in L1(P). Here with limM→∞RM(t) = 0 a.s. and in L1(P). Here (132) Σ(t) := 1 M −1 X i ∥Xi t −¯xM t ∥2 ! 1 2 + 1 M −1 X i ∥ˆXi t −¯xt∥2 ! 1 2 . (132) Similarly, Similarly, (133) ∥QM t −Qt∥F ≤2(1 + ∥h∥Lip)Σ(t) 1 M −1 X i ∥Xi t −ˆXi t∥2 ! 1 2 + SM(t) (133) with limM→∞SM(t) = 0 a.s. and in L1(P). with limM→∞SM(t) = 0 a.s. and in L1(P). Remark 20. Note that the factor Σ(t) is locally bounded in t due to Lemma 14 and an appropriate generalization of Lemma 4. c⃝2018 SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license LONG-TIME BEHAVIOR OF THE ENSEMBLE KALMAN FILTER LONG-TIME BEHAVIOR OF THE ENSEMBLE KALMAN FILTER 1177 Proof of Lemma 19. First, note that we can decompose Proof of Lemma 19. First, note that we can decompose Proof of Lemma 19. First, note that we can decompose Proof of Lemma 19. First, note that we can decompose (134) P M t −Pt = 1 M −1 M X i=1 Xi t −¯xM t Xi t −¯xM t T −E ˆXt −¯xt ˆXt −¯xt T = 1 M −1 M X i=1 Xi t −¯xM t − ˆXi t −¯xt Xi t −¯xM t T + 1 M −1 M X i=1 ˆXi t −¯xt Xi t −¯xM t − ˆXi t −¯xt T + 1 M −1 M X i=1 ˆXi t −¯xt ˆXi t −¯xt T −E ˆXt −¯xt ˆXt −¯xt T (134) ownloaded 08/17/18 to 134.225.109.120. Redistribution subject to CCBY license = I + II + III. In particular, ∥P M t −Pt∥F ≤∥I∥F + ∥II∥F + ∥III∥F. Term I can be estimated from above by (135) In particular, ∥P M t −Pt∥F ≤∥I∥F + ∥II∥F + ∥III∥F. Term I can be estimated from above by (135) ( ) ∥I∥F ≤ 1 M −1 M X i=1 ∥Xi t −¯xM t − ˆXi t −¯xt ∥2 !1/2 1 M −1 M X i=1 ∥Xi t −¯xM t ∥2 !1/2 ≤   1 M −1 M X i=1 ∥Xi t −ˆXi t∥2 !1/2 + r M M −1∥xM t −¯xt∥   1 M −1 M X i=1 ∥Xi t −¯xM t ∥2 !1/2 ≤  2 1 M −1 M X i=1 ∥Xi t −ˆXi t∥2 !1/2 + r M M −1 1 M M X i=1 ˆXi t −E h ˆXi t i   × 1 M −1 M X i=1 ∥Xi t −¯xM t ∥2 !1/2 . Similarly !1/2 !1/2 Similarly, (136) ∥II∥F ≤  2 1 M −1 M X i=1 ∥Xi t −ˆXi t∥2 !1/2 + r M M −1 1 M M X i=1 ˆXi t −E h ˆXi t i   × 1 M −1 M X i=1 ∥ˆXi t −¯xt∥2 !1/2 . JANA DE WILJES, SEBASTIAN REICH, AND WILHELM STANNAT JANA DE WILJES, SEBASTIAN REICH, AND WILHELM STANNAT 1178 Adding up all terms we arrive at the estimate Adding up all terms we arrive at the estimate Adding up all terms we arrive at the estimate (138) ∥P M t −Pt∥F ≤2Σ(t) 1 M −1 M X i=1 ∥Xi t −ˆXi t∥2 !1/2 + RM(t) (138) with the remainder (139) RM(t) = Σ(t) r M M −1 1 M M X i=1 ˆXi t −E h ˆXi t i + ∥III∥F . (139) The strong law of large numbers now implies that limM→∞RM(t) = 0 in a.s. and in L1(P). The proof of the second estimate is done similarly. The strong law of large numbers now implies that limM→∞RM(t) = 0 in a.s. and in L1(P). The proof of the second estimate is done similarly. Lemma 21. Let ˆXi t, 1 ≤i ≤M, M ≥2, be the solution of (11) with initial conditions i.i.d. (π0) and suppose that π0 has bounded support contained in a ball with radius K. Then for all T > 0 there exist δ0 > 0 and κ0 > 0 depending on T, but independent of M, such that (140) E h eδ0 R t 0 1 M PM i=1 ∥ˆ Xi s∥2dsi ≤e2κ0 K2 M +∥h(Xref 0:T)∥ 2 ∞ < +∞ ∀t ≤T . (140) Here, the expectation is taken also w.r.t. the distribution of {Ys}. Proof. First, note that Itˆo’s formula and (11) imply that Proof. First, note that Itˆo’s formula and (11) imply that (141) d 1 M M X i=1 ∥ˆXi t∥2 ! = 2 M M X i=1 ⟨f ˆXi t , ˆXi t⟩dt + 2 M M X i=1 ⟨DP−1 t ˆXi t −¯xt, , ˆXi t⟩dt −1 M M X i=1 ⟨QtR−1h ˆXi t , ˆXi t⟩dt −1 M M X i=1 ⟨QtR−1¯ht, ˆXi t⟩dt + 2 M M X i=1 ⟨ˆXi t, QtR−1dYt⟩+ 1 M tr QtR−1Qt dt . (141) Using Lipschitz continuity of f and h and the Lemmata 14 and 15, the right-hand side can be estimated from above for t ≤T by Using Lipschitz continuity of f and h and the Lemmata 14 and 15, the right-hand side can be estimated from above for t ≤T by (142) C(T) 1 + 1 M M X i=1 ∥ˆXi t∥2 ! LONG-TIME BEHAVIOR OF THE ENSEMBLE KALMAN FILTER Fi ll ∥II∥F ≤  2 1 M −1 M X i=1 ∥Xi t −ˆXi t∥2 !1/2 + r M M −1 1 M M X i=1 ˆXi t −E h ˆXi t i   (136) (136)  i=1 ! × 1 M −1 M X i=1 ∥ˆXi t −¯xt∥2 !1/2 .  × 1 M −1 M X i=1 ∥ˆXi t −¯xt∥2 ! Finally, y (137) (137) ∥III∥F = 1 M M X i=1 ˆXi t( ˆXi t)T −E h ˆXi t( ˆXi t)Ti + 1 M(M −1) ˆXi t −¯xt ˆXi t −¯xt T F ≤ 1 M M X i=1 ˆXi t( ˆXi t)T −E h ˆXi t( ˆXi t)Ti F + 1 M 1 M −1 M X i=1 ˆXi t −¯xt ˆXi t −¯xt T F . c⃝2018 SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license c⃝2018 SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license c⃝2018 SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license c⃝2018 SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license JANA DE WILJES, SEBASTIAN REICH, AND WILHELM STANNAT + 2 M M X i=1 ⟨ˆXi t, QtR−1dYt⟩ (142) for some uniform constant C(T). Since dYt = h Xref t dt + R−1/2dBt we can further estimate from above for t ≤T for some uniform constant C(T). Since dYt = h Xref t dt + R−1/2dBt we can further estimate from above for t ≤T (143) C(T) 1 + h Xref t 2 + 1 M M X i=1 ∥ˆXi t∥2 ! + 2 M M X i=1 ⟨ˆXi t, QtR−1/2dBt⟩ (143) c⃝2018 SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license LONG-TIME BEHAVIOR OF THE ENSEMBLE KALMAN FILTER LONG-TIME BEHAVIOR OF THE ENSEMBLE KALMAN FILTER 1179 for some possibly diﬀerent constant C(T). Itˆo’s product rule now implies for α := 1 + C(T) and t ≤T for some possibly diﬀerent constant C(T). Itˆo’s product rule now implies for α := 1 + C(T) and t ≤T (144) d e−αt 1 M M X i=1 ∥ˆXi t∥2 ! ≤e−αtC(T) 1 + h Xref t 2 dt −e−αt 1 M M X i=1 ∥ˆXi t∥2 ! dt + e−αt 2 M M X i=1 ⟨ˆXi t, QtR−1/2dBt⟩, (144) (144) + e−αt 2 M M X i=1 ⟨ˆXi t, QtR−1/2dBt⟩, which implies that which implies that (145) Z t 0 e−αs 1 M M X i=1 ∥ˆXi s∥2ds ≤1 M M X i=1 ∥ˆXi 0∥2 + C(T) 1 + h Xref 0:T 2 ∞ + Z t 0 e−αs 2 M M X i=1 ⟨ˆXi s, QtR−1/2dBs⟩. (145) To simplify notation in the following, let To simplify notation in the following, let To simplify notation in the following, let (146) Mt := Z t 0 e−αs 2 M M X i=1 ⟨ˆXi s, QtR−1/2dBs⟩ (146) and observe that the quadratic variation ⟨M⟩t can be estimated from above by (147) ⟨M⟩t = 4 M2 M X i=1 Z t 0 e−2αs∥R−1/2QT s ˆXi s∥2ds ≤ 4∥R−1/2∥2 F∥h∥2 LipC(T)2 M Z t 0 e−αs 1 M M X i=1 ∥ˆXi s∥2ds , (147) (147) ≤ 4∥R−1/2∥2 F∥h∥2 LipC(T)2 M Z t 0 e−αs 1 M M X i=1 ∥ˆXi s∥2ds , (148) ∥Qs∥2 F ≤∥h∥2 LipE h ∥ˆXs −¯xs∥2i ≤∥h∥2 LipC(T)2 (148) and Lemma 14. The assumption on the initial condition now implies for δ > 0 and Lemma 14. ⃝2018 SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license JANA DE WILJES, SEBASTIAN REICH, AND WILHELM STANNAT 1180 thereby using the inequality thereby using the inequality thereby using the inequality (150) E h eδMti = E h e 1 2(2δMt−2δ2⟨M⟩t)e 1 2(2δ2⟨M⟩t)i ≤E h e2δMt−2δ2⟨M⟩ti1/2 E h e2δ2⟨M⟩ti1/2 = E h e2δ2⟨M⟩ti1/2 . (150) E h eδMti = E h e 1 2(2δMt−2δ2⟨M⟩t)e 1 2(2δ2⟨M⟩t)i ≤E h e2δMt−2δ2⟨M⟩ti1/2 E h e2δ2⟨M⟩ti1/2 = E h e2δ2⟨M⟩ti1/2 . (150) Hence for δ0 > 0 with Hence for δ0 > 0 with Hence for δ0 > 0 with (151) δ0 8∥R−1/2∥2 F∥h∥2 LipC(T)2 M < 1 (151) (152) E h eδ0 R t 0 e−αs 1 M PM i=1 ∥ˆ Xi s∥2dsi ≤e2δ0 K2 M +C(T) 1+∥h(Xref 0:T)∥ 2 ∞ < e2κ0 K2 M +∥h(Xref 0:T)∥ 2 ∞ < +∞ oaded 08/17/18 to 134.225.109.120. Redistribution subject to CCBY license for a suitable κ0 > 0. for a suitable κ0 > 0. c⃝2018 SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license LONG-TIME BEHAVIOR OF THE ENSEMBLE KALMAN FILTER The assumption on the initial condition now implies for δ > (149) E h eδ R t 0 e−αs 1 M PM i=1 ∥ˆ Xi s∥2 dsi ≤eδ K2 M +C(T) 1+∥h(Xref 0:T)∥ 2 ∞ E h eδMti ≤eδ K2 M +C(T) 1+∥h(Xref 0:T)∥ 2 ∞ E h e2δ2⟨M⟩ti1/2 ≤eδ K2 M +C(T) 1+∥h(Xref 0:T)∥ 2 ∞ (149) ≤eδ K2 M +C(T) 1+∥h(Xref 0:T)∥ 2 ∞ E h e2δ2⟨M⟩ti1/2 ≤eδ K2 M +C(T) 1+∥h(Xref 0:T)∥ 2 ∞ × E " e2δ 4∥R−1/2∥2 F∥h∥2 LipC(T )2 M δ R t 0 e−αs 1 M PM i=1 ∥ˆ Xi s∥2ds #1/2 , (149) ≤e × E " e2δ 4∥R−1/2∥2 F∥h∥2 LipC(T )2 M δ R t 0 e−αs 1 M PM i=1 ∥ˆ Xi s∥2ds #1/2 , c⃝2018 SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license JANA DE WILJES, SEBASTIAN REICH, AND WILHELM STANNAT c⃝2018 SIAM. Published by SIAM under the terms of the Creative Commons 4.0 licen REFERENCES [AKIR14] J. Amezcua, E. Kalnay, K. Ide, and S. Reich, Ensemble transform Kalman-Bucy ﬁlters, Q. J. R. Meteorol. Soc., 140 (2014), pp. 995–1004. 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Appl., 20 (1999), pp. 800–819, https://doi.org/10.1137/S0895479897330182. P. Del Moral, A. Kurtzmann, and J. Tugaut, On the Stability and the Uniform Propagation of Chaos of a Class of Extended Ensemble Kalman-Bucy Filters, Technical Report, INRIA Bordeaux Research Center, Talence, France, https://arxiv.org/abs/1606.08256, 2016. [Eve06] G. Evensen, Data Assimilation. The Ensemble Kalman Filter, Springer-Verlag, New York, 2006. F. Le Gland, V. Monbet, and V.D. Tran, Large sample asymptotics for the ensemble Kalman ﬁlter, in The Oxford Handbook of Nonlinear Filtering, Oxford University Press, Oxford, 2011, pp. 598–631. C. Gonz´alez-Tokman and B.R. Hunt, Ensemble data assimilation for hyperbolic systems, Phys. D, 243 (2013), pp. 128–142. A.H. Jazwinski, Stochastic Processes and Filtering Theory, Academic Press, New York, 1970. A.H. Jazwinski, Stochastic Processes and Filtering T D.T. Kelly, K.J.H. Law, and A. Stuart, Well-posedness and accuracy of the ensemb Kalman ﬁlter in discrete and continuous time, Nonlinearity, 27 (2014), pp. 2579–2604. E. Kwiatowski and J. Mandel, Convergence of the square root ensemble Kalman ﬁlter in the large ensemble limit, SIAM/ASA J. Uncertain. Quantif., 3 (2015), pp. 1–17, https://doi.org/ 10.1137/140965363. ⃝2018 SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license LONG-TIME BEHAVIOR OF THE ENSEMBLE KALMAN FILTER 1181 D. Kelly, A.J. Majda, and X.T. Tong, Concrete ensemble Kalman ﬁlters with rigorous catastrophic ﬁlter divergence, Proc. Natl. Acad. Sci. USA, 112 (2015), pp. 10589–10594. ( ) H. Kwakernaak and R. Sivan, Linear Optimal Control Systems, Wiley Interscience, New York, 1972. [Lor63] E.N. Lorenz, Deterministic non-periodic ﬂows, J. Atmos. Sci., 20 (1963), pp. 130–141. K. Law, A. Stuart, and K. Zygalakis, Data Assimilation: A Mathematical Introduction, Springer-Verlag, Cham, 2015. A. Majda and J. Harlim, Filtering Complex Turbulent Systems, Cambridge University Press, Cambridge, 2012. P. Del Moral, Mean Field Simulation for Monte Carlo Integration, CRC Press, Boca Raton, FL, 2013. Øksendal, Stochastic Diﬀerential Equations, 5th ed., D. Ocone and E. Pardoux, Asymptotic stability of the optimal ﬁlter with respect to its ini- tial condition, SIAM J. Control Optim., 34 (1996), pp. 226–243, https://doi.org/10.1137/ S0363012993256617. S. Reich and C.J. Cotter, Probabilistic Forecasting and Bayesian Data Assimilation, Cam- bridge University Press, New York, 2015. S. Reich, A dynamical systems framework for intermittent data assimilation, BIT, 51 (2011), pp. 235–249. F. Rellich, Perturbation Theory for Linear Operators, Gordon and Breach, New York, 1969. X.T. Tong, A.J. Majda, and D. Kelly, Nonlinear stability and ergodicity of ensemble based Kalman ﬁlters, Nonlinearity, 29 (2016), pp. 657–691. ( ) X.T. Tong, Performance Analysis of Local Ensemble Kalman Filter, Technical Report, National University of Singapore, Singapore, https://arxiv.org/abs/1705.10598, 2017. c⃝2018 SIAM. Published by SIAM under the terms of the Creative Commons 4.0 license
https://openalex.org/W3215636253	https://upcommons.upc.edu/bitstream/2117/359913/1/symmetry-13-02244.pdf	English	null	Racetrack Microtron—Pushing the Limits	Symmetry	2,021	cc-by	14,723	symmetry S S symmetry S S symmetry S S S S symmetry Article Maxim Borisov 1,2,, Andrey Ermakov 2,3, Vadim Khankin 2,3, Yuri Kubyshin 4 and Vasiliy Shvedunov 2,3,5 Symmetry 2021, 13, 2244. https:// doi.org/10.3390/sym13122244 Academic Editor: Vitalii A. Okorokov Keywords: electron accelerator; classical microtron; racetrack microtron; multisection LINAC; longi- tudinal phase space Maxim Borisov 1,2,, Andrey Ermakov 2,3, Vadim Khankin 2,3, Yuri Kubyshin 4 and Vasiliy Shvedunov 2,3,5 Maxim Borisov 1,2,, Andrey Ermakov 2,3, Vadim Khankin 2,3, Yuri Kubyshin 4 and Vasiliy Shvedunov 2,3,5 1 Faculty of Physics, Lomonosov Moscow State University, Leninskie Gory, 119991 Moscow, Russia y y y y 2 Laboratory of Electron Accelerators MSU Ltd., Leninskie Gory, 119992 Moscow, Russia; a_ermak1978@mail.ru (A.E.); v-k32@yandex.ru (V.K.); shved@depni.sinp.msu.ru (V.S.) 3 2 Laboratory of Electron Accelerators MSU Ltd., Leninskie Gory, 119992 Moscow, Russia; a ermak1978@mail.ru (A.E.); v-k32@yandex.ru (V.K.); shved@depni.sinp.msu.ru (V.S.) Laboratory of Electron Accelerators MSU Ltd., Leninskie Gory, 119992 Moscow, Russia; a_ermak1978@mail.ru (A.E.); v-k32@yandex.ru (V.K.); shved@depni.sinp.msu.ru (V.S.) 3 Skobeltsyn Institute of Nuclear Physics, Lomonosov Moscow State University, Leninskie Gory, 119991 Moscow, Russia ( ) y ( ) p p ( ) 3 Skobeltsyn Institute of Nuclear Physics, Lomonosov Moscow State University, Leninskie Gory, 119991 Moscow, Russia 4 Institute of Energy Technologies, Technical University of Catalonia, 08028 Barcelona, Spain; iouri.koubychine@upc.edu 4 Institute of Energy Technologies, Technical University of Catalonia, 08028 Barcelona, Spain; iouri.koubychine@upc.edu y p 5 P.N. Lebedev Physical Institute of the Russian Academy of Sciences, Leninskiy Prospect 53, 119991 Moscow, Russia 5 P.N. Lebedev Physical Institute of the Russian Academy of Sciences, Leninskiy Prospect 53, 119991 Moscow, Russia Correspondence: borisov.ma14@physics.msu.ru Abstract: We consider three types of electron accelerators that can be used for various applications, such as industrial, medical, cargo inspection, and isotope production applications, and that require small- and medium-sized machines, namely classical microtron (CM), race-track microtron (RTM), and multisection linac. We review the principles of their operation, the speciﬁc features of the beam dynamics in these machines, discuss their advantages and weak points, and compare their technical characteristics. In particular, we emphasize the intrinsic symmetry of the stability region of microtrons. We argue that RTMs can be a preferable choice for medium energies (up to 100 MeV) and that the range of their potential applications can be widened, provided that the beam current losses are signiﬁcantly reduced. In the article, we analyze two possible solutions in detail, namely increasing the longitudinal acceptance of an RTM using a higher-order harmonic accelerating structure and improving beam matching at the injection. Citation: Borisov, M.; Ermakov, A.; Khankin, V.; Kubyshin, Y.; Shvedunov, V. Racetrack Microtron—Pushing the Limits. Symmetry 2021, 13, 2244. https:// doi.org/10.3390/sym13122244 Academic Editor: Vitalii A. Okorokov Received: 25 October 2021 Accepted: 17 November 2021 Published: 24 November 2021 Citation: Borisov, M.; Ermakov, A.; Khankin, V.; Kubyshin, Y.; Shvedunov, V. Racetrack Microtron—Pushing the Limits. 1. Introduction https://doi.org/10.3390/sym13122244 Symmetry 2021, 13, 2244 2 of 17 of this hem to structures, is the MAMI cascade of three RTMs and one double-sided continuous wave (CW) microtron with normal conducting accelerating structures, which have a ﬁnal energy of 1.5 GeV and an average current of 100 µA for fundamental research in the ﬁeld of nuclear physics [8–11]. energy of several tens of MeV; these parameters are necessary for the production of med ical isotopes through the use of photonuclear reactions [12–14]. Particular emphasis can be placed on methods for reducing beam current losses during acceleration by increasing the longitudinal acceptance and the better matching of the injected longitudinal beam emittance with the RTM acceptance. structures, is the MAMI cascade of three RTMs and one double-sided continuous wave (CW) microtron with normal conducting accelerating structures, which have a ﬁnal energy of 1.5 GeV and an average current of 100 µA for fundamental research in the ﬁeld of nuclear physics [8–11]. energy of several tens of MeV; these parameters are necessary for the production of med ical isotopes through the use of photonuclear reactions [12–14]. Particular emphasis can be placed on methods for reducing beam current losses during acceleration by increasing the longitudinal acceptance and the better matching of the injected longitudinal beam emittance with the RTM acceptance. Figure 1. (a) Classical microtron. (b) Racetrack microtron. (c) Multisection linear accelerator. (1)—electron gun (electron source), (2)—accelerating structure (resonator), (3)—RF energy source (magnetron, klystron) with a high-voltage power supply (modulator), (4)—bending magnet(s), (5)—beam extraction magnet (or system), (6)—injection magnet, (7)—quad- rupole lenses. Figure 1. (a) Classical microtron. (b) Racetrack microtron. (c) Multisection linear accelerator. (1)—electron gun (elec- tron source), (2)—accelerating structure (resonator), (3)—RF energy source (magnetron, klystron) with a high-voltage power supply (modulator), (4)—bending magnet(s), (5)—beam extraction magnet (or system), (6)—injection magnet, (7)—quadrupole lenses. Figure 1. (a) Classical microtron. (b) Racetrack microtron. (c) Multisection linear accelerator. (1)—electron gun (electron source), (2)—accelerating structure (resonator), (3)—RF energy source (magnetron, klystron) with a high-voltage power supply (modulator), (4)—bending magnet(s), (5)—beam extraction magnet (or system), (6)—injection magnet, (7)—quad- rupole lenses. Figure 1. (a) Classical microtron. (b) Racetrack microtron. (c) Multisection linear accelerator. (1)—electron gun (elec- tron source), (2)—accelerating structure (resonator), (3)—RF energy source (magnetron, klystron) with a high-voltage power supply (modulator), (4)—bending magnet(s), (5)—beam extraction magnet (or system), (6)—injection magnet, (7)—quadrupole lenses. 1. Introduction This paper is based on the experience of developing and constructing various types of RTM at the Skobeltsyn Institute of Nuclear Physics at Lomonosov Moscow State Uni- versity in cooperation with research groups from Russia and other countries [10,15–20]. 2. Comparison of Classical and Racetrack Microtrons 2.1. RTM Advantages The following features are usually indicated when comparing CM and RTM: Because of the design geometry in a CM, only one resonator with a moderate electric field strength can be installed (see Figure 1a); therefore, the synchronous energy gain per turn ∆ܧ௦ is quite low and is typically equal to the electron rest mass, ∆ܧ௦≈0.5 MeV. In the case of the CMs and RTMs for the resonance conditions of acceleration to be In this article, we compare CMs, RTMs, and multisection linear accelerators (LINACs), such as those shown schematically in Figure 1, in terms of design complexity, dimensions, cost, and capability of achieving the main beam parameters such as energy, energy spread, pulse and average current, and beam stability. The ultimate goal of this analysis is to formulate lines of improvement for pulsed RTMs that would allow them to produce beams with an average beam current that is higher than 100 µA and with an energy of several tens of MeV; these parameters are necessary for the production of medical isotopes through the use of photonuclear reactions [12–14]. Particular emphasis can be placed on methods for reducing beam current losses during acceleration by increasing the longitudinal acceptance and the better matching of the injected longitudinal beam emittance with the RTM acceptance. fulfilled, the magnetic field ܤ଴ of the bending magnet and the synchronous energy gain per turn must be related through the following formula [3–5]: 2ߨ∆ܧ p This paper is based on the experience of developing and constructing various types of RTM at the Skobeltsyn Institute of Nuclear Physics at Lomonosov Moscow State University in cooperation with research groups from Russia and other countries [10,15–20]. 1. Introduction Since its invention, the pulsed racetrack microtron (RTM) has been the attracting atten- tion of potential users due to the possibility of its use resulting in obtaining an accelerated electron beam of sufﬁciently high energy using a seemingly simple, compact, and quite economic machine. However, the number of RTMs built and that are in operation is in- comparably less than the number of linear accelerators, which have an average accelerated beam current that is too low for many potential applications. In the article, we analyze the problems that led to this situation and outline solutions that are aimed at increasing the average current of RTMs. Received: 25 October 2021 Accepted: 17 November 2021 Published: 24 November 2021 Received: 25 October 2021 Accepted: 17 November 2021 Published: 24 November 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. g The idea of a classical microtron (CM) (Figure 1a) was ﬁrst proposed in 1944 in the pioneering article by V.I. Veksler that was devoted to the principle of phase stability in cyclic accelerators [1]. The idea of an RTM (Figure 1b), as indicated in Ref. [2], belongs to J.S. Schwinger. The main period of CM and RTM development fell within the 1960s–1990s of the last century. There is a number of books and review articles in which various versions of these types of accelerators are described in detail and where references to original articles are given, see, for example, Refs. [3–7]. The majority of CM and RTM applications are in the ﬁelds of nuclear physics research, radiation therapy, radiography, injection into syn- chrotrons, gamma-activation analysis, research on the production of medical isotopes, and the detection of explosives. The pinnacle of microtron development, which is unlikely to be surpassed because of the development of the technology of superconducting accelerating Copyright: © 2021 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/symmetry Symmetry 2021, 13, 2244. ܤ଴= ݁ߥܿߣ, where e is the elementary electric charge, λ is the wavel 2. Comparison of Classical and Racetrack Microtrons 2.1. RTM Advantages For an energy of, for example, 50 MeV, the number of orbits will be close to 100, the last orbit radius will be approximately 1.7 m, and the outer diameter of the magnet will be about 4 m. In practice, CMs are operated at larger value of the synchronous energy gain, which makes it possible to decrease the dimensions of the accelerator slightly. However, this also reduces the stability of the resonator operation, and for an energy of tens of MeV, the dimensions of the accelerator and the number of orbits are still large. In an RTM, a multi-cavity accelerating structure can be placed in the drift space between the bending magnets (see Figure 1b) so that the energy gain per turn can be increased considerably. Therefore, if, for example, ∆Es = 5 MeV, ν = 1, λ = 0.1 m, and the ﬁnal energy is 50 MeV, then the magnetic ﬁeld will increase to 1 T, and consequently, the maximum orbit radius will decrease to 0.17 m. Thus, the RTM design makes it possible to build signiﬁcantly more compact accelerators that are able to produce higher energy beams. Another advantage of RTMs is the possibility of implementing various beam focusing schemes. In CMs, the problem of focusing in the vertical plane (a plane perpendicular to the plane of the orbits) is partially solved by introducing RF quadrupole focusing by means of elliptical resonator beam holes [22,23]. The main disadvantages of this approach are the dependence of the optical power on the phase of the particle relative to the accelerating ﬁeld that leads to the coupling of the longitudinal and transverse planes and the appearance of defocusing in the horizontal plane. RTM design creates different possibilities for electron injection, thus being more ﬂexible than the CM design. In fact, a large number of injection schemes for both low- energy beams from an electron gun and relativistic beams from a linear pre-accelerator can be implemented in RTMs. To stress the importance of optimal injection, we would like to note that CMs became widespread in various applications after the invention of new injection schemes in the 1960s and 1970s that made it possible to increase the accelerated beam current signiﬁcantly [24]. ܤ଴= ݁ߥܿߣ, where e is the elementary electric charge, λ is the wavel 2. Comparison of Classical and Racetrack Microtrons 2.1. RTM Advantages Finally, it is straightforward to obtain electrons of different energies in an RTM by simply extracting the beam from the different orbits; a review of these extraction schemes can be seen in Ref. [4]. ܤ଴= ݁ߥܿߣ, where e is the elementary electric charge, λ is the wavel 2. Comparison of Classical and Racetrack Microtrons 2.1. RTM Advantages free space, ν is the increase of the harmonic number per turn, and c is the speed of light. The following features are usually indicated when comparing CM and RTM: Because of the design geometry in a CM, only one resonator with a moderate electric ﬁeld strength can be installed (see Figure 1a); therefore, the synchronous energy gain per turn ∆Es is quite low and is typically equal to the electron rest mass, ∆Es ≈0.5 MeV. In the case of the CMs and RTMs for the resonance conditions of acceleration to be fulﬁlled, the magnetic ﬁeld B0 of the bending magnet and the synchronous energy gain per turn must be related through the following formula [3–5]: B0 = 2π∆Es eνcλ , (1) (1) where e is the elementary electric charge, λ is the wavelength of the accelerating ﬁeld in free space, ν is the increase of the harmonic number per turn, and c is the speed of light. We would like to note that ν is essentially the number of the accelerating ﬁeld wavelengths where e is the elementary electric charge, λ is the wavelength of the accelerating ﬁeld in free space, ν is the increase of the harmonic number per turn, and c is the speed of light. We would like to note that ν is essentially the number of the accelerating ﬁeld wavelengths Symmetry 2021, 13, 2244 3 of 17 3 of 17 by which the length of the orbit increases from turn to turn. The maximum width of the region of stable phase oscillations is achieved for ν = 1 [21]. Thus, for a typical value of λ = 0.1 m, the ﬁeld in the CM magnet is B0 ≈0.1 T. For an energy of, for example, 50 MeV, the number of orbits will be close to 100, the last orbit radius will be approximately 1.7 m, and the outer diameter of the magnet will be about 4 m. by which the length of the orbit increases from turn to turn. The maximum width of the region of stable phase oscillations is achieved for ν = 1 [21]. Thus, for a typical value of λ = 0.1 m, the ﬁeld in the CM magnet is B0 ≈0.1 T. 2.2. Common Problems of CM and RTM CM and RTM share a number of common problems. The most serious one is the narrow stable phase oscillation region. For ν = 1, the region of stability is at its maximum for the synchronous phase ϕs = 16◦and is of only 32◦width in phase, whereas its size in energy is given by \|δEn/∆Es\| < 0.05. Here, δEn = En −Es,n is the deviation of the beam energy at the nth orbit from the synchronous particle energy Es,n = Einj + n∆Es, and Einj is the injection energy. The problem that is caused by the stable phase oscillation region being narrow is aggravated by the presence of nonlinear resonances. The most dangerous nonlinear resonances causing beam losses are the third-order and forth-order resonances situated for ν = 1 at the synchronous phase values ϕs = 25.5◦and ϕs = 17.7◦, respectively. If the number of orbits is not small, 10 or more, and if ϕs is close to the resonance, then the stable phase oscillation region narrows sharply [25–27]. Previous studies have also revealed quite non-trivial properties of the stable phase oscillation region in RTMs. It turns out to be symmetrical with respect to certain symmetry lines, whose shape is determined by the accelerating voltage as a function of the phase. Additionally, the stability region includes stable elliptic islands that are separated from the longitudinal acceptance which, in terms of the theory of dynamical systems, is a connected to a local stability domain that includes the synchronous trajectory (see Ref. [27] for details). Symmetry 2021, 13, 2244 4 of 17 Within the framework of the present work, we studied a possibility of expanding the region of stable phase oscillations by linearizing the time dependence of the accelerating ﬁeld by adding a second harmonic (see Section 4.1). Another problem that is common for both types of accelerators is a high-current longitudinal instability that can occur for a high total load of the resonator or accelerating structure by the beam current [28–30]. To a certain extent, this instability is similar to the Robinson instability in synchrotrons [31], but there are also signiﬁcant differences: ﬁrst, in CMs and RTMs the accelerating structure is simultaneously loaded by the beam current from all of the orbits and second, the stable phase oscillation region in the microtrons is considerably smaller than that of the synchrotrons. y y In Ref. 2.2. Common Problems of CM and RTM [30], a model describing the behavior of a beam current-loaded accelerating structure fed by a klystron and longitudinal beam dynamics in an RTM is considered. Computer simulations show that the threshold current is 20–30 mA with an instability development time of the order of 1 µs for a 70 MeV pulsed RTM with 14 orbits. The threshold current can be increased by reducing the effective shunt impedance of the accel- erating structure, diminishing the number of orbits, optimizing the coupling factor of the accelerating structure with the feeding waveguide, and increasing the size of the effective aperture (i.e., the aperture that takes into account ﬁeld errors and the misalignments of machine elements) in the horizontal plane on the return orbits. Finally, the acceleration of bunches with a large charge and a small phase length both in CMs and RTMs leads to an increase of the beam emittance due to the coherent synchrotron radiation in the bending magnets [32]. According to results obtained in Refs. [16,32] for the wavelengths of λ~0.1 m, if no special measures to suppress this radiation are taken, then the upper limit of the bunch charge for which no signiﬁcant increase of the emittance occurs is about 10 pC. This corresponds to a pulse current of approximately 30 mA. The suppression of the coherent synchrotron radiation can be achieved by reducing the height of the vacuum chamber section inside of the bending magnets to a value close to the bunch length [33]. However, in this case, the vertical aperture may decrease, leading to a decrease of the transverse acceptance of the accelerator. 2.3. RTM Problems As it has been explained above, dividing the CM magnet into two 180◦dipoles and separating them by a drift space allows a multi-cavity accelerating structure to be placed within a common orbit. However, this also leads to new problems that complicate the beam dynamics and that limit the value of the accelerated beam current in RTMs. We consider these problems below. 2.3.1. Beam Defocusing and Distortion by the Magnet Fringe Field 2.3.1. Beam Defocusing and Distortion by the Magnet Fringe Field In contrast to the CM magnet, the RTM magnets have a fringe ﬁeld that exerts a strong defocusing effect in the vertical plane on the beam. To compensate it, an additional narrow dipole magnet is installed at the entrance of the main bending magnet that has a ﬁeld of polarization that is opposite to that of the main ﬁeld [34]. The focal length f0 of this magnetic system is determined by the expression [8]: 1 f0 = R l 0(B(z)/B0 )2dz −R l 0(B(z)/B0 )dz R2 (2) (2) where B(z) is the magnetic ﬁeld proﬁle as a function of the distance z = 0 . . . l in the direction orthogonal to the edge of the magnet, and B0 and R are the value of the ﬁeld and bending radius in the region of the uniform ﬁeld of the main dipole, respectively. g g y By properly selecting the position, shape, and magnitude of the inverse ﬁeld, it is possible to adjust the focal length of the 2-dipole magnetic system over a wide range. It is also possible to achieve the closure of the orbit and reﬂection of the beam back to the accelerating structure after the ﬁrst acceleration, and in this way, it is possible solve Symmetry 2021, 13, 2244 5 of 17 beam the problem of the beam bypassing the accelerating structure in RTMs; see Ref. [4] for more details. length from the median plane given by the formula [8]: 2 ଶ׬ ቀܤᇱ(ݖ)ൗ ቁ ଶ ݀ ௟ The beam defocusing the fringe ﬁeld also causes the nonlinear distortion of the beam phase space due to aberrations and an increase in the vertical effective emittance. One of the manifestations of this effect is the contribution to the deviation of the quadratic focal length from the median plane given by the formula [8]: 1 ݂ଵ = 2ݕଶ׬ ቀܤ(ݖ) ܤ଴ ൗ ቁ݀ݖ ௟ ଴ 3ܴଶ (3) where y is the particle vertical displacement and ܤᇱ(ݖ) is derivative of the magnetic field profile It is clear that generally the aberrations in the magnetic system with an inverse 1 f1 = 2y2 R l 0(B′(z)/B0)2dz 3R2 (3) y g y than in an ordinary main bending magnet. 2.3.1. Beam Defocusing and Distortion by the Magnet Fringe Field Indeed, the inte- former case, there are two regions with varying magnetic ro derivative, namely the main magnet fringe field region (3) e netic i where y is the particle vertical displacement and B′(z) is derivative of the magnetic ﬁeld proﬁle. It is clear that generally, the aberrations in the magnetic system with an inverse dipole will be much stronger than in an ordinary main bending magnet. Indeed, the integrand is positive, and in the former case, there are two regions with varying magnetic ﬁelds and therefore a non-zero derivative, namely the main magnet fringe ﬁeld region and the inverse ﬁeld one, whereas in the latter case, there is just one fringe ﬁeld region. and the inverse field one, whereas in the latter case, there is just one fringe field region. The contribution of aberrations to the emittance growth is determined by the ratio of the vertical beam size ߪ௬ to the pole gap height h. For example, in the cascade of high energy CW RTMs [8–11], ߪ௬≪ℎ, and as a consequence, the aberrations are small. How- ever, an increase in the pole gap height leads to a proportional increase of the current in the magnet coils and an increase in the size and mass of the magnet that is not acceptable The contribution of aberrations to the emittance growth is determined by the ratio of the vertical beam size σy to the pole gap height h. For example, in the cascade of high energy CW RTMs [8–11], σy ≪h, and as a consequence, the aberrations are small. However, an increase in the pole gap height leads to a proportional increase of the current in the magnet coils and an increase in the size and mass of the magnet that is not acceptable in the case of accelerators for applied purposes because it results in a signiﬁcant increase in the size and cost of the whole machine. Therefore, for energies of up to 100 MeV in pulsed RTMs, a usual choice is h = 2–3 cm with the vertical beam size in the region of the fringe ﬁeld reaching σy ≈1 cm, leading to strong nonlinear distortions of the phase space and noticeable losses of the beam current. in the case of accelerators for applied purposes because it results in a significant increase in the size and cost of the whole machine. 2.3.1. Beam Defocusing and Distortion by the Magnet Fringe Field Therefore, for energies of up to 100 MeV in pulsed RTMs, a usual choice is h = 2–3 cm with the vertical beam size in the region of the fringe field reaching ߪ௬≈ 1 cm, leading to strong nonlinear distortions of the phase space and noticeable losses of the beam current. As an example, in Figure 2, the results of the numerical simulations of the beam dy- namics in the 55 MeV RTM exit are shown [18]. In this case, the pole gap height of the magnets is h = 2 cm. Nonlinear distortions of the phase space are clearly seen in the vertical plane. The results discussed here show that when choosing the shape of the fringe field, one As an example, in Figure 2, the results of the numerical simulations of the beam dynamics in the 55 MeV RTM exit are shown [18]. In this case, the pole gap height of the magnets is h = 2 cm. Nonlinear distortions of the phase space are clearly seen in the vertical plane. g p g , should minimize the aberrations given by Equation (3). Moreover, the pole gap height should be at least 3 cm, and the optics of the RTM should be optimized with the minimi- zation criteria for the vertical beam size and should be in the region of the fringe field of the magnets. (a) (b) Figure 2. Results of numerical simulations of the beam dynamics of the 55 MeV RTM [18]; (a) phase space in the vertical plane, (b) beam spot at the RTM exit. The pole gap height of the magnet is 2 cm. (a) (a) (b) (b) (b) (a) Figure 2. Results of numerical simulations of the beam dynamics of the 55 MeV RTM [18]; (a) phase space in the vertical plane, (b) beam spot at the RTM exit. The pole gap height of the magnet is 2 cm. The results discussed here show that when choosing the shape of the fringe ﬁeld, one should minimize the aberrations given by Equation (3). Moreover, the pole gap height should be at least 3 cm, and the optics of the RTM should be optimized with the The results discussed here show that when choosing the shape of the fringe ﬁeld, one should minimize the aberrations given by Equation (3). 2.3.1. Beam Defocusing and Distortion by the Magnet Fringe Field Moreover, the pole gap height should be at least 3 cm, and the optics of the RTM should be optimized with the Symmetry 2021, 13, 2244 6 of 17 minimization criteria for the vertical beam size and should be in the region of the fringe ﬁeld of the magnets. minimization criteria for the vertical beam size and should be in the region of the fringe ﬁeld of the magnets. 2.3.2. Phase Slip Because of the presence of the drift space between the magnets, electrons with even a slight deviation in their velocity from the speed of light experience a signiﬁcant phase slip with respect to the synchronous phase. The slip decreases from orbit to orbit, as the electron energy increases and approaches ultrarelativistic values. In addition, the fringe ﬁeld of the bending magnets causes a deviation in the electron trajectory from the ideal circular ﬁeld, which also contributes to the phase shift that changes from orbit to orbit. The variable phase slip in the drift space and phase shift in the fringe ﬁeld limit the minimum allowable energy of electrons in the ﬁrst orbit, which is determined by the length of the drift space and the shape of the fringe ﬁeld. As shown in Refs. [35,36], if the energy of the electrons in the ﬁrst orbit exceeds this threshold value, then it is possible to provide an asymptotic approach of the phase of a particle to the synchronous phase ϕs through the correct choice of the injection phase. j p Mitigating the phase slip effect of a low-energy beam injected from an electron gun can be achieved in a scheme with reﬂection back to the beam in the bending magnet after the ﬁrst acceleration and the subsequent acceleration of that beam to an energy that is approximately equal to twice the synchronous energy gain. The beam of this energy will be further accelerated with less phase slip. The exact value of the energy and injection phase of bunches into the microtron acceleration regime are controlled by the choice of the position of the bending magnet that reﬂects the beam (see, for example, [4] and references therein). g g ( p ) A phase shifter installed in the ﬁrst return orbit can be used to correct the phase slip effect. Since insufﬁcient beam energy leads to a lag of the bunch phase with respect to the optimal one, such a device should change the phase by more than 360◦. A simple chikane cannot provide a phase shift that is this large. In Ref. [37], a phase shifter that was able to control the bunch phase over a large range was described, and its successful implementation in the 70 MeV RTM was reported in Ref. [17]. 2.3.3. Errors in Magnets Manufacturing and Magnet Poles Positioning 2.3.3. Errors in Magnets Manufacturing and Magnet Poles Positioning The poles of a CM magnet can be manufactured and positioned relative to each other with high accuracy that, in principle, allows high ﬁeld uniformity to be obtained in the entire working area and a high degree of symmetry with respect to the vertical plane passing through the center of the resonator gap and the center of the magnet. Nevertheless, in practice, a problem related to the non-uniformity of the ﬁeld exists due to inaccuracies in the manufacturing and assembling of the magnet, the effects of steel saturation, the inhomogeneity of its magnetic properties, and the deformation of the poles under the inﬂuence of atmospheric pressure as well as other factors that require the use of correcting coils or the introduction of shims [7]. In the case of RTMs, the situation is noticeably more complicated. In the presence of a drift space of about 1 m, an error in the bending angle of 1 mrad caused by the ﬁrst magnet of an electron after crossing the drift space arrives at the entrance of the second magnet with a displacement of about 1 mm. Such displacements grow from turn to turn, resulting with a complete beam loss. It is important to keep in mind that in a 180◦bending magnet, an error of 1 mrad means a relative difference of the ﬁeld integrals along the two halves of the trajectory in the magnet at a level of just 3 × 10−4. Usually, in RTMs for applied purposes, the ﬁeld inhomogeneity that is primarily caused by edge effects and a strayed ﬁeld in the drift space is noticeably higher [38]. In addition to errors in the bending angle, other errors that are caused by imprecisions in the magnet positioning are of great importance. g In RTMs, beam steering along the orbits is often provided by pairs of steering coils that displace the beam in both the horizontal and vertical planes. To correct both the offset and slope of the beam trajectory, two pairs of coils need to be installed in each orbit. This Symmetry 2021, 13, 2244 7 of 17 inevitably leads to the appearance of apertures in the drift space between the magnets, on which the beam current can be lost. Keep in mind that these are sections with a signiﬁcant increase in the horizontal beam size due to the dispersion. 2.3.4. Beam Blow-Up Along with the aforementioned longitudinal instability, which can arise in both CMs and RTMs, the RTM may also exhibit transverse instability, namely a beam blow-up that is associated with an excitation of parasitic modes with a transverse magnetic ﬁeld on the axis in the accelerating structure. The threshold current for this instability depends on the effective shunt impedance of the parasitic modes, the optics of the RTM, and a number of other parameters [39,40]. p Accurate estimates of the threshold current and for the time in which instability devel- ops for speciﬁc characteristics of the RTM and its accelerating structure can be obtained by numerical simulations using codes such as, for example, HBBU (stationary case) [40] and TDBBU (time evolution of the process) [41]. In Ref. [40], the following formula is given for the conservative estimate of the threshold current for the stationary case obtained under the assumption of the resonance between the parasitic mode and transverse beam oscillations: Ithr = 4∆Esλtr rtrπLβ f × 1 Nln Ef /Ei (4) (4) where λtr is the wavelength of the parasitic mode with a transverse magnetic ﬁeld on the axis, rtr is its effective shunt impedance per unit length, L is the length of the accelerating structure, β f is the value of the RTM beta-function at the center of the accelerating structure, N is the number of orbits, Ei is the injection energy, and Ef is the energy at the exit of the accelerator. The beta function depends on the RTM’s optical scheme, which should have the relationship β f ∼= (0.5 −2)C, where C is the orbit length, which can be used for estimates. As an example, let us consider the 55 MeV pulsed RTM described in Ref. [18]. Its parameters are ∆Es = 5 MeV, L = 0.4 m, N = 9, β f = 3 m, λtr = 0.06 m, rtr = 18 MΩ/m, Ef = 55 MeV, and Ei = 10 MeV in the RTM mode. In this case, after the ﬁrst acceleration, a 5 MeV beam is reﬂected back into the accelerating structure by the bending magnet and is consequently accelerated to 10 MeV energy. Equation (4) provides the estimate Ithr ≈7 mA. A more accurate result for the threshold current obtained in Ref. 2.3.3. Errors in Magnets Manufacturing and Magnet Poles Positioning The mitigation of the effects of these errors is achieved by increasing the accuracy of manufacturing and assembling the magnets and the supports on which they are positioned, the better alignment of the magnets, improving the ﬁeld uniformity, and expanding the uniform ﬁeld region by shimming. Active and passive magnetic screens to control the strayed ﬁeld in the drift space are also used. Other important aspects of the design, building, and adjusting of an RTM magnetic system include high accuracy ﬁeld measurements in manufactured magnets and beam tracing in the measured ﬁelds that is conducted using existing programs for numerical simulations of the beam dynamics that take into account the position of the axis of the accelerating structure [38]. If necessary, the pole shimming is used as a local ﬁeld correction technique. Finally, the design of the beam steering coils and their placement should only limit the aperture in the horizontal and vertical planes minimally. 2.3.4. Beam Blow-Up [40] through the numerical simulations with the real optics parameters for the RTM produces a value that exceeds the conservative estimate by about an order of magnitude. Hence, in the design of a concrete RTM project, in order to achieve estimates for the current and time of instability development thresholds, it is advisable to rely on numerical simulations of the beam blow-up phenomenon. Symmetry 2021, 13, 2244 8 of 17 3. Comparison of RTM and LINAC An RTM can be regarded as a LINAC with non-isochronous beam recirculation in many aspects. In this respect, an RTM with N orbits replaces a LINAC with an accelerating structure with N sections, each section being fed by a separate klystron (see Figure 1b,c). Thus, there is an obvious gain in the size of the accelerator and, possibly, in its cost, since the cost of a klystron, its power supply, a modulator, and the accelerating structure make a decisive contribution to the cost budget of the entire machine. Of course, normally conducting electron LINACs for energies up to 100 MeV, as a rule, are built following a different scheme, namely less but more powerful klystrons and longer accelerating sections are used. Moreover, most LINACs operate in a traveling wave mode; however, this makes little difference on the results in the analysis of RF power consumption by these two types of accelerators given below. Beam blow-up caused by the excitation of the hybrid modes with a transverse mag- netic ﬁeld on the axis can also occur in LINACs [42]. However, for the range of energies of the accelerated beam that is discussed here and, accordingly, for a small number of relatively short accelerating sections, the threshold current turns out to be a few hun- dreds of milliamperes, especially if dedicated suppression measures are taken for the parasitic modes. p An important advantage of RTMs over linear accelerators is high energy stability and the energy spectrum of the accelerated beam, which is quite narrow ∼10−3 , which are due to speciﬁc features of the longitudinal dynamics of the RMT. Achieving similar values in a LINAC requires a complex injection system that forms short bunches and an expensive RF system with high parameter stability. We would like to note that energy stability and a narrow energy spectrum are important for accelerators that are dedicated to the production of isotopes where a magnetic system for switching the beam between targets is required. When estimating the gain in the cost of the accelerator, one must take into account the total consumption of the RF power for the beam acceleration. 3. Comparison of RTM and LINAC This not only leads to an increase in the RF power consumption, but it also leads to a high radiation background from the accelerator and to the appearance of significantly induced activity on the accelerator elements. In the subsequent parts of this article, possible approaches for reducing beam current losses that have not been previously tested in RTMs are discussed p y However, in the most of the previously built pulsed RTMs with a low-energy injection from an electron gun, large beam current losses take place at the orbits. This not only leads to an increase in the RF power consumption, but it also leads to a high radiation background from the accelerator and to the appearance of signiﬁcantly induced activity on the accelerator elements. In the subsequent parts of this article, possible approaches for reducing beam current losses that have not been previously tested in RTMs are discussed. reducing beam current losses that have not been previously tested in RTMs are discussed. As a conclusion of this section, we would like to note that to accelerate a significantly larger average beam current where both a high duty cycle and a large pulse current are required there is certainly no alternative to LINACs As a conclusion of this section, we would like to note that to accelerate a signiﬁcantly larger average beam current where both a high duty cycle and a large pulse current are required, there is certainly no alternative to LINACs. required, there is certainly no alternative to LINACs. Another observation is pertinent here. An attractive option is to accelerate a beam with a moderately high average current using a superconducting LINAC. In this case, the losses of the RF power in the walls of the accelerating structure are negligible, and the electronic efficiency will be larger than that of the RTM. However, the complexity and cost of the injection system, which should form short bunches of electrons with a sufficiently high energy to allow acceleration in a structure with β = 1, as well as the high cost of an accelerating structure with a cryostat, result in this LINAC design not being competitive ith RTM Another observation is pertinent here. An attractive option is to accelerate a beam with a moderately high average current using a superconducting LINAC. 3. Comparison of RTM and LINAC For example, for Ib = 20 mA and beam energy 50 MeV, the RTM electronic efﬁciency is 54% and the total RF pulse power consumption is about 1.9 MW, whereas in the case of the LINAC, these parameters are 11% and 8.8 MW, respectively. Taking into account these estimates and the effects limiting the pulse beam current discussed above, it can be argued that RTMs have undoubted advantages over LINACs in those applications for which the required beam energy is of tens of MeV and for which a pulse current about 20 mA is sufficient. Taking into account these estimates and the effects limiting the pulse beam current discussed above, it can be argued that RTMs have undoubted advantages over LINACs in those applications for which the required beam energy is of tens of MeV and for which a pulse current about 20 mA is sufﬁcient. p One potential application of RTMs is the production of medical isotopes, which re- quires an average current of at least 100 μA. To achieve this value with a pulse current of 20 mA, the RTM RF system must operate with a duty cycle D > 0.5%. Thus, to produce a beam with the energy 50 MeV, the pulse current ܫ௕ = 20 mA and the average current 100 μA of the RTM RF power source must provide a pulse and an average RF power that is about 2 MW and 10 kW, respectively. One potential application of RTMs is the production of medical isotopes, which requires an average current of at least 100 µA. To achieve this value with a pulse current of 20 mA, the RTM RF system must operate with a duty cycle D > 0.5%. Thus, to produce a beam with the energy 50 MeV, the pulse current Ib = 20 mA and the average current 100 µA of the RTM RF power source must provide a pulse and an average RF power that is about 2 MW and 10 kW, respectively. about 2 MW and 10 kW, respectively. However, in the most of the previously built pulsed RTMs with a low-energy injec- tion from an electron gun, large beam current losses take place at the orbits. 3. Comparison of RTM and LINAC If we take the following typical RTM values as an example: ∆Es = 5 MeV, N = 10, Ze = 80 MΩ/m, L = 0.4 m, and ϕs = 16◦, then we obtain the plots of ηRTM e , ηLinac e and PRTM Σ , PLinac Σ /PRTM Σ as functions of the beam current, as shown in Figure 3a,b. ߟ௘ ௕/ ఀ ( ) If we take the following typical RTM values as an example: ∆ܧ௦ = 5 MeV, ܰ = 10, ܼ௘ = 80 MΩ/m, ܮ = 0.4 m, and ߮௦ = 16° , then we obtain the plots of ߟ௘ ோ்ெ, ߟ௘ ௅௜௡௔௖ and ܲఀ ோ்ெ, ܲఀ ௅௜௡௔௖/ܲఀ ோ்ெ as functions of the beam current, as shown in Figure 3a,b. (a) (b) (a) (b) Figure 3. (a) Electronic efficiency of the RTM and LINAC as functions of the pulse beam current ܫ௕; (b) ratio ܲఀ ௟௜௡௔௖/ܲఀ ோ்ெ of the LINAC and RTM total RF power consumption and the RTM total RF power consumption ܲఀ ோ்ெ as functions of ܫ௕. Figure 3. (a) Electronic efﬁciency of the RTM and LINAC as functions of the pulse beam current Ib; (b) ratio Plinac Σ /PRTM Σ of the LINAC and RTM total RF power consumption and the RTM total RF power consumption PRTM Σ as functions of Ib. As it can be seen from Figure 3, for a pulse beam current ܫ௕ < 100 mA in the LINAC, most of the consumed RF power is spent on building-up an accelerating field so that the RTM obviously has advantages in terms of efficiency and total RF power consumption. For example, for ܫ௕ = 20 mA and beam energy 50 MeV, the RTM electronic efficiency is 54% and the total RF pulse power consumption is about 1.9 MW, whereas in the case of the LINAC, these parameters are 11% and 8.8 MW, respectively. As it can be seen from Figure 3, for a pulse beam current Ib < 100 mA in the LINAC, most of the consumed RF power is spent on building-up an accelerating ﬁeld so that the RTM obviously has advantages in terms of efﬁciency and total RF power consumption. 3. Comparison of RTM and LINAC In the case of RTM, the RF power consumption that is required to build-up the accelerating ﬁeld is given by the formula: 2 PRTM w = ∆Es e cos ϕs 2 ZeL (5) (5) where Ze is the effective shunt impedance of the fundamental mode per unit length. For a LINAC, which shown in Figure 1c, with an acceleration that is in the phase of the maximum ﬁeld 2 PLinac w = N ∆E2 s e2ZeL (6) (6) The total RF power consumption for the accelerating ﬁeld that is building-up and the beam acceleration neglecting current losses is given by PRTM Σ = ∆Es cos ϕs 2 e2ZeL + Pb (7) PLinac Σ = N (∆Es)2 e2ZeL + Pb (8) (7) PLinac Σ = N (∆Es)2 e2ZeL + Pb (8) (8) where Pb = (N∆EsIb)/e is the pulse beam power, and Ib is the pulse beam current. The electronic efﬁciencies of the RTM and LINAC are determined by the expressions: ηRTM e = Pb/PRTM Σ (9) ηlinac e = Pb/Plinac Σ (10) (9) (10) Symmetry 2021, 13, 2244 9 of 17 If we take the following typical RTM values as an example: ∆Es = 5 MeV, N = 1 Ze = 80 MΩ/m, L = 0.4 m, and ϕs = 16◦, then we obtain the plots of ηRTM e , ηLinac e an PRTM Σ , PLinac Σ /PRTM Σ as functions of the beam current, as shown in Figure 3a,b. ߟ௘ ௟௜௡௔௖= ܲ௕/ܲఀ ௟௜௡௔௖ (10) If we take the following typical RTM values as an example: ∆ܧ௦ = 5 MeV, ܰ = 10, ܼ = 80 MΩ/m, ܮ = 0.4 m, and ߮௦ = 16° , then we obtain the plots of ߟ௘ ோ்ெ, ߟ௘ ௅௜௡௔௖ and ܲఀ ோ்ெ, ܲఀ ௅௜௡௔௖/ܲఀ ோ்ெ as functions of the beam current, as shown in Figure 3a,b. (a) (b) Figure 3. (a) Electronic efficiency of the RTM and LINAC as functions of the pulse beam current ܫ௕; (b) ratio ܲఀ ௟௜௡௔௖/ܲఀ ோ்ெ of the LINAC and RTM total RF power consumption and the RTM total RF power consumption ܲఀ ோ்ெ as functions of ܫ௕. Figure 3. (a) Electronic efﬁciency of the RTM and LINAC as functions of the pulse beam current Ib; (b) ratio Plinac Σ /PRTM Σ of the LINAC and RTM total RF power consumption and the RTM total RF power consumption PRTM Σ as functions of Ib. 3. Comparison of RTM and LINAC In this case, the losses of the RF power in the walls of the accelerating structure are negligible, and the electronic efﬁciency will be larger than that of the RTM. However, the complexity and cost of the injection system, which should form short bunches of electrons with a sufﬁciently high energy to allow acceleration in a structure with β = 1, as well as the high cost of an accelerating structure with a cryostat, result in this LINAC design not being competitive with RTM. Symmetry 2021, 13, 2244 10 of 17 10 of 17 4. Approaches for Minimizing Beam Current Losses in RTM In the case, the beam current in an RTM is lower than the threshold value determined by the longitudinal and transverse instabilities; the main reason for the loss of the beam current along the orbits is the small value of the longitudinal and transverse acceptances. Of course, if an injected beam is relativistic and if its transverse and longitudinal emittances are much smaller than the acceptance values, then the current loss can be reduced to almost zero. A good example is the above-mentioned cascade of CW microtrons with the ﬁnal beam energy of 1.5 GeV [8–11]. It includes a low-energy line for the formation of short bunches and has a small transverse emittance as well as a 3.5 MeV CW LINAC injector. Another example is the 35 MeV pulsed RTM with bunches formed by an RF gun that has a photocathode and a 5 MeV booster-accelerating structure [16]. However, because of the design complexity and high cost of such approaches, their use is not appropriate when using pulsed RTM for applied purposes. From previous vast experience in the design, construction, and operation of RTMs the following lines of improvement for the machine beam dynamics and for the optimization of its elements with the aim of minimizing current losses at the orbits and increasing the electronic efﬁciency can be suggested: (1) increasing the threshold of the current due to the development of longitudinal and transverse instabilities; (2) increasing the longitudinal and transverse acceptances; (3) matching the longitudinal and transverse emittances of the injected beam with the corresponding acceptances; and (4) setting limits on the accuracy of the machining, tuning, and positioning of RTM elements and the stability of the accelerating and magnetic ﬁelds to ensure the passage of the beam along the orbits with the minimal trajectory corrections. j y Here, we discuss two of these approaches, namely increasing the longitudinal ac- ceptance of the RTM through the linearization of the accelerating ﬁeld time dependence and through the matching of the longitudinal emittance of the injected beam with the acceptance by optimizing the initial part of the accelerating structure. 4.1. Increasing the Longitudinal Acceptance of the RTM by Linearizing the Accelerating Field The width of the stable phase oscillation region in a cyclic accelerator is limited by the harmonic law of variation of the accelerating ﬁeld. In early works devoted to the theory of synchrotrons [43,44], it was shown that the linearization of the time dependence of the accelerating ﬁeld can widen the stable phase oscillation region signiﬁcantly with the addition of higher harmonics. g In the case of CMs, a theoretical proof for the possibility of expanding the stable phase oscillation region with the addition of a second or third harmonic to the ﬁeld was given in Ref. [45]. An experiment with a third harmonic resonator added to the main resonator that was reported in Ref. [46] did not produce tangible results, which was primarily due to the complexity of placing this additional resonator. Nevertheless, it was shown that the excitation of the third harmonic resonator by the beam leads to a certain increase of the accelerated current. Using its drift space, an RTM offers signiﬁcantly more options for placing a higher harmonic resonator. To assess this possibility of increasing the longitudinal acceptance in RTMs, we performed computer simulations of the phase motion for the following two cases: (1) with the fundamental harmonic ﬁeld only and (2) with the fundamental and second harmonics. The voltage across the inﬁnitely narrow gap of the equivalent resonator in the presence of the second harmonic is given by (11) U(t) = U1 cos ωt + U2 cos(2ωt + θ) (11) where U1 and U2 are the amplitudes of the fundamental and second harmonics, ω is the angular frequency of the fundamental harmonic, and θ is the phase of the second harmonic with respect to the fundamental one. The simulations were conducted for a fundamental frequency of 2856 MHz. To calculate the acceptance in the presence of the fundamental harmonic alone, the value Symmetry 2021, 13, 2244 11 of 17 11 of 17 U1 = 5.2 MV was chosen so that the synchronous energy gain of 5 MeV would be achieved for the synchronous phase ϕs = 16◦. In the case of two harmonics the parameters U1, U2, and θ were optimized with the condition of obtaining the maximum acceptance, while the magnetic ﬁeld remained the same as it did in the case for the fundamental harmonic on its own. 4.1. Increasing the Longitudinal Acceptance of the RTM by Linearizing the Accelerating Field (a) Voltage across the resonator gap as a function of the phase ϕ = ωt given by Equation (11) for one (U2 = 0) and two harmonics; (b) longitudinal acceptance as functions of δϕ; the deviation of the phase from the synchronous phase ϕs for the cases of harmonics one (ϕs = 16◦) and two (ϕs = 2.8◦) in the accelerating ﬁeld. In Figure 4b, the longitudinal acceptances for the case of one (orange region) and two (blue region) harmonics are shown. The vertical axis is the ratio ߜܧ௜௡௝⁄ ∆ܧ௦ of the devia- tion of the particle energy at the injection from the synchronous particle energy to the synchronous energy gain. One can see that the addition of the second harmonic leads to a significant, approximately 20-fold, increase in the area of the acceptance region. From the comparison of the plots in Figure 4a,b, it can be concluded that this increase in the stable phase oscillation region is due to the flat part in the voltage plot in the in phase interval between −50° and 70°. In Figure 4b, the longitudinal acceptances for the case of one (orange region) and two (blue region) harmonics are shown. The vertical axis is the ratio δEinj∆Es of the deviation of the particle energy at the injection from the synchronous particle energy to the synchronous energy gain. One can see that the addition of the second harmonic leads to a signiﬁcant, approximately 20-fold, increase in the area of the acceptance region. From the comparison of the plots in Figure 4a,b, it can be concluded that this increase in the stable phase oscillation region is due to the ﬂat part in the voltage plot in the in phase interval between −50◦and 70◦. The installation of an additional active accelerating structure with its own RF power supply system operating at the second harmonic would significantly complicate the de- sign of the RTM. In cases where the microtron would operate at a fixed beam current, a passive accelerating structure mounted on the axis of the main structure can be used to solve the problem. The voltage in the passive cavities was excited by the beam itself; thus, no external RF source is required. 4.1. Increasing the Longitudinal Acceptance of the RTM by Linearizing the Accelerating Field 5.2 MV was chosen so that the synchronous energy gain of 5 MeV would be achieved for the synchronous phase ߮௦= 16௢. In the case of two harmonics the parameters ܷଵ, ܷଶ, and θ were optimized with the condition of obtaining the maximum acceptance, while the magnetic field remained the same as it did in the case for the fundamental harmonic on its own. The results of the computer simulations are shown in Figure 4. In Figure 4a plots of the voltage across the equivalent resonator gap as a function of the phase ϕ = ωt for one and two harmonics is given. In the case of two harmonics, the maximum acceptance is obtained for U1 = 7.17 MV, U2 = 2.3 MV, θ = 155◦, and ϕs = 2.8◦. The results of the computer simulations are shown in Figure 4. In Figure 4a plots of the voltage across the equivalent resonator gap as a function of the phase ߮= ߱ݐ for one and two harmonics is given. In the case of two harmonics, the maximum acceptance is obtained for ܷଵ = 7.17 MV, ܷଶ = 2.3 MV, θ = 155°, and ߮௦ = 2.8°. (a) (b) Figure 4. (a) Voltage across the resonator gap as a function of the phase ߮= ߱ݐ given by Equation (11) for one (ܷଶ= 0) and two harmonics; (b) longitudinal acceptance as functions of ߜ߮; the deviation of the phase from the synchronous phase ߮௦ for the cases of harmonics one (߮௦= 16௢) and two (߮௦= 2.8௢) in the accelerating field. Figure 4. (a) Voltage across the resonator gap as a function of the phase ϕ = ωt given by Equation (11) for one (U2 = 0) and two harmonics; (b) longitudinal acceptance as functions of δϕ; the deviation of the phase from the synchronous phase ϕs for the cases of harmonics one (ϕs = 16◦) and two (ϕs = 2.8◦) in the accelerating ﬁeld. (b) (a) (b) (a) Figure 4. (a) Voltage across the resonator gap as a function of the phase ߮= ߱ݐ given by Equation (11) for one (ܷଶ= 0) and two harmonics; (b) longitudinal acceptance as functions of ߜ߮; the deviation of the phase from the synchronous phase ߮௦ for the cases of harmonics one (߮௦= 16௢) and two (߮௦= 2.8௢) in the accelerating field. Figure 4. 4.1. Increasing the Longitudinal Acceptance of the RTM by Linearizing the Accelerating Field In the absence of the structure coupling with the exter- nal loads, it is equal to [47]: The installation of an additional active accelerating structure with its own RF power supply system operating at the second harmonic would signiﬁcantly complicate the design of the RTM. In cases where the microtron would operate at a ﬁxed beam current, a passive accelerating structure mounted on the axis of the main structure can be used to solve the problem. The voltage in the passive cavities was excited by the beam itself; thus, no external RF source is required. In the absence of the structure coupling with the external loads, it is equal to [47]: ܷଶ= −ܫ௕ ఀܼ௘ (ଶ)ܮ(ଶ) ܿ݋ݏ߰(ଶ) (12) U2 = −IΣ b Z(2) e L(2) cos ψ(2) (12) 12) (12) where ܫ௕ ఀ is the sum of the beam currents from all of the RTM orbits, ܼ௘ (ଶ) and ܮ(ଶ) are the effective shunt impedance per unit length and length of the second harmonic acceler- ating structure, respectively, and ߰(ଶ) is the detuning angle determined by the difference between the second harmonic of the bunch repetition rate and the operating frequency of the structure. The negative sign in the right-hand side of Equation (12) is due to the fact that the accelerating field in the structure is induced by the beam in the decelerating phase. where IΣ b is the sum of the beam currents from all of the RTM orbits, Z(2) e and L(2) are the effective shunt impedance per unit length and length of the second harmonic accelerating structure, respectively, and ψ(2) is the detuning angle determined by the difference between the second harmonic of the bunch repetition rate and the operating frequency of the structure. The negative sign in the right-hand side of Equation (12) is due to the fact that the accelerating ﬁeld in the structure is induced by the beam in the decelerating phase. To Symmetry 2021, 13, 2244 12 of 17 12 of 17 ensure the required phase ratio of the ﬁrst and second harmonics, the detuning angle in Formula (11) should be (2) ensure the required phase ratio of the ﬁrst and second harmonics, the detuning angle in Formula (11) should be ( ) ψ(2) = θ −π (13) ψ(2) = θ −π (13) For the example considered above, the second harmonic frequency is 5712 MHz, and the accelerating structure is described in Refs. 4.1. Increasing the Longitudinal Acceptance of the RTM by Linearizing the Accelerating Field [48,49], which allows the use of an effective shunt impedance of 98 MΩ/m For an accelerated current of 20 mA and for neglecting beam losses in 10 beam passages through the accelerating structure, the total current IΣ b = 0.2 A. For the second harmonic phase θ = 155◦, the detuning angle is ψ(2) = 25◦, and for U2 = 2.3 MV, we obtain the length L(2) = 0.13 m. The period of the accelerating structure is 2.625 cm; thus, it must contain ﬁve accelerating cells. Since it is the beam that excites the ﬁeld in these cells, the coupling between them is not necessary and the accelerating structure is composed of six properly detuned and uncoupled cavities. As we have shown, the addition of the second harmonic to the accelerating ﬁeld enlarges the longitudinal acceptance; in particular, the RTM accepts a relative deviation ∆E/∆Es of the energy from the synchronous one by up to 30% with respect to the syn- chronous energy gain (see Figure 4b). However, such a large deviation in energy can lead to a loss of particles because of the limited size of the aperture in the horizontal plane in the return orbits. This limitation is due to installed steering coils, quadrupole lenses, or beam current monitors. The deviation ∆x of the particle trajectory from the synchronous one due to the particle momentum offset ∆p with respect to the synchronous value ps is equal to ∆x = 2Rs∆pps , where Rs is the radius of the synchronous orbit. If the relative energy deviation in energy is within the range \|∆E∆Es \| < 0.3, then for the shift of the entire beam to be within the acceptance, the aperture radius must satisfy the inequality a > νλ π × ∆p ∆Es ≈νλ π × ∆E ∆Es . Thus, for ν = 1, a > 0.3λ π in the presence of the second harmonic. For the RF ﬁeld wavelength λ = 10 cm, the aperture radius a > 0.95 cm, which is a quite feasible condition. Notice that νλ π is the distance between the RTM orbits. 4.2. Matching the Longitudinal Emittance of the Injected Beam with the RTM Acceptance by Optimizing the Initial Part of the Accelerating Structure 4.2. Matching the Longitudinal Emittance of the Injected Beam with the RTM Acceptance by Optimizing the Initial Part of the Accelerating Structure In addition, schemes with an external pre-buncher complicate the accelerator RF system signiﬁcantly and require a dispersion-free injection system. phases from the synchronous particle values that are too large and that are outside of RTM acceptance. In addition, schemes with an external pre-buncher complicate the accelerator RF system significantly and require a dispersion-free injection system. Figure 5. Theoretical and measured capture efficiency for the first passage of the beam through the accelerating structure as functions of the pre-buncher field phase [53]. Figure 5. Theoretical and measured capture efﬁciency for the ﬁrst passage of the beam through the accelerating structure as functions of the pre-buncher ﬁeld phase [53]. Figure 5. Theoretical and measured capture efficiency for the first passage of the beam through the accelerating structure as functions of the pre-buncher field phase [53]. We have studied the possibility of increasing the RTM capture efficiency by optimiz- ing the initial part of the accelerating structure with the aim of maximizing the fraction of electrons injected from the gun within the longitudinal acceptance. In Figure 6a, the loca- tion of the longitudinal beam emittance after the first acceleration for a beam with zero transverse emittance in both planes with respect to the acceptance of the 55 MeV RTM with its accelerating structure described in Ref. [18] is shown. Figure 6b shows the emit- Figure 5. Theoretical and measured capture efficiency for the first passage of the beam through the accelerating structure as functions of the pre-buncher field phase [53]. Figure 5. Theoretical and measured capture efﬁciency for the ﬁrst passage of the beam through the accelerating structure as functions of the pre-buncher ﬁeld phase [53]. tion of the longitudinal beam emittance after the first acceleration for a beam with zero transverse emittance in both planes with respect to the acceptance of the 55 MeV RTM with its accelerating structure described in Ref. [18] is shown. Figure 6b shows the emit- We have studied the possibility of increasing the RTM capture efficiency by optimiz- ing the initial part of the accelerating structure with the aim of maximizing the fraction of electrons injected from the gun within the longitudinal acceptance. In Figure 6a, the loca- tion of the longitudinal beam emittance after the first acceleration for a beam with zero transverse emittance in both planes with respect to the acceptance of the 55 MeV RTM with its accelerating structure described in Ref. 4.2. Matching the Longitudinal Emittance of the Injected Beam with the RTM Acceptance by Optimizing the Initial Part of the Accelerating Structure [18] is shown. Figure 6b shows the emit- tance in the case of the optimized accelerating structure. The accelerating field amplitudes for both variants of the accelerating structure are shown in Figure 7. Finally, plots depict- ing the decay to the beam current along the orbits for the considered variants of the accel- erating structure are given in Figure 8. As one can clearly see, the optimization of the initial cells allows the RTM capture efficiency to increase from 21 to 81%. We have studied the possibility of increasing the RTM capture efﬁciency by optimizing the initial part of the accelerating structure with the aim of maximizing the fraction of electrons injected from the gun within the longitudinal acceptance. In Figure 6a, the location of the longitudinal beam emittance after the ﬁrst acceleration for a beam with zero transverse emittance in both planes with respect to the acceptance of the 55 MeV RTM with its accelerating structure described in Ref. [18] is shown. Figure 6b shows the emittance in the case of the optimized accelerating structure. The accelerating ﬁeld amplitudes for both variants of the accelerating structure are shown in Figure 7. Finally, plots depicting the decay to the beam current along the orbits for the considered variants of the accelerating structure are given in Figure 8. As one can clearly see, the optimization of the initial cells allows the RTM capture efﬁciency to increase from 21 to 81%. tance in the case of the optimized accelerating structure. The accelerating field amplitudes for both variants of the accelerating structure are shown in Figure 7. Finally, plots depict- ing the decay to the beam current along the orbits for the considered variants of the accel- erating structure are given in Figure 8. As one can clearly see, the optimization of the initial cells allows the RTM capture efficiency to increase from 21 to 81%. With a nonzero transverse beam emittance and in the presence of magnetic field er- rors and errors in the RTM elements, positioning particles with parameters near the ac- ceptance boundary can be lost during the acceleration. The addition of the second har- monic of the field, as discussed in Section 4.1, moves the acceptance boundary farther away, allowing a high capture efficiency to be maintained and reducing the particle cur- rent loss that may take place during the acceleration. 4.2. Matching the Longitudinal Emittance of the Injected Beam with the RTM Acceptance by Optimizing the Initial Part of the Accelerating Structure When developing modern electron LINACs for applied purposes with beam injection from an electron gun without intermediate systems for beam bunching and focusing, the initial part of the accelerating structure is optimized with conditions to provide a high capture efﬁciency up to 60–90% (see, for example, [49–52]). As a rule, for this aim, it is sufﬁcient to choose the correct lengths and ﬁeld amplitudes of the ﬁrst three accelerating cells. Their function is beam bunching and focusing, and they also provide a sufﬁcient increase in the energy of the particles in order to further their acceleration in subsequent regular β = 1 cells. However, this optimization principle has not been applied to the accelerating struc- tures of the existing RTMs. Usually, the ﬁeld amplitude of the ﬁrst accelerating cell is set close to that of the regular cells, and its length is chosen to be approximately equal to λ/4. With this design, the beam current at the exit of the accelerating structure is 30–40% of the injected current and, according to numerical simulations, only 20% of the injected particles reach the RTM beam exit. Taking into account the magnetic ﬁeld errors and errors in the positioning of the accelerator elements discussed above, the fraction of the electrons at the end of acceleration turns out to be even lower. In the 70 MeV RTM described in Ref. [17], a pre-buncher was installed at the exit of the electron gun in order to increase the capture efﬁciency. In Figure 5, theoretical and measured values of the ratio Ib1/Igun, where Ib1 is the current at the exit of the accelerating structure after the ﬁrst beam passage, and Igun is the electron gun current, is shown [53]. It can be seen that in principle, the capture efﬁciency can be increased by up to 60–70%. However, in this case, a signiﬁcant number of particles have energy deviations and phases from the synchronous particle values that are too large and that are outside of RTM acceptance. Symmetry 2021, 13, 2244 13 of 17 13 of 17 In addition, schemes with an external pre-buncher complicate the accelerator RF system signiﬁcantly and require a dispersion-free injection system. phases from the synchronous particle values that are too large and that are outside of RTM acceptance. In addition, schemes with an external pre-buncher complicate the accelerator RF system significantly and require a dispersion-free injection system. 4.2. Matching the Longitudinal Emittance of the Injected Beam with the RTM Acceptance by Optimizing the Initial Part of the Accelerating Structure On-axis distribution of the accelerating ﬁeld for the accelerating structure of the 55 MeV RTM [18] (blue line) and for the structure with the optimized initial part (orange line). Figure 7. On-axis distribution of the accelerating field for the accelerating structure of the 55 MeV RTM [18] (blue line) and for the structure with the optimized initial part (orange line). Figure 8. Decay of the beam current along the orbits for the two variants of the accelerating structure considered in Section 4.2. Figure 8. Decay of the beam current along the orbits for the two variants of the accelerating structure considered in Section 4.2. Figure 8. Decay of the beam current along the orbits for the two variants of the accelerating structure considered in Section 4.2. Figure 8. Decay of the beam current along the orbits for the two variants of the accelerating structure considered in Section 4.2. Figure 8. Decay of the beam current along the orbits for the two variants of the accelerating structure considered in Section 4.2. Figure 8. Decay of the beam current along the orbits for the two variants of the accelerating structure considered in Section 4.2. 5. Conclusions The analysis presented in this article shows that for applications requiring relatively small pulse currents (20–30 mA) and energies up to tens of MeV, RTMs have clear ad- vantages over LINACs in terms of efficiency, size, and cost. An average current of about 100 μA can be achieved by operating an RTM RF system with a high duty cycle. However, 5. Conclusions The analysis presented in this article shows that for applications requiring relatively small pulse currents (20–30 mA) and energies up to tens of MeV, RTMs have clear ad- vantages over LINACs in terms of efficiency, size, and cost. An average current of about 100 μA can be achieved by operating an RTM RF system with a high duty cycle. However, th i ifi t b t l th t t k l d i th l ti th t With a nonzero transverse beam emittance and in the presence of magnetic ﬁeld errors and errors in the RTM elements, positioning particles with parameters near the acceptance boundary can be lost during the acceleration. 4.2. Matching the Longitudinal Emittance of the Injected Beam with the RTM Acceptance by Optimizing the Initial Part of the Accelerating Structure With a nonzero transverse beam emittance and in the presence of magnetic field rors and errors in the RTM elements, positioning particles with parameters near the ceptance boundary can be lost during the acceleration. The addition of the second h monic of the field, as discussed in Section 4.1, moves the acceptance boundary fart away, allowing a high capture efficiency to be maintained and reducing the particle c rent loss that may take place during the acceleration. (a) (b) Figure 6. Longitudinal phase space (green and red dotted line) of the beam after the ﬁrst acceleration superimposed on the RTM acceptance (a) for the accelerating structure of the 55 MeV RTM [18]; (b) for the structure with optimized initial part. e beam emittance and in the presence of magnetic f ements, positioning particles with parameters near t during the acceleration. The addition of the secon ed in Section 4.1, moves the acceptance boundary efficiency to be maintained and reducing the partic uring the acceleration. (b) rors and errors in the RTM ceptance boundary can be monic of the field, as discu away, allowing a high captu rent loss that may take plac (a) (b) (a) Figure 6. Longitudinal phase space (green and red dotted line) of the beam after the ﬁrst acceleration superimposed on the RTM acceptance (a) for the accelerating structure of the 55 MeV RTM [18]; (b) for the structure with optimized initial part. 14 of 17 on ial on tial Symmetry 2021, 13, 2244 Figure 6. Longitu the RTM acceptan Figure 6. Longitu the RTM acceptan Figure 7. On-axis distribution of the accelerating field for the accelerating structure of the 55 MeV RTM [18] (blue line) and for the structure with the optimized initial part (orange line). Figure 7. On-axis distribution of the accelerating ﬁeld for the accelerating structure of the 55 MeV RTM [18] (blue line) and for the structure with the optimized initial part (orange line). Figure 7. On-axis distribution of the accelerating field for the accelerating structure of the 55 MeV RTM [18] (blue line) and for the structure with the optimized initial part (orange line). Figure 7. On-axis distribution of the accelerating field for the accelerating structure of the 55 MeV RTM [18] (blue line) and for the structure with the optimized initial part (orange line). Figure 7. 4.2. Matching the Longitudinal Emittance of the Injected Beam with the RTM Acceptance by Optimizing the Initial Part of the Accelerating Structure The addition of the second harmonic of the ﬁeld, as discussed in Section 4.1, moves the acceptance boundary farther away, allowing a high capture efﬁciency to be maintained and reducing the particle current loss that may take place during the acceleration. the significant be in pulsed RTMs g in pulsed RTMs l l 5. Conclusions p g g y accelerator elements. The article describes a number of approaches that aim to reduce the losses of the beam current. These include decreasing the fringe field aberrations of the magnets; minimizing the inhomogeneity of the magnetic field in the area of the orbits; reducing the strayed magnetic field in the drift space; increasing the accuracy of the ma- chining and positioning of the magnets; expanding the aperture dimensions on the return accelerator elements. The article describes a number of approaches that aim to reduce the losses of the beam current. These include decreasing the fringe field aberrations of the magnets; minimizing the inhomogeneity of the magnetic field in the area of the orbits; reducing the strayed magnetic field in the drift space; increasing the accuracy of the ma- chining and positioning of the magnets; expanding the aperture dimensions on the return The analysis presented in this article shows that for applications requiring relatively small pulse currents (20–30 mA) and energies up to tens of MeV, RTMs have clear ad- vantages over LINACs in terms of efﬁciency, size, and cost. An average current of about 100 µA can be achieved by operating an RTM RF system with a high duty cycle. However, the signiﬁcant beam current losses that can take place during the acceleration that occurs in pulsed RTMs lead to a signiﬁcant radiation background and induced activity on the accelerator elements. The article describes a number of approaches that aim to reduce the losses of the beam current. These include decreasing the fringe ﬁeld aberrations of the magnets; minimizing the inhomogeneity of the magnetic ﬁeld in the area of the orbits; Symmetry 2021, 13, 2244 15 of 17 15 of 17 reducing the strayed magnetic ﬁeld in the drift space; increasing the accuracy of the ma- chining and positioning of the magnets; expanding the aperture dimensions on the return orbits; increasing the current thresholds due to the longitudinal and transverse instability; enlarging the RTM longitudinal acceptance by adding the second harmonic of the accelerat- ing ﬁeld; and matching the emittance of the injected beam with the RTM acceptance better by optimizing the initial part of the accelerating structure. We also explored the possibility of enlarging the stability of the phase oscillation region using its intrinsic symmetry, which was mentioned in Section 2.2. References 1. Veksler, V.I. 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Author Contributions: Conceptualization, V.K.; methodology, V.S.; software, M.B.; validation, V.S. and Y.K.; formal analysis, V.S. and M.B.; investigation, A.E. and V.K.; writing—original draft prepa- ration, V.S., M.B. and Y.K; writing—review and editing, V.S., M.B. and Y.K; visualization, V.S. and M.B.; supervision, V.S.; project administration, V.S. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Acknowledgments: The authors are grateful to Mamoru Fujiwara, whose interest in a racetrack microtron with a high average beam current stimulated this work. Acknowledgments: The authors are grateful to Mamoru Fujiwara, whose interest in a racetrack microtron with a high average beam current stimulated this work. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. References (In Russian) 24. 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https://openalex.org/W2007620217	https://www.scielo.br/j/pci/a/pRQcQGQHwTQNHXsmhbSbGWm/?lang=pt&format=pdf	Portuguese	null	O uso da informação e a prática clínica de médicos residentes	Perspectivas em Ciência da Informação	2,011	cc-by	10,718	O uso da informação e a prática clínica de médicos residentes O uso da informação e a prática clínica de médicos residentes O uso da informação e a prática clínica de médicos id t Maria Gorete Monteguti Savi; Edna Lucia da Silva O uso da informação e a prática clínica de médicos residentes Maria Gorete Monteguti Savi Maria Gorete Monteguti Savi Mestre em Ciência da Informação pela Universidade Federal de Santa Catarina.Bibliotecária da Universidade Federal de Santa Catarina, Perspectivas em Ciência da Informação, v.16, n.3, p.232-254, jul./set. 2011 Edna Lucia da Silva Doutora em Ciência da Informação IBICT- UFRJ.Professora do Departamento de Ciência da Informação e do Programa de Pós-Graduação em Ciência da Informação da Universidade Federal de Santa Catarina Relato de pesquisa que aborda as necessidades informacionais e o uso de informação dos médicos residentes, na prática clínica. Contextualiza a pesquisa realizada no Hospital Universitário da UFSC, que teve como participantes os médicos residentes. Levanta as necessidades informacionais na prática clínica dos médicos residentes, que atuam nos ambulatórios do hospital. Mapeia o processo de escolha das fontes de informação e indica as mais utilizadas. Os resultados mostram que: durante sua prática ambulatorial, o médico residente se depara com questões classificadas como foreground, mais ligadas a aspectos clínicos do tratamento do paciente, em detrimento de questões ligadas a aspectos básicos, tais como epidemiologia e etiologia; utilizam como principais fontes de informação: periódicos, textbooks e handbooks, guidelines e preceptores, quase que de maneira equitativa; a Internet foi o meio mais utilizado na busca de informação; dentre os sistemas informatizados utilizados com frequência, destacam-se: UptoDate, Portal de Periódicos da CAPES e PubMed/Medline. Os resultados permitem inferir que os médicos residentes estão conscientes da importância do uso da informação para uma melhor prática clínica. Palavras-chave: Uso da informação; Prática clínica; Médicos residentes; Hospital universitário. 232 Perspectivas em Ciência da Informação, v.16, n.3, p.232-254, jul./set. 2011 O uso da informação e a prática clínica de médicos residentes O uso da informação e a prática clínica de médicos residentes Maria Gorete Monteguti Savi; Edna Lucia da Silva O uso da informação e a prática clínica de médicos id Information use and clinical practice by resident physicians This paper describes the study of information requirements and use of information by resident physicians in clinical practice. It contextualizes the study undertaken at UFSC University Hospital of resident physicians and outlines the information requirements in clinical practice by the resident physicians in the hospital. It then maps the process of choosing the information sources to be used and shows those most widely used. The results suggest that: in the course of their work resident physicians are faced with issues classified as foreground issues which are more connected with the clinical aspects of patient treatment, to the detriment of issues connected with basic aspects such as epidemiology and etiology; for their main sources of information they use periodicals, textbooks and handbooks, guidelines and other manuals in an almost equal way; the internet was the most used method for information searches; within those information systems the most used were: UptoDate, the CAPES journal portal and PubMed/Medline. These results suggest that the resident physicians are aware of the importance of information use for more efficient clinical practice. Keywords: Information Use; Clinical Practice; Resident Physicians; University Hospital. Recebido em 08.06.2009 Aceito em 30.07.2011 Keywords: Information Use; Clinical Practice; Resident Physicians; University Hospital. Recebido em 08.06.2009 Aceito em 30.07.2011 Recebido em 08.06.2009 Aceito em 30.07.2011 Perspectivas em Ciência da Informação, v.16, n.3, p.232-254, jul./set. 2011 1 Introdução No cenário da sociedade da informação e de um mundo globalizado, no qual o desenvolvimento da ciência e tecnologia tem como consequência para a área médica a criação incessante de novos métodos diagnósticos, terapêuticos e preventivos, o acesso à informação para a prática clínica é essencial. Nesta pesquisa, procurou-se identificar as necessidades informacionais dos médicos residentes e as fontes de informação utilizadas no processo de busca de solução para as questões clínicas, surgidas no atendimento ambulatorial de um hospital. Os resultados foram descritos e analisados a partir da coleta de dados obtidos através de questionário e entrevista aplicados aos médicos residentes do Hospital Universitário (HU) da UFSC. As informações obtidas nas entrevistas, realizadas através da técnica do incidente crítico, 233 Perspectivas em Ciência da Informação, v.16, n.3, p.232-254, jul./set. 2011 O uso da informação e a prática clínica de médicos residentes Maria Gorete Monteguti Savi; Edna Lucia da Silva Maria Gorete Monteguti Savi; Edna Lucia da Silva foram agrupadas por semelhança de conteúdo (categorias) e foram analisadas juntamente com as respostas dos questionários que apresentavam pertinência temática. Algumas questões elencaram respostas com indicação de prioridades, sendo que foi analisado até o nível de prioridade 3, por ter sido considerado o nível médio respondido nessas questões. Os dados transcritos farão indicação ao código alfanumérico da entrevista correspondente, compreendido entre E01 e E12. A análise e a interpretação dos dados realizaram-se privilegiando duas categorias: necessidades informacionais e fontes de informação. Os resultados mais pontuais foram confrontados com pesquisas realizadas no Brasil e no exterior, a fim de se estabelecerem paralelos e de se identificarem semelhanças ou diferenças. 2 Contextualização da pesquisa Tabela 1- Distribuição de horas dedicadas a RM, ao atendimento ambulatorial, às atividades teóricas e indicação do número de pacientes atendidos nos ambulatórios pelos médicos residentes do da UFSC Medidas Horas/dia dedicados a RM Horas/dia de atendimento ambulatorial Horas/dia de atividades teóricas Pacientes/dia atendidos nos ambulatórios Média 10 4 2 7 Mínimo 8 0 0,5 0 Máximo 18 9 6 16 Fonte: Dados da pesquisa. Medidas Horas/dia dedicados a RM Horas/dia de atendimento ambulatorial Horas/dia de atividades teóricas Pacientes/dia atendidos nos ambulatórios Média 10 4 2 7 Mínimo 8 0 0,5 0 Máximo 18 9 6 16 Fonte: Dados da pesquisa. Perspectivas em Ciência da Informação, v.16, n.3, p.232-254, jul./set. 2011 2 Contextualização da pesquisa Para melhor entendimento das questões aqui tratadas, faz-se necessário a caracterização do lócus da pesquisa e seus dos participantes. A pesquisa foi realizada com 38 médicos residentes, que atuam na prática clínica ambulatorial do Hospital Universitário Professor Polydoro Ernani de São Thiago da UFSC, sendo 22 mulheres (57,89%), com idade mínima de 24 anos e máxima de 31 anos, e 16 homens (42,10%), com idade mínima de 25 anos e máxima de 32 anos. Com relação à distribuição dos pesquisados por ano, no Programa de Residência Médica, verificou-se que 42,11% (n=16) encontravam-se no 1º. ano (R1); 42,11% (n=16) no 2º. ano (R2); e 15,79% (n=6) no 3º. ano (R3).Quanto aos anos de graduação acadêmica dos médicos residentes, verificou-se que estavam compreendidos no período de 1996 a 2006, destacando-se o ano de 2005, com 12 médicos residentes. Em relação à instituição em que se graduaram, observa-se que a UFSC formou a grande maioria dos médicos, 28 pesquisados (73,68%); 6 (15,78%) eram provenientes de outras universidades do Estado de Santa Catarina: UNISUL e FURB, ambas com três (7,89%); quatro eram provenientes de universidades brasileiras de outros estados: UNICAMP, UFMG, UFPel e UFRGS com um pesquisado para cada universidade (2,63%). Normalmente, as atividades dos médicos residentes ligados às áreas clínicas se dividem entre o atendimento ambulatorial, o atendimento nas unidades de internação, o atendimento na emergência e atividades teóricas. Os médicos residentes participantes desta pesquisa, indicaram que utilizavam a média de dez horas/dia (desvio padrão 1,77), com mínimo de oito horas/dia e máximo de 18 horas/dia, para suas atividades profissionais. Sendo que, para o atendimento ambulatorial, utilizavam a média de quatro horas/dia (desvio padrão 1,90), oscilando entre nenhum tempo e, no máximo, nove horas/dia (TAB. 1). Nesta pesquisa, os dados referentes ao número de pacientes atendidos, diariamente, se ateve ao atendimento ambulatorial. Assim, com relação a Perspectivas em Ciência da Informação, v.16, n.3, p.232-254, jul./set. 2011 234 O uso da informação e a prática clínica de médicos residentes Maria Gorete Monteguti Savi; Edna Lucia da Silva essa variável, obteve-se a média de sete pacientes/dia (desvio-padrão 3,36), que oscilou entre zero e 16 pacientes/dia. 3 As necessidades informacionais na prática clínica Portanto, temos a média de 21% de respostas não atendidas, ou seja, de cada cinco questões clínicas que foram realizadas diariamente, uma ficava sem resposta. foi de 79% (desvio padrão de 15,20), com mínimo de 30% e máximo de 100% (TAB. 2). Portanto, temos a média de 21% de respostas não atendidas, ou seja, de cada cinco questões clínicas que foram realizadas diariamente, uma ficava sem resposta. Resultado praticamente igual foi levantado por Green, Ciampi e Ellis (2000), que detectaram que os médicos residentes atenderam a 80% das questões. E, por Ramos, Linscheid e Schafer (2003), cujos resultados mostram que os médicos residentes atenderam a 74% e os professores a 39%, ressaltando-se, nesta última pesquisa, o índice dos médicos residentes. Já na pesquisa de Covell, Uman e Manning (1985), os autores observaram um índice muito inferior, pois somente 30% das necessidades de informação dos médicos foram satisfeitas. Com relação ao tempo gasto na resolução de cada questão clínica, foi indicado o tempo médio de 20 minutos (desvio padrão 15,46), com tempo mínimo de dois minutos e máximo de 60 minutos (TAB. 2). Na literatura estrangeira, verifica-se que existe diferença no tempo médio utilizado para resolução da questão clínica. Os dados da pesquisa de Woolf e Benson (1989) indicam que 49% dos médicos e estudantes de medicina levavam entre 10 e 30 minutos para a resolução de cada questão clínica. Os dados de Gorman (2001) indicam um tempo médio de 12 minutos; os de Green, Ciampi e Ellis (2000) a média é de 15 minutos; e na de Ramos, Linscheid e Schafer (2003) entre 5 e 15 minutos. Já contrastando com esse resultado, a pesquisa de Ely et al. (1999) indica um tempo muito inferior, ou seja, cada médico levava apenas dois minutos para responder a uma questão clínica. Essas três variáveis (número de questões clínicas, percentual de respostas encontradas e tempo gasto para resposta) foram calculadas com 37 pesquisados, já que um deles não informou esses dados. 3 As necessidades informacionais na prática clínica Na prática clínica, o médico se depara com um número variável de questões que necessitam de respostas. Essas questões resultantes do fluxo informacional, que se estabelece na relação médico/paciente, e que geram uma necessidade de informação, deverão ser claras e objetivas, para que possam ser efetivamente respondidas. Para responder às questões clínicas, torna-se necessário que o médico busque informações para subsidiar esse processo, que deverão ser identificadas, interpretadas, integradas à realidade do paciente e aplicadas à decisão clínica. Green, Ciampi e Ellis (2000, p. 220, tradução nossa) definiram “a necessidade informacional do médico como a exigência para novos conhecimentos médicos no que se refere ao cuidado de um paciente ou de um grupo de pacientes”. Para Osheroff et al. (1991), as necessidades informacionais do médico são variáveis e complexas, e diversas fontes de informação são passíveis de consulta. Outras variáveis também são apontadas na literatura, como fatores intervenientes nas necessidades informacionais. Para Leckie, Pettigrew e Sylvain (1996), os fatores demográficos, o contexto, a frequência, o grau de importância e a complexidade, podem direcionar as necessidades informacionais. Na pesquisa realizada, foi identificado que os médicos residentes do HU da UFSC formulavam a média diária de 5,25 questões (desvio padrão 2,13), tendo sido indicado o mínimo de uma e o máximo de dez questões por dia. Considerando que a média de atendimento ambulatorial é de sete pacientes ao dia, conclui-se que os médicos residentes elaboravam 0,75 questão por paciente. q p p Alguns autores da literatura estrangeira apontam resultados aproximados. Dee e Blazek (1993) apontaram 0,3 necessidades por paciente; Gorman e Helfand (1995) e Green, Ciampi e Ellis (2000) 0,7; Cogdill et al. (2000) indicaram 0,29, quando o médico estava na presença de estudantes de medicina e 0,42, quando estava sozinho no atendimento clínico; Ramos, Linscheid e Schafer (2003) indicaram que entre os residentes o índice foi 1,5 e, entre os professores, de 0,8. p Procurou-se saber, também, qual o percentual de respostas encontradas para as questões clínicas formuladas. A média de respostas 235 Perspectivas em Ciência da Informação, v.16, n.3, p.232-254, jul./set. 2011 O uso da informação e a prática clínica de médicos residentes Maria Gorete Monteguti Savi; Edna Lucia da Silva Maria Gorete Monteguti Savi; Edna Lucia da Silva foi de 79% (desvio padrão de 15,20), com mínimo de 30% e máximo de 100% (TAB. 2). Perspectivas em Ciência da Informação, v.16, n.3, p.232-254, jul./set. 2011 Perspectivas em Ciência da Informação, v.16, n.3, p.232-254, jul./set. 2011 3 As necessidades informacionais na prática clínica Tabela 2- Indicação do número de questões clínicas formuladas, do percentual de respostas encontradas e do tempo gasto em cada resposta pelos médicos residentes do HU da UFSC Tabela 2- Indicação do número de questões clínicas formuladas, do percentual de respostas encontradas e do tempo gasto em cada resposta pelos médicos residentes do HU da UFSC Variável Média Mínima Máxima Nº de respondentes Questões clínicas (indicação diária) 5,25 01 10 37 Respostas encontradas (percentual) 79% 30% 100% 37 Tempo gasto em cada resposta (minutos) 20 02 60 37 Fonte: Dados da pesquisa Variável Média Mínima Máxima Nº de respondentes Questões clínicas (indicação diária) 5,25 01 10 37 Respostas encontradas (percentual) 79% 30% 100% 37 Tempo gasto em cada resposta (minutos) 20 02 60 37 Fonte: Dados da pesquisa Com relação à frequência com que os médicos buscavam informação para resolver suas questões, a pesquisa apontou que 42,10% (n=16) indicaram quase todos os dias, 31,58% (n=12) duas ou três vezes por semana, 23,68% (n=9) buscavam todos os dias, e 2,63% (n=1) menos de uma vez por semana. A opção “uma vez por semana” não foi indicada por nenhum dos pesquisados. 236 Perspectivas em Ciência da Informação, v.16, n.3, p.232-254, jul./set. 2011 O uso da informação e a prática clínica de médicos residentes O uso da informação e a prática clínica de médicos residentes Maria Gorete Monteguti Savi; Edna Lucia da Silva Maria Gorete Monteguti Savi; Edna Lucia da Silva A elaboração da questão clínica deve considerar o enfoque do problema, que pode estar relacionado a aspectos básicos, tais como causa, fisiopatologia; ou a aspectos clínicos, como diagnóstico, tratamento ou prognóstico. Nesta pesquisa, foi identificado que a maioria (73,68%, n=28) dos médicos residentes buscava resposta para questões clínicas direcionadas para tratamento, seguidas de questões relacionadas com diagnóstico (21,05%, n=8) e, por último, com prevenção (5,26%, n=2). Questões relacionadas a prognóstico, etiologia, epidemiologia e outros não tiveram nenhuma indicação. ç Este resultado em que o alto índice das questões está relacionado com a terapêutica também fica explicitado nas falas dos pesquisados: [...] eu utilizo o critério da fonte de informação, aquilo que chega mais próximo possível [...] da melhor terapêutica que existe no momento (E01). [São usados os] consensos pra ajudar na decisão terapêutica d i ( 0 ) [...] eu utilizo o critério da fonte de informação, aquilo que chega mais próximo possível [...] da melhor terapêutica que existe no momento (E01). 3 As necessidades informacionais na prática clínica Gráfico 1 - Aplicabilidade da solução da questão clínica pelos médicos residentes do HU da UFSC Gráfico 1 - Aplicabilidade da solução da questão clínica pelos médicos residentes do HU da UFSC 0 10 20 30 40 50 60 70 80 90 100 Intervencão no paciente Recomendacões ao paciente Programar retorno do paciente Aumentar conhecimento Modificar decisão Outros Percentual de respostas Frequentemente Ás vezes Pouco Não utiliza Fonte: Dados da pesquisa. Fonte: Dados da pesquisa. No item “às vezes”, os resultados obtidos pelas opções de modificar decisão anterior, programar retorno do paciente, passar recomendações para o paciente e definição de intervenção no paciente ficaram mais equilibrados, entre 36,84% e 57,89% (entre 14 e 22 indicações). O alto índice (86,84%, n=33) da opção “outros” como não utilizados pelos médicos residentes indicou que todas as opções sobre a aplicabilidade da resposta da questão clínica foram sugeridas. As variáveis indicadas nas opções “pouco” e “não utiliza” foram, de maneira geral, inexpressivas. Portanto, os médicos residentes do HU da UFSC elaboraram suas questões clínicas para problemas relacionados ao tratamento do paciente, seguido do diagnóstico. A solução das questões clínicas está frequentemente relacionada com o aumento do nível de conhecimento, ou seja, os médicos precisam de mais informações para melhor direcionar a prática clínica. Perspectivas em Ciência da Informação, v.16, n.3, p.232-254, jul./set. 2011 3 As necessidades informacionais na prática clínica [...] eu utilizo o critério da fonte de informação, aquilo que chega mais próximo possível [...] da melhor terapêutica que existe no momento (E01). [São usados os] consensos pra ajudar na decisão terapêutica dos pacientes. (E05). [São usados os] consensos pra ajudar na decisão terapêutica dos pacientes. (E05). Também surgem nos depoimentos, questões relacionadas ao diagnóstico, segundo item indicado pelos médicos residentes do HU da UFSC, conforme as falas ilustrativas transcritas abaixo: Pesquiso o que tem mais recente na literatura, em relação aos consensos na prática clínica, em relação àquela doença que estou suspeitando no paciente (E05). Contribuíram pra diagnóstico. O diagnóstico estava difícil pra se fazer. Através de testes que eu aprendi nestes artigos, deu pra definir o diagnóstico do paciente (E07). Na literatura nacional, a pesquisa de Martinez-Silveira (2005) aponta que as questões mais importantes estavam relacionadas ao diagnóstico, seguido do tratamento. Alguns resultados presentes em literatura estrangeira corroboram com os resultados encontrados nesta pesquisa, quando indicam que as questões estão relacionadas com a terapêutica e o diagnóstico (DEE; BLAZEK, 1993; ELY et al., 1999; GREEN; CIMPI; ELLIS, 2000). Smith (1996), em trabalho de revisão que incluiu 13 pesquisas, concluiu que a maioria das questões clínicas estava relacionada com o tratamento do paciente. Para os médicos residentes do HU, a solução da questão clínica (GRÁF. 1) está frequentemente relacionada ao aumento do seu nível de conhecimento (86,84%, n=33), definição de intervenção no paciente (63,16%,n=24), passar recomendações para o paciente (28,95%, n=11), a modificar decisão anterior (10,53%, n=4) e para programar retorno do paciente (7,89%, n=3). 237 Perspectivas em Ciência da Informação, v.16, n.3, p.232-254, jul./set. 2011 O uso da informação e a prática clínica de médicos residentes Maria Gorete Monteguti Savi; Edna Lucia da Silva Gráfico 1 - Aplicabilidade da solução da questão clínica pelos médicos residentes do HU da UFSC 0 10 20 30 40 50 60 70 80 90 100 Intervencão no paciente Recomendacões ao paciente Programar retorno do paciente Aumentar conhecimento Modificar decisão Outros Percentual de respostas Frequentemente Ás vezes Pouco Não utiliza Fonte: Dados da pesquisa. 4 A seleção de fontes e de canais de informação Atualmente, a grande quantidade de informação disponível na área médica tem exigido do médico o desenvolvimento de sua capacidade crítica para avaliar as fontes de informação disponíveis, a fim de selecionar as que possam responder mais adequadamente suas questões clínicas, seja através de canais formais ou informais, de fontes de informação impressas ou eletrônicas. Para os médicos residentes do HU da UFSC que realizam atendimento ambulatorial, a informação é ponto de destaque na prática clínica, assumindo um papel primordial. A grande maioria, ou seja, 37 (97,36%) dos médicos residentes, considerou que a informação científica é muito importante na prática clínica e apenas um deles (2,63%) considerou “importante”. As opções que indicavam “pouco importante” e “não considera importante” não tiveram nenhuma indicação. 238 Perspectivas em Ciência da Informação, v.16, n.3, p.232-254, jul./set. 2011 O uso da informação e a prática clínica de médicos residentes O uso da informação e a prática clínica de médicos residentes O uso da informação e a prática clínica de médicos residentes Maria Gorete Monteguti Savi; Edna Lucia da Silva Maria Gorete Monteguti Savi; Edna Lucia da Silva Maria Gorete Monteguti Savi; Edna Lucia da Silva Os depoimentos, abaixo, reforçam a constatação de que a informação é muito importante para os médicos residentes, seja para esclarecer ou para decidir qual conduta médica tomar: Contribuíram pra tomar a conduta, não necessariamente pra mudar uma decisão anterior (E10). Contribui bastante. Porque a gente estava numa contradição, porque uma especialidade, no caso a [...] sugeria uma via de parto diferente da que a gente estava pensando pra esta paciente. E realmente no Cochrane a gente pode comprovar por metanálise que um número muito grande de pacientes com o mesmo problema igual à nossa [paciente], que a via de parto que a gente optou era a melhor pra aquela paciente. Foi bem legal, bem importante (E11). De acordo com alguns depoimentos, percebe-se que a importância das fontes de informação está muito ligada à confirmação da resposta clínica, o que Smith (1996) indicou em sua pesquisa como a necessidade informacional que ultrapassa o conhecimento médico e se estende para uma sustentação psicológica, uma confirmação da resposta: Acho que sim [importância da informação]. Principalmente, pra gente ter certeza daquilo que a gente está fazendo (E06). Contribuíram bastante [as fontes de informação]. Só depois que a gente consultou [fontes de informação] é que foi tomada uma decisão. Perspectivas em Ciência da Informação, v.16, n.3, p.232-254, jul./set. 2011 4 A seleção de fontes e de canais de informação Antes disso, a gente estava bem reticente quanto a iniciar o tratamento, e foi depois desta consulta que foi começado (E08). A questão referente aos principais meios que os médicos residentes utilizavam no processo de busca da informação (GRÁF. 2), para resolver suas questões clínicas, foi elucidada com base na indicação de prioridades. Com prioridade 1 (mais importante), a Internet foi apontada por 68,42% (n=26), seguida da consulta ao acervo particular, com 13,16% (n=5) e da junção das opções: bibliotecas, contato com pares e contato com médicos da instituição, incluindo preceptores, com 18,41% (n=7). A opção “contato com médicos fora da instituição” não foi indicada com prioridade 1. 239 Perspectivas em Ciência da Informação, v.16, n.3, p.232-254, jul./set. 2011 O uso da informação e a prática clínica de médicos residentes Maria Gorete Monteguti Savi; Edna Lucia da Silva Gráfico 2 – Busca da informação: canais de informação utilizados pelos médicos residentes do HU da UFSC 0 10 20 30 40 50 60 70 80 Prioridade 1 Prioridade 2 Prioridade 3 Percentual de respostas Internet Bibliotecas Pares M édicos da instituição M édicos fora da instituição Acervo Particular Outros Fonte: Dados da pesquisa. Gráfico 2 – Busca da informação: canais de informação utilizados pelos médicos residentes do HU da UFSC Fonte: Dados da pesquisa. Este dado referente ao expressivo uso da Internet como canal de acesso à informação entre os profissionais da área da saúde já foi apontado em pesquisas mais recentes, que abordam exclusivamente esse tema, tais como a de Devitt e Murphy (2004), Cuenca e Tanaka (2005), Oliveira e Silva (2005), Renwick (2005) e Gómez-Tello et al.(2006). Nas entrevistas realizadas com os médicos do HU, o uso da Internet como canal de informação é claramente evidenciado: Quando eu precisei, a última vez que eu consultei foi a Internet (E04). Quando eu precisei, a última vez que eu consultei foi a Internet (E04). [...] mas na maioria das vezes [a consulta] foi na fonte on-line (E06). Eu tenho procurado bastante em Internet, mas nos meios que eu considero confiáveis, sites de revistas: New England e Jama, ou então o UptoDate. Ou acabo consultando os colegas pela facilidade, afinal de contas está todo mundo junto e acaba discutindo as coisas (E08) Internet (E04). [...] mas na maioria das vezes [a consulta] foi na fonte on-line (E06). [...] mas na maioria das vezes [a consulta] foi na fonte on-line (E06). Perspectivas em Ciência da Informação, v.16, n.3, p.232-254, jul./set. 2011 4 A seleção de fontes e de canais de informação [...] mas na maioria das vezes [a consulta] foi na fonte on-line (E06). Eu tenho procurado bastante em Internet, mas nos meios que eu considero confiáveis, sites de revistas: New England e Jama, ou então o UptoDate. Ou acabo consultando os colegas pela facilidade, afinal de contas está todo mundo junto e acaba discutindo as coisas (E08). Eu tenho procurado bastante em Internet, mas nos meios que eu considero confiáveis, sites de revistas: New England e Jama, ou então o UptoDate. Ou acabo consultando os colegas pela facilidade, afinal de contas está todo mundo junto e acaba discutindo as coisas (E08). Porém, com relação à Internet, mesmo tendo sido considerada pelos médicos residentes do HU como a fonte mais utilizada, percebe-se que a questão da confiabilidade da informação encontrada nesse ambiente é apontada como fator preocupante pelos médicos residentes. Com prioridade 2 prevaleceu o contato com os pares (outros médicos residentes) e com os médicos do próprio hospital, nos quais se enquadra a figura do preceptor. Eles foram os mais representativos, respectivamente com 28,94% (n=11) e 23,68% (n=9), conforme Gráfico 2. Esse dado é compatível com os dados indicados na pesquisa de Haug (1997), que também apontou os pares como a opção mais indicada com prioridade 2. 240 Perspectivas em Ciência da Informação, v.16, n.3, p.232-254, jul./set. 2011 O uso da informação e a prática clínica de médicos residentes O uso da informação e a prática clínica de médicos residentes Maria Gorete Monteguti Savi; Edna Lucia da Silva Maria Gorete Monteguti Savi; Edna Lucia da Silva As fontes de informação ajudam, nesse caso, a formar uma opinião clínica abalizada e permitem que a consulta e a discussão do caso, com profissionais mais experientes, sejam mais produtivas. Nas falas abaixo, essa afirmação é ratificada, já que os pesquisados inicialmente fazem menção ao uso da fonte de informação, para, só depois, indicarem a procura pelos preceptores e pares: Bom, uma coisa que tenho utilizado bastante é o Uptodate [...] Também procurei, no caso, o staff e o residente mais graduado (E08). Bom, uma coisa que tenho utilizado bastante é o Uptodate [...] Também procurei, no caso, o staff e o residente mais graduado (E08). A gente consulta guidelines, também a opinião dos chefes. E é basicamente isto. A gente consulta banco de dados [...] quando tem casos mais específicos tem que ir fazer a pesquisa (E09). 1 Nas pesquisas indicadas, categorizou-se por “outro médico” os pares, os médicos da instituição e médicos de outras instituições. 4 A seleção de fontes e de canais de informação Em relação à consulta que os médicos residentes fazem aos pares, somente em um segundo momento, parece estar associado à responsabilidade que pode gerar para os pacientes uma decisão equivocada, já que, mesmo após a informação ter sido repassada por outro médico residente, o médico procura referendá-la na literatura. Abaixo, destacam-se algumas falas que ilustram essa situação: [A informação] sugerida por colegas não era bem o que estava escrito nas fontes de informação (E01). Colegas [consulto] mais pela facilidade. Tenho medo de pegar alguma coisa errada e trazer para a prática. Confiabilidade em primeiro lugar [...] Foi depois de olhar esta literatura um pouco mais que achei o artigo no Uptodate, mas foi um residente mais graduado que achou uma revisão, se não me engano foi no Chest, uma revista um pouco mais conceituada, e acabamos usando aquela informação e depois fui olhar no site para ver se a informação batia. Mas, é bem comum quando alguém toma uma decisão ou pra corroborar com o que a gente fez, dar uma olhadinha depois (E08). No Brasil, a pesquisa de Breglia (1989) indica que os pares e outros médicos tiveram baixa expressão como fonte de informação, mas são muito procurados, especialmente os preceptores, como um guia para indicar qual a melhor fonte de informação a ser utilizada. Os dados da pesquisa de Martínez-Silveira (2005) apontam os preceptores e os pares como os mais consultados, seguidos da coleção particular; este último item coincide com esta pesquisa, que também indicou o acervo particular como o segundo mais importante meio de acesso às fontes de informação. Na pesquisa de Martínez-Silveira (2005), a Internet foi indicada como o terceiro canal de informação mais utilizado, enquanto que os médicos residentes do HU a apontaram como o mais utilizado. Na literatura estrangeira, algumas pesquisas (STINSON; MUELLER, 1980; DEE; BLAZEK, 1993; RAMOS; LINSCHEID; SCHAFER, 2003) deixam Perspectivas em Ciência da Informação, v.16, n.3, p.232-254, jul./set. 2011 241 O uso da informação e a prática clínica de médicos residentes Maria Gorete Monteguti Savi; Edna Lucia da Silva Maria Gorete Monteguti Savi; Edna Lucia da Silva claro que o contato com outro médico1 é indicado como o canal de informação mais importante, sendo que outras pesquisas, tais como Woolf e Benson (1989), Osherhoff et al. (1991), Cogdill et al. (2000) e Green, Ciampi e Ellis (2000) consideram como segunda opção mais importante. Perspectivas em Ciência da Informação, v.16, n.3, p.232-254, jul./set. 2011 4 A seleção de fontes e de canais de informação claro que o contato com outro médico1 é indicado como o canal de informação mais importante, sendo que outras pesquisas, tais como Woolf e Benson (1989), Osherhoff et al. (1991), Cogdill et al. (2000) e Green, Ciampi e Ellis (2000) consideram como segunda opção mais importante. Diferentemente desta pesquisa, a de Gorman (2001) identificou que os médicos que atendiam em comunidades rurais e urbanas pouco utilizavam os computadores e as bases de dados, preferindo contato com os pares. Na prioridade 3, conforme mostra o GRÁF. 2, obtiveram indicações: consulta ao acervo particular com dez incidências (26,32%), consulta aos médicos da instituição com nove (23,68%), contato com colegas com oito (21,05%), consulta à Internet e à biblioteca, ambas com cinco cada (13,16%) e consulta a médicos de fora da instituição com uma (2,63%). O baixo índice de utilização da biblioteca indicado nesta pesquisa já foi percebido em pesquisas anteriores. Na pesquisa de Martínez-Silveira (2005), a biblioteca ocupou o 6º. lugar na preferência dos médicos residentes. Na literatura estrangeira, Stinson e Mueller (1980) e Dee e Blazek (1993) também constataram a baixa utilização das bibliotecas. Quando se depara com dificuldades para encontrar informações que possam responder sua questão clínica, ou seja, em um segundo momento do processo de busca de informação, o médico residente recorre a agentes que possam ajudá-lo. Na pesquisa, foram apontados como agentes desse processo (prioridade 1): preceptores (63,15%, n=24), pares (34,31%, n=13) e outros médicos da instituição (2,63%, n=1), conforme GRÁF. 3. Com relação à importância do preceptor no processo de busca da informação, novamente ele aparece como mais importante, mas na hora da dificuldade. Em análise anterior, quando foram colocados em questão os meios utilizados na busca de informação, o preceptor não obteve dados expressivos dentre os indicados como prioridade 1, mas assumiu a primeira posição dentre os indicados na prioridade 2. Os bibliotecários só foram indicados na prioridade 3, com frequência de duas respostas (5,26%). Portanto, novamente, obteve-se outro indicador que leva ao questionamento da posição da biblioteca e do bibliotecário nesse processo. A opção “outros” não foi indicada por nenhum dos pesquisados. 242 Perspectivas em Ciência da Informação, v.16, n.3, p.232-254, jul./set. 4 A seleção de fontes e de canais de informação 2011 O uso da informação e a prática clínica de médicos residentes Maria Gorete Monteguti Savi; Edna Lucia da Silva O uso da informação e a prática clínica de médicos residentes O uso da informação e a prática clínica de médicos residentes Maria Gorete Monteguti Savi; Edna Lucia da Silva Gráfico 3 - Agentes de apoio informacional dos médicos residentes do HU da UFSC 0 10 20 30 40 50 60 70 80 Pares Preceptores Professionais da instituição Profissionais externos Bibliotecários Outros Percentual de respostas Prioridade 1 Prioridade 2 Prioridade 3 Fonte: Dados da pesquisa. Gráfico 3 - Agentes de apoio informacional dos médicos residentes do HU da UFSC Fonte: Dados da pesquisa. Na questão referente ao grau de frequência com que os médicos residentes utilizam as fontes de informação (GRÁF. 4), obteve-se nesta pesquisa, a indicação de uso frequente para os periódicos (84,21%), textbooks e handbooks (73,68%), guidelines (73,68%) e preceptores (73,68%) quase que de maneira eqüitativa, apenas com um pouco mais de ênfase nos periódicos. p Na prática clínica, as questões podem ser classificadas como básicas (background) e aplicadas (foreground). As respostas para questões básicas dependem de conhecimentos comumente relacionadas com etiologia, patogênese de doenças, mecanismos de ação e efeitos adversos de medicamentos. A questão aplicada (foreground) tem relação mais direta com a conduta a ser adotada, seja a conduta terapêutica ou conduta diagnóstica e, também, pode estar relacionada com o conhecimento sobre fatores indicadores de prognóstico. (BERNARDO; NOBRE; JATENE, 2003). De maneira geral, os dados da pesquisa apontam que, para os médicos residentes do HU da UFSC resolverem suas questões clínicas, são necessárias informações em nível de foreground em maior escala, geralmente disponíveis em periódicos técnico-científicos e guidelines e, em nível de background, geralmente disponíveis em textbooks (exceção para os baseados em evidência) e handbooks. Sobre o uso frequente dos periódicos, constata-se que, ao longo do tempo, eles sempre assumiram uma posição de destaque na comunidade científica, porém, foram, paulatinamente, sendo incorporados no ambiente da prática profissional. Em relação ao uso dos periódicos no metiêr médico, a afirmação de Coutinho e Li (2004, p. 30) respalda o resultado desta pesquisa: O processo de formação e transmissão do conhecimento sofreu uma mudança radical nas últimas décadas. Se, no passado, o tratado médico era a fonte de referência por excelência, hoje os periódicos científicos tomaram seu lugar, dada a grande rapidez na expansão do conhecimento. Perspectivas em Ciência da Informação, v.16, n.3, p.232-254, jul./set. 2011 4 A seleção de fontes e de canais de informação O processo de formação e transmissão do conhecimento sofreu uma mudança radical nas últimas décadas. Se, no passado, o tratado médico era a fonte de referência por excelência, hoje os periódicos científicos tomaram seu lugar, dada a grande rapidez na expansão do conhecimento. 243 Perspectivas em Ciência da Informação, v.16, n.3, p.232-254, jul./set. 2011 O uso da informação e a prática clínica de médicos residentes O uso da informação e a prática clínica de médicos residentes Maria Gorete Monteguti Savi; Edna Lucia da Silva Maria Gorete Monteguti Savi; Edna Lucia da Silva Os preceptores (73,68%), pares (60,53%) e outros especialistas (50,00%) também são consultados frequentemente. Porém, quando se leva em consideração os dados analisados anteriormente, pode-se concluir que os mesmos são consultados com frequência, mas não em um primeiro momento. Na literatura estrangeira, a pesquisa de Green, Ciampi e Ellis (2000) indicou que 28% das questões eram resolvidas com o uso de literatura de background e 66% de foreground. De maneira geral, as mesmas fontes de informação indicadas na presente pesquisa aparecem como mais utilizadas na literatura revisada, porém, não necessariamente na mesma ordem. Gráfico 4 - Fontes de informação: uso pelos médicos residentes do HU da UFSC 0 10 20 30 40 50 60 70 80 90 Frequentemente Às vezes Muito pouco Não consulta Percentual de respostas Textbooks/Handbooks Artigos de periódicos Guidelines Eventos Teses Boletins Videos Outros recursos eletrônicos Pares Preceptores Especialistas Outras fontes Fonte: Dados da pesquisa. Fonte: Dados da pesquisa. No Brasil, nas pesquisas de Martínez-Silveira (2005) e de Lima (2005), os livros aparecem como a fonte de informação mais consultada, seguidos dos periódicos. Porém, deve ser ressaltado que, em sua pesquisa, Lima (2005) colocou em uma única listagem as fontes de informação em seus diferentes suportes (por exemplo: CD-ROMs, slides, fitas de videocassete), juntamente com bases de dados específicas (por exemplo: Medline, Lilacs, Cochrane, SciELO), o que torna os resultados pouco reveladores. Breglia (1989) aponta que a fonte mais utilizada foram os textbooks, seguidos das bibliografias. Algumas pesquisas utilizam “livros” para designar os textbboks, handbooks, guidelines e outras publicações avulsas. A revisão de 12 pesquisas realizadas por Haug (1997), entre o período de 1975 a e 1992, indica que os periódicos e os livros eram as fontes de informação mais consultadas. Coumou (2006), em revisão realizada no período de 1992 a 2005, indica os pares e as fontes de informação impressa. Perspectivas em Ciência da Informação, v.16, n.3, p.232-254, jul./set. 2011 4 A seleção de fontes e de canais de informação A pesquisa de Green, Ciampi e Ellis (2000) indica os textbooks, seguidos dos periódicos e dos preceptores. 244 Perspectivas em Ciência da Informação, v.16, n.3, p.232-254, jul./set. 2011 O uso da informação e a prática clínica de médicos residentes O uso da informação e a prática clínica de médicos residentes Maria Gorete Monteguti Savi; Edna Lucia da Silva Maria Gorete Monteguti Savi; Edna Lucia da Silva Na presente pesquisa, na opção “às vezes” são consultadas, foram indicadas as fontes de informação: recursos eletrônicos on-line, como imagens e sites institucionais com 52,63%, pares e especialistas ambos com 31,58%, e guidelines e textbooks/handbooks ambos com 26,32%. Dentre as fontes indicadas como “muito pouco” consultadas, temos as teses (65,79%), os trabalhos apresentados em eventos (57,89%) e os boletins informativos (50%). Entre as indicações de fontes não consultadas, temos outras fontes (84,21%), os vídeos (39,47%), os boletins informativos (31,58%), as teses, dissertações e trabalhos acadêmicos (26,32%), os trabalhos apresentados em eventos (18,42%) e outros recursos eletrônicos on-line (2,63%). O alto índice de indicação dos médicos residentes do HU para a opção “não consulta outras fontes de informação” (84,21%), indica que as fontes de informação utilizadas, em maior ou menor escala, são as referendadas na questão. Os que responderam que utilizam outras fontes de informação com a opção “às vezes” (2,63%) e com consulta “muito pouco” (10,53%), não fizeram indicação de quais são essas outras fontes utilizadas. Pelos dados desta pesquisa, em relação ao baixo índice de uso das teses, dissertações e trabalhos acadêmicos (indicação de “muito pouca” consulta – 65,79%), pode-se inferir que não existe uma tradição de uso dessas fontes pelos médicos residentes do HU da UFSC. Esse mesmo resultado foi observado na pesquisa realizada por Lima (2005), na qual o uso das teses e dissertações impressas ocupou a 13ª colocação, e o uso das teses e dissertações eletrônicas a 16ª. colocação. Na pesquisa de Breglia (1989), as teses e dissertações não foram incluídas entre as fontes consultadas em um primeiro momento; somente em um segundo momento elas aparecem, com apenas 0,9%. Na literatura estrangeira analisada, essa variável não faz parte das abordagens realizadas. Gráfico 5 - Médicos residentes do HU da UFSC e o uso de sistemas informatizados de informação 4 A seleção de fontes e de canais de informação O processo de busca da informação, por meio de bases de dados, tem sido alvo de muitas pesquisas, desde o início deste século (por exemplo: DEVITT; MURPHY, 2004; REWINCK, 2005, CUENCA; TANAKA, 2003; 2005 e GÓMEZ-TELLO et al., 2006), considerando o momento ímpar liderado pelo desenvolvimento das TIC e pela quantidade de informação técnico-científica produzida e disponibilizada diariamente. Para Bernardo, Nobre e Jatene (2004, p. 106), “as bases de dados informatizadas e acessadas pela Internet têm se constituído na principal fonte de busca de informação” para o médico. A pesquisa com os médicos residentes do HU da UFSC sobre o uso de bases de dados (GRÁF. 5) indicou acesso frequente ao UptoDate (71,05%), Portal de Periódicos da CAPES (65,79%), PubMed – incluindo MEDLINE (63,16%), bases de dados da BIREME (39,47%), ferramentas de busca tipo Google, Cadê, Yahoo (26,32%), SciELO (23,68%), Cochrane Library (21,05%), bases de dados Web of Science e EBMR (ambas com 5,26% cada) e MD Consult (2,63%). Gráfico 5 - Médicos residentes do HU da UFSC e o uso de sistemas informatizados de informação 245 Perspectivas em Ciência da Informação, v.16, n.3, p.232-254, jul./set. 2011 O uso da informação e a prática clínica de médicos residentes Maria Gorete Monteguti Savi; Edna Lucia da Silva O uso da informação e a prática clínica de médicos residentes O uso da informação e a prática clínica de médicos residentes Maria Gorete Monteguti Savi; Edna Lucia da Silva 0 10 20 30 40 50 60 70 80 90 Frequentemente Às vezes Muito Pouco Não consulta Percentual de respostas Bireme PubMed Cochrane Portal de Periódicos/ CAPES Scielo Web of Science UpToDate EBMR ACP Medicine MD_Consult Ferramenta de busca Outros Fonte: Dados da pesquisa Fonte: Dados da pesquisa Na entrevistas, o uso do Uptodate e PubMed foi referenciado como fontes de informação consultadas para resolver uma questão clínica relevante: No PubMed apareceu vários artigos que me interessavam, selecionei os mais recentes e procurei fontes européias, porque já conhecia as americanas, para comparar as duas (E03). [Consulto] na Internet, via PubMed. O que mais uso na minha prática clínica é usar o PubMed para ver o que tenho mais dúvida (E05). Nas fontes que geralmente eu consulto [...] PubMed ou UptoDate e algumas coisas em livros (E06). Bom, uma coisa que eu tenho utilizado bastante é o Uptodate, que o hospital assinou há pouco tempo. Perspectivas em Ciência da Informação, v.16, n.3, p.232-254, jul./set. 2011 4 A seleção de fontes e de canais de informação Primeiro por ser confiável e segundo por estar disponível facilmente em qualquer computador do hospital, e está disponível também no nosso quarto [quarto dos residentes], onde tem uma impressora e é muito fácil de imprimir. [...] UptoDate, que é um site idôneo que até hoje eu não vi comentário de erro nas revisões deles (E08). Como a base de dados Uptodate, cujo conteúdo é conciso e dirigido especialmente para quem atua na prática clínica, foi assinada no ano de 2007 pela UFSC e disponibilizada nos computadores do HU, deve ter desencadeado seu alto índice de uso. Na pesquisa de Ospina, Herault e Cardona (2005), foi revelado que os médicos acessavam cinco vezes mais as bases de dados gratuitas do que as que requerem pagamento. Além disso, outros diferenciais da base de dados Uptodate, que estão contidos em uma das falas citadas acima, referem-se à questão da confiabilidade da informação e à sua disponibilidade de acesso. 246 Perspectivas em Ciência da Informação, v.16, n.3, p.232-254, jul./set. 2011 O uso da informação e a prática clínica de médicos residentes O uso da informação e a prática clínica de médicos residentes Maria Gorete Monteguti Savi; Edna Lucia da Silva Maria Gorete Monteguti Savi; Edna Lucia da Silva Em relação ao PubMed, serviço criado pela NLM, que inclui as bases de dados MEDLINE, é importante ressaltar que o nome desse serviço, para a grande maioria dos médicos, é sinônimo da base de dados MEDLINE. O alto índice de utilização do Portal de Periódicos da CAPES demonstra que esse instrumento está, cada vez mais, sendo incorporado no meio acadêmico e profissional. As bases de dados com literatura nacional e latino-americana disponíveis no site da BIREME são utilizadas com certa cautela. As bases de dados da BIREME e a SciELO, mesmo que tenham obtido, respectivamente, a quarta e a sexta indicações de uso frequente, apresentaram uma grande diferença nos percentuais, quando comparadas com o uso do UptoDate e do PubMed. O depoimento de um entrevistado pode ser um indicador de motivo dessa diferença: Ah! O Scielo que é outra fonte [consultada] e outra a Bireme, às vezes não tem artigos bons como na literatura inglesa2 (E03). Na pesquisa de Martínez-Silveira (2005), a base de dados MEDLINE teve o mais alto índice de utilização (71%), seguida pela LILACS e Cochrane Library, ambas com 12%. 2 Nota dos autores: “literatura inglesa”, referindo-se à literatura disponível em inglês. 4 A seleção de fontes e de canais de informação Já na de Lima (2005), considerando- se somente as bases de dados acessadas, tem-se: MEDLINE (ocupa a 3ª. posição na listagem geral, que inclui também fontes impressas), LILACS (6ª. posição), Cochrane (7ª. posição), Outras bases da Bireme (8ª. posição), SciELO (14ª. posição), OVID (15ª. posição) e ProBE (16ª. – última posição). p ç ) Entre os sistemas informatizados indicados como “às vezes” consultados, temos as ferramentas de busca (47,37%), Cochrane Library (44,74%), BIREME (39,470%), PubMed e SciELO (ambos com 28,95% cada) e Portal de Periódicos da CAPES (23,68%) e UptoDate (21,05%). Percebe-se que as fontes de informação que foram indicadas como as mais utilizadas aparecem com os menores índices na opção “às vezes”. Um dado que surpreendeu foi a indicação de ferramentas de busca como a primeira opção em “às vezes” é consultada, além de constarem entre os usados “frequentemente”, indicando que, mesmo utilizando fontes de informação de caráter científico, existia a procura de informação em mecanismos gerais de busca. Porém, considerando o que foi apontado anteriormente sobre a questão da confiabilidade dos sites, supõe-se que os médicos residentes, quando recorriam às ferramentas de busca, deviam empregar algum critério de seleção para uso das informações encontradas. Dentre os sistemas indicados como “pouco consultados”, os mais apontados foram Web of Science e SciELO, respectivamente com 47,37% e 36,84%. Os sistemas de informação não consultados pelos médicos residentes do HU da UFSC tiveram alto índice para ACP Medicine e para outros sistemas (ambos com 84,21%), sendo seguidos do MD Consult Perspectivas em Ciência da Informação, v.16, n.3, p.232-254, jul./set. 2011 247 O uso da informação e a prática clínica de médicos residentes Maria Gorete Monteguti Savi; Edna Lucia da Silva Maria Gorete Monteguti Savi; Edna Lucia da Silva (68,42%), EBMR (60,53%) e Web of Science (36,84%). Como as bases de dados ACP Medicine, MD Consult e EBMR não são bases de dados de acesso livre e a UFSC não mantém assinatura, este pode ser outro indicador de que a disponibilidade gratuita da informação é fator primordial na seleção das fontes de informação, conforme observado anteriormente. (68,42%), EBMR (60,53%) e Web of Science (36,84%). Como as bases de dados ACP Medicine, MD Consult e EBMR não são bases de dados de acesso livre e a UFSC não mantém assinatura, este pode ser outro indicador de que a disponibilidade gratuita da informação é fator primordial na seleção das fontes de informação, conforme observado anteriormente. Perspectivas em Ciência da Informação, v.16, n.3, p.232-254, jul./set. 2011 Perspectivas em Ciência da Informação, v.16, n.3, p.232-254, jul./set. 2011 4 A seleção de fontes e de canais de informação Ospina, Herault e Cardona (2005), em pesquisa com médicos latino- americanos dos países de língua espanhola, identificaram algumas fontes de informação mais consultadas, que são: MEDLINE, motores de busca geral (Google, Yahoo e Altavista) e BIREME. g ( g ) O médico pode fazer uso da informação que foi selecionada e analisada antecipadamente, normalmente representada pelas fontes terciárias de informação, ou ele próprio realizar a seleção e análise da informação, através da consulta às fontes primárias ou secundárias. Na pesquisa, quando questionados sobre o tema, 26 (70,27%) dos médicos residentes do HU da UFSC informaram que utilizavam a informação já selecionada e analisada, e 11 (29,72%) preferiam realizar a seleção e análise da informação. Dessa forma, a maioria estava preferindo consultar fontes de informação terciárias, em detrimento do uso de publicações primárias e secundárias, que envolvem um processo mais complexo de seleção e análise da informação. Esse dado é confirmado na análise da questão anterior, quando se detectou que, dentre os sistemas informatizados de recuperação da informação, o UptoDate, base de dados que dispõe da informação já selecionada e analisada, apresentou o maior índice de utilização freqüente. Essa fonte foi complementada pelo PubMed que, dependendo dos critérios da estratégia de busca, pode, também, apresentar a informação já selecionada e analisada, também pelo acesso à Cochrane Library, que não teve forte indicação de uso frequente, mas que ocupou o segundo lugar na opção “às vezes”. Outro ponto enfocado na pesquisa está relacionado aos critérios que os médicos residentes utilizavam para escolher as fontes de informação. De acordo com o Gráfico 6, pode-se verificar que as respostas indicaram como muito importante a atualidade da informação com 84,21% (n=32), a confiabilidade da informação com 73,68% (n=28), a facilidade de acesso à informação com 71,05% (n=27), a facilidade de uso com 44,74% (n=17) e os custos da informação com 34,21% (n=13). Pelos dados analisados nas questões anteriores, já foi possível perceber, especialmente pelas entrevistas, a incidência de três variáveis preponderantes nos critérios de seleção de fontes de informação, que agora são confirmadas como as mais importantes: atualidade, confiabilidade e acessibilidade. Na opção “importante”, por ordem de indicação obteve-se: custos da informação representando 47,37% (n=18), facilidade de uso com 44,74% (n=17), confiabilidade com 23,68% (n=9), acessibilidade com 21,05% (n=8) e atualidade da informação com 15,79% (n=7). 248 Perspectivas em Ciência da Informação, v.16, n.3, p.232-254, jul./set. 4 A seleção de fontes e de canais de informação A busca da informação na prática clínica interage com uma diversidade de motivos que impulsionam o médico a priorizar a busca de respostas. p Nesta pesquisa, verificou-se que os médicos residentes buscavam informação para resolver uma questão clínica, considerando como fator máximo de prioridade a urgência do caso clínico em 47,37% (n=18), o interesse pelo caso em 28,95% (n=11), dúvidas sobre os procedimentos no caso clínico em 19,53% (n=4), caso raro em 7,89% (n=3) e receio de não realizar uma boa prática e receio de cometer erro médico em 2,63% cada (n=1 cada), conforme indicado no GRÁF. 7. Gráfico 7 - Busca da informação: fatores priorizados pelos médicos residentes do HU da UFSC 0 5 10 15 20 25 30 35 40 45 50 Urgência Expectativa do paciente Interesse pelo caso Caso raro Dúvida Receio prática Receio erro Outros Sem prioridade Percentual de respostas Prioridade 1 Prioridade 2 Prioridade 3 Fonte: Dados da pesquisa Pesquisa anterior, realizada na Bahia por Martínez-Silveira (2005), aponta que a dúvida ou insegurança no caso clínico (72,6%), a existência de caso raro (71,2%), a apresentação do caso clínico em reunião (67,1%) e o interesse pelo caso do paciente (65,8%) eram os principais motivos que levavam os médicos residentes à busca de informação. Na literatura estrangeira, Green, Ciampi e Ellis (2000) constataram que os motivos estavam relacionados com a expectativa do paciente e o receio de cometer erro médico, este último item podendo ter estreita ligação com o rigor da legislação americana para casos dessa natureza. Na pesquisa de Gorman e Helfand (1995), foi identificado o mesmo resultado da presente pesquisa, ou seja, a urgência do caso clínico foi o fator que mais contribuiu para a busca da informação. 4 A seleção de fontes e de canais de informação 2011 O uso da informação e a prática clínica de médicos residentes O uso da informação e a prática clínica de médicos residentes O uso da informação e a prática clínica de médicos residentes Maria Gorete Monteguti Savi; Edna Lucia da Silva A maioria dos médicos residentes do HU da UFSC, exatamente 32 respondentes (84,21%), indicou que não considera importante outros critérios além dos indicados na pesquisa. Gráfico 6 - Critérios de seleção das fontes de informação dos médicos residentes HU/UFSC 0 10 20 30 40 50 60 70 80 90 Muito importante Importante Pouco importante Não considera importante Percentual de respostas Acessibilidade Confiabilidade Atualidade Facilidade Custo Outros Fonte: Dados da pesquisa Fonte: Dados da pesquisa Nos preceitos da metodologia da MBE, quando se busca na literatura, especialmente nos artigos de periódicos, respostas para as questões clínicas, o médico se depara com diferentes tipos de pesquisa ou desenhos de pesquisa e necessita determinar a sua validade e sua aplicabilidade para o problema em questão. Quando instigados sobre o tema, os médicos residentes do HU da UFSC indicaram como mais importantes os artigos de revisão com 44,74% (n=17), as revisões sistemáticas e/ou metanálises com 28,95%(n=11), os ensaios clínicos com 23,68% (n=9) e 2,63% (n=1) informou não ter preferências. Pelas entrevistas, percebe-se que os artigos de revisão e as metanálises predominam nas falas dos entrevistados: Geralmente [o que consulto] são artigos de revisão. [...] Geralmente são revisões ou grandes metanálises (E04). Dou preferência para os livros de revisão, metanálises. Teoricamente são melhores (E06). [Utilizo] porque são artigos de revisão. Já selecionaram vários outros estudos, eles pegam os principais pra fazer os artigos de revisão. Por isto (E07). [Utilizo] Portal que apresenta metanálise, [porque] estão uma seleção de vários trabalhos que passam por critérios rigorosos de metodologia, que foram selecionados pra montar uma fonte um pouco maior (E11). O ensaio clínico, terceiro mais indicado pelos médicos residentes, teve seu uso ressaltado por um dos entrevistados: 249 Perspectivas em Ciência da Informação, v.16, n.3, p.232-254, jul./set. 2011 Maria Gorete Monteguti Savi; Edna Lucia da Silva O uso da informação e a prática clínica de médicos residentes Maria Gorete Monteguti Savi; Edna Lucia da Silva Geralmente eu utilizo o critério da fonte de informação, aquilo que chega mais próximo possível do ensaio clínico randomizado controlado duplo cego com placebo ou com a melhor terapêutica que existe no momento (E01). Perspectivas em Ciência da Informação, v.16, n.3, p.232-254, jul./set. 2011 5 Considerações finais Notadamente, os médicos residentes do HU da UFSC estão conscientizados da importância da informação científica e já incorporaram o seu uso, como fator determinante do processo de decisão clínica. Dos resultados alcançados, foi possível perceber que, durante sua prática ambulatorial, o médico residente se depara com questões classificadas como foreground, mais ligadas a aspectos clínicos do tratamento do paciente, em detrimento de questões ligadas a aspectos básicos, tais como epidemiologia e etiologia. Tais resultados permitem inferir que o formando de medicina está saindo para o mercado de trabalho com um bom nível de conhecimentos, que suprem suas necessidades informacionais, relacionadas a aspectos básicos da profissão. p p Dentre as fontes de informação utilizadas com mais frequência pelos médicos residentes do HU da UFSC, foram indicados os periódicos, com pequena vantagem sobre os textbooks e handbooks, os guidelines e os preceptores. Dentre as fontes com menor indicação de uso estavam os vídeos, os boletins informativos, as teses, dissertações e os trabalhos acadêmicos e os apresentados em eventos. Com relação aos sistemas informatizados utilizados frequentemente, verificou-se a preferência pelo UptoDate, Portal de Periódicos da CAPES e PubMed/Medline. A preferência por informação já selecionada e analisada em idioma inglês, a confiabilidade da fonte de informação e a gratuidade no acesso foram, em maior ou menor grau, determinantes na indicação das bases de dados. As bases de dados nacionais, como as da BIREME e a SciELO, eram utilizadas com mais cautela. O uso de fontes terciárias teve predominância, através da indicação de uso frequente da base de dados UptoDate, sendo que também tiveram destaque as fontes primárias, através do Portal de Periódicos da CAPES, e as fontes secundárias, com a indicação do PubMed. Com relação ao uso das bases de dados, verificou-se que o médico residente do HU da UFSC dava preferência à busca por termos livres e estava deixando de usar os recursos disponíveis para tornar a recuperação da informação mais produtiva, em termos de relevância e pertinência. Essa constatação abre espaço para as bibliotecas oferecerem treinamento a usuários para uso das bases de dados, especialmente para explorar as potencialidades que cada sistema oferece. Da análise empreendida, mesmo não sendo o foco principal desta pesquisa, vale destacar a pouca relevância dada à biblioteca nesse processo. 4 A seleção de fontes e de canais de informação Gráfico 7 - Busca da informação: fatores priorizados pelos médicos residentes do HU da UFSC Gráfico 7 - Busca da informação: fatores priorizados pelos médicos residentes do HU da UFSC 0 5 10 15 20 25 30 35 40 45 50 Urgência Expectativa do paciente Interesse pelo caso Caso raro Dúvida Receio prática Receio erro Outros Sem prioridade Percentual de respostas Prioridade 1 Prioridade 2 Prioridade 3 Fonte: Dados da pesquisa Fonte: Dados da pesquisa Fonte: Dados da pesquisa Pesquisa anterior, realizada na Bahia por Martínez-Silveira (2005), aponta que a dúvida ou insegurança no caso clínico (72,6%), a existência de caso raro (71,2%), a apresentação do caso clínico em reunião (67,1%) e o interesse pelo caso do paciente (65,8%) eram os principais motivos que levavam os médicos residentes à busca de informação. Na literatura estrangeira, Green, Ciampi e Ellis (2000) constataram que os motivos estavam relacionados com a expectativa do paciente e o receio de cometer erro médico, este último item podendo ter estreita ligação com o rigor da legislação americana para casos dessa natureza. Na pesquisa de Gorman e Helfand (1995), foi identificado o mesmo resultado da presente pesquisa, ou seja, a urgência do caso clínico foi o fator que mais contribuiu para a busca da informação. 250 Perspectivas em Ciência da Informação, v.16, n.3, p.232-254, jul./set. 2011 O uso da informação e a prática clínica de médicos residentes O uso da informação e a prática clínica de médicos residentes Maria Gorete Monteguti Savi; Edna Lucia da Silva Maria Gorete Monteguti Savi; Edna Lucia da Silva Maria Gorete Monteguti Savi; Edna Lucia da Silva Perspectivas em Ciência da Informação, v.16, n.3, p.232-254, jul./set. 2011 5 Considerações finais Os resultados mostram que parece haver um importante papel a ser desempenhado pelo bibliotecário que atua na área médica, de modo que o planejamento dos serviços da biblioteca seja estruturado a partir de estudos de usuários que enfoquem as necessidade informacionais e as dificuldades encontradas pelo usuário na busca e recuperação da informação. Para tal, faz-se necessário investir no desenvolvimento de habilidades e de conhecimentos específicos do bibliotecário para essa atuação. Perspectivas em Ciência da Informação, v.16, n.3, p.232-254, jul./set. 2011 251 Maria Gorete Monteguti Savi; Edna Lucia da Silva O uso da informação e a prática clínica de médicos residentes O uso da informação e a prática clínica de médicos residentes Referências BERNARDO, W. M.; NOBRE, M. R. C.; JATENE, F. B. A prática clínica baseada em evidências: parte II - buscando as evidências em fontes de informação. Revista da Associação Médica Brasileira, Rio de Janeiro, v. 50, n. 1, p.104-108, jan./mar. 2004. Disponível em: <http://www.sci.br/pdf/ramb/v50n1/ a45v50n1.pdf>. Acesso em: 20 jul. 2006. BREGLIA, V. L. A. A comunicação da informação na residência médica. 1989. 203 f. Dissertação (Mestrado em Ciência da Informação)- IBICT/Universidade Federal do Rio de Janeiro, Rio de Janeiro, 1989. BREGLIA, V. L. A. A comunicação da informação na residência médica. 1989. 203 f. Dissertação (Mestrado em Ciência da Informação)- IBICT/Universidade Federal do Rio de Janeiro, Rio de Janeiro, 1989. COGDILL, K. W. et al. Information needs and information seeking in community medical education. Academic Medicine, v. 75, n. 5, p. 484- 486, 2000. COUMOU, H. C. H. How do primary care physicians seek answers to clinical questions? A literature review. Journal of the Medical Library Association, Chicago, v. 94, n. 1, p. 55-60, jan. 2006. COUTINHO, M.; LI, S. M. Como ter acesso à literatura médica. In: DRUMMOND, J. P.; SILVA, E.; COUTINHO, M. Medicina baseada em evidências: novo paradigma assistencial e pedagógico. 2. ed. São Paulo: Atheneu, 2004. cap. 2. COVELL, D. G.; UMAN, G. C.; MANNING, P. R. Information needs in office practice: are they being met? Annals of Internal Medicine, Philadelphia, v. 103, n. 4, p.596-599, Oct. 1985. CUENCA, A. M. B.; TANAKA, A. C. D. As novas tecnologias na comunicação científica: o uso da Internet pela comunidade de docentes da área de saúde pública. In: ENCONTRO NACIONAL DE PESQUISA EM CIÊNCIA DA INFORMAÇÃO, 5., 2003, Belo Horizonte. Anais... Belo Horizonte: UFMG, 2003. CUENCA, A. M. B.; TANAKA, A. C. D. As novas tecnologias na comunicação científica: o uso da Internet pela comunidade de docentes da área de saúde pública. In: ENCONTRO NACIONAL DE PESQUISA EM CIÊNCIA DA INFORMAÇÃO, 5., 2003, Belo Horizonte. Anais... Belo Horizonte: UFMG, 2003. ______. Influência da internet na comunidade acadêmico-científica da área de saúde pública. Revista de Saúde Pública, São Paulo, v. 39, n. 5, p. 840-846, 2005. ______. Influência da internet na comunidade acadêmico-científica da área de saúde pública. Revista de Saúde Pública, São Paulo, v. 39, n. 5, p. 840-846, 2005. DEE, C.; BLAZEK, R. Information needs of the rural physician: a descriptive study. Bulletin of the Medical Library Association, Chicago, v. 81, n. 3, p. 259-264, 1993. Referências DEE, C.; BLAZEK, R. Information needs of the rural physician: a descriptive study. Bulletin of the Medical Library Association, Chicago, v. 81, n. 3, p. 259-264, 1993. DEVITT, N.; MURPHY, J. A survey of the information management and technology training needs of doctors in an acute NHS trust in the United Kingdom. Health Information and Libraries Journal, v. 21, n. 3, p. 164- 172, 2004. ELY, J. W. et al. Analysis of questions asked by family doctors regarding patient care. British Medical of Journal, London, v.319, p. 358-361, Aug. 1999. Disponível em: <http://www.bmj.com/cgi/reprint/319/7206/358>. Acesso em: 27 mar. 2007. ELY, J. W. et al. Analysis of questions asked by family doctors regarding patient care. British Medical of Journal, London, v.319, p. 358-361, Aug. 1999 Disponível em: <http://www bmj com/cgi/reprint/319/7206/358> patient care. British Medical of Journal, London, v.319, p. 358-361, Aug. 1999. Disponível em: <http://www.bmj.com/cgi/reprint/319/7206/358>. Acesso em: 27 mar. 2007. Perspectivas em Ciência da Informação, v.16, n.3, p.232-254, jul./set. 2011 252 O uso da informação e a prática clínica de médicos residentes Maria Gorete Monteguti Savi; Edna Lucia da Silva GOMÉZ-TELLO, V. et al. Uso de Internet y recursos electrónicos entre médicos intensivistas españoles: primera encuesta nacional. Medicina Intensiva, Madrid, v. 30, n. 6, Aug./Sept. 2006. GOMÉZ-TELLO, V. et al. Uso de Internet y recursos electrónicos entre médicos intensivistas españoles: primera encuesta nacional. Medicina Intensiva, Madrid, v. 30, n. 6, Aug./Sept. 2006. GORMAN, P. N. Information needs in primary care: a survey of rural and nonrural primary care physicians. Medinfo, Edmonton, v. 10, pt. 1, p. 338-342, 2001. GORMAN, P. N.; HELFAND, M. Information seeking in primary care: how physicians choose which clinical questions to pursue and which to leave unanswered. Medical Decision in Making, v. 15, n. 2, p.113-119, 1995. GREEN, M. L.; CIAMPI, M. A.; ELLIS, P. J. Resident’s medical information needs in clinic; are they being met? American Journal of Medicine, Newton, v. 109, p. 218-223, Aug. 2000. HAUG, J. D. Physicians’ preferences for information sources: a meta- analytic study. Bulletin of Medical Library Association, Chicago, v.85, n.3, p.223-232, July 1997. Disponível em: http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=226263&blobtype =pdf>. Acesso em: 26 mar. 2007. LECKIE, G. J..; PETTIGREW, K. E.; SYLVAIN, C. Modeling the information seeking of professional: a general model derived from research on engineers, health care professionals and lawyers. Library Quartely, v. 66, n. 2, p. 161-193, 1996. LIMA, H. M. C. Experiências em buscas de informações por residentes de medicina. Referências Revista Brasileira de Educação Médica, Rio de Janeiro, v. 29, n. 1, p. 13-21, jan./abr. 2005. MARTINEZ-SILVEIRA, M. S. A informação científica na prática médica: estudo do comportamento informacional do médico residente. 2005. 184 f. Dissertação (Mestrado em Ciência da Informação) - Programa de Pós Graduação em Ciência da Informação, IBICT/Universidade Federal da Bahia, 2005. OLIVEIRA, A. M.; SILVA, I. Study of information channels utilized by associated doctors of UNIMED - Ponta Grossa, Paraná-Brazil. 2005. In: CONGRESSO MUNDIAL DE INFORMAÇÃO EM SAÚDE E BIBLIOTECAS, 9., Salvador, 2005. Anais... Salvador: [s.n.], 2005. Disponível em: <http://www.icml9.org/>. Acesso em: 12 fev. 2007. OSHEROFF, J. A . et al. Physicians’ information needs: analysis of questions posed during clinical teaching. Annals of Internal Medicina, v. 114, n. 7, p. 576-581, 1991. OSHEROFF, J. A . et al. Physicians’ information needs: analysis of questions posed during clinical teaching. Annals of Internal Medicina, v. 114, n. 7, p. 576-581, 1991. OSHEROFF, J. A . et al. Physicians’ information needs: analysis of questions posed during clinical teaching. Annals of Internal Medicina, v. 114, n. 7, p. 576-581, 1991. OSPINA, E. G.; REVEIZ HERAULT, L.; CARDONA, A. F. Uso de bases de datos bibliográficas por investigadores biomédicos latinoamericanos hispanoparlantes: estudio transversal. Revista Panamericana de Salud Pública, Washington, v. 17, n. 4, p. 230-236, Apr. 2005. Disponível em: <http://www.ingentaconnect.com/content/paho/pajph;jsessionid=299c5v3x7kl9n.alice>. Acesso em: 04 jun. 2008. OSPINA, E. G.; REVEIZ HERAULT, L.; CARDONA, A. F. Uso de bases de datos bibliográficas por investigadores biomédicos latinoamericanos hispanoparlantes: estudio transversal. Revista Panamericana de Salud Pública, Washington, v. 17, n. 4, p. 230-236, Apr. 2005. Disponível em: <http://www.ingentaconnect.com/content/paho/pajph;jsessionid=299c5v3x7kl9n.alice>. Acesso em: 04 jun. 2008. 253 Perspectivas em Ciência da Informação, v.16, n.3, p.232-254, jul./set. 2011 Maria Gorete Monteguti Savi; Edna Lucia da Silva O uso da informação e a prática clínica de médicos residentes RAMOS, K.; LINSCHEID, R.; SCHAFER, S. Real-time information-seeking behavior of residence physicians. Family Medicine, v. 35, n. 4, p. 257- 260, Apr. 2003. RENWICK, S. Knowledge and use of electronic information resources by medical sciences faculty at the University of the West Indies. Journal of Medical Library Association, Chicago, v. 93, n.1, p. 21-31, jan. 2005. Disponível em: <http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=545116& blobtype=pdf>. Acesso em: 16 fev. 2007. SMITH, R. What clinical information do doctors need? British Medical Journal, London, v. 313, n. 7064, p. 1062-1068, Oct. 1996. STINSON, E. R.; MUELLER, D. A. Perspectivas em Ciência da Informação, v.16, n.3, p.232-254, jul./set. 2011 Referências Survey of health professionals’ information habits and needs: conducted through personal interviews. The Journal of the American Medical Association, Chicago, v. 243, n. 2, p. 140- 143, 1980. WOOLF, S. H.; BENSON, D. A. The medical information needs of internists and pediatricians at an academic medical center. Bulletin of the Medical Library Association, Chicago, v. 77, n. 4, p. 372-380, 1989. WOOLF, S. H.; BENSON, D. A. The medical information needs of internists and pediatricians at an academic medical center. Bulletin of the Medical Library Association, Chicago, v. 77, n. 4, p. 372-380, 1989. 254
https://openalex.org/W2982989659	https://periodicos.uffs.edu.br/index.php/FRCH/article/download/8051/5224	Portuguese	null	Big Science e Tecnologia no século XX	Fronteiras	2,018	cc-by	9,088	1 Este artigo foi originalmente escrito pelo autor para a Fronteiras: revista catarinense de História, e traduzido livremente pelo Prof. Dr. Jo Klanovicz (PPGIDC/LHAG/UNICENTRO). O tradutor manteve a expressão Big Science inalterada por entender que esse é o termo corrente utilizado na literatura internacional no campo de história da tecnologia. 2 Professor de História no Colby College, Maine, EUA, e da Universidade Estatal de Tomsk, Rússia. Doutor pelo Massachussets Institute of Technology, mestre em estudos soviéticos pela Universidade de Harvard. Campos de atuação: História da Ciência e da Tecnologia no século XX, História Soviética e Russa e História Ambiental. E-mail: paul.josephson@colby.edu. Big Science and Technology in the Twentieth Century Paul Robert Josephson2 Resumo: Neste artigo argumento que um dos caminhos mais profícuos para pensar histórias da ciência e da tecnologia no século XX é considerás-las como constructos humanos de larga escala, nunca como objetos em si, mas sim grandes sistemas de instituições políticas, econômicas, sociais e de engenharia. Abstract: In this essay I argue that one of the most fruitful ways to consider the history of science and technology in the twentieth century is to consider them as large scale human constructs, never as objects in themselves, but extensive systems of political, engineering, economic, and social institutions. Ciência, Palavras-chave: Tecnologia, Século XX. Keywords: Science, Technology, 20th century. Keywords: Science, Technology, 20th century. 149 Neste artigo argumento que um dos caminhos mais profícuos para pensar histórias da ciência e da tecnologia no século XX é considerás-las como constructos humanos de larga escala, nunca como objetos em si, mas sim grandes sistemas de instituições políticas, econômicas, sociais e de engenharia. Uma hidrelétrica obviamente nasceu de esforços de oficiais de governo, de engenheiros e de trabalhadores que represaram um rio, construíram uma barrage, moldaram dezenas de milhares de metros cúbicos de concreto, implantaram turbogeradores e linhas de transmissão e enviaram energia para uma rede de indústrias e cidades – comumente localizadas muito longe e servindo a pessoas igualmente distantes. Um computador – e seus microcircuitos – é uma tecnologia de larga escala que nasceu da corrida especial entre a URSS e os EUA e agora alcança a maior parte do globo no seu abraço apaixonante. A história da ciência no século XX trabalha com coisas que são muito grandes, outras que são muito pequenas e rápidas – como fótons e partículas elementares. Astrônomos e agências espaciais foram muito além do sistema solar, enquanto físicos buscam detectar neutrinos subatômicos com vistas a explicar o decaimento beta em multimilionários projetos. Engenheiros continuam a construir reatores nucleares e usinas, manipulando moléculas e elétrons em física do estado sólido, pesquisa que resulta em aparelhos de comunicação miniaturizados e de alta velocidade e que promete avanços nanotecnológicos. Eles usam aminoácidos na engenharia genética com aplicações na agricultura, na medicina e em outros setores. Big Science and Technology in the Twentieth Century Mas, mesmo quando especialistas lidam com o que é muito pequeno, quase sempre o fazem em laboratórios grandiosos, industriais, governamentais ou de instituições de ensino superior, em times gabaritados de pesquisadores – no CERN, em Genebra, no Instituto Kurtchátov de Energia Atômica (Moscou), no Instituto Nacional de Pesquisas da Amazônia (INPA), em Manaus, ou na Johns Hopkins University, em Baltimore, Maryland (EUA) com suas 10 divisões e fundos de pesquisa que ultrapassam US$3,2 bilhões. A big science e a tecnologia da pesquisa em química aplicada, em agronomia, em hidreletricidade, em projetos de bombas atômicas, na exploração genômica tornou-se paradigma de pesquisa no século XX. Se, num momento, cientistas tenderam a trabalhar sozinhos em pequenos laboratórios que eram autofinanciados em grupos íntimos de universidades e ocasionalmente projetos de pesquena escala em conexão com academias de ciência que foram estabelecidas na virada do século XVIII para o XIX, então, por volta dos anos 1920, eles passaram a fazer parte de grupos mais amplos de especialistas, o que refletiu a aglomeração de interesses de governo, da engenharia, do mundo financeiro e científico e, frequentemente, de novos interesses e organizações. A pesquisa de larga escala necessitou 150 não apenas laboratórios e institutos científicos grandiosos mas também apoio governamental, fundos privados e demanda industrial ou de consumo, e uma visão de mundo baseada na crença que a tecnologia e os governos deveriam trabalhar juntos para promover o fortalecimento militar, a melhoria da saúde pública, a economia, a exploração de recursos e o binômio Pesquisa e Desenvolvimento a alto custo. De muitas formas, a big science e a tecnologia no século XX são diluídas num mundo crescentemente industrial e refletem um ethos industrial. 3 BUCK, Peter. Adjusting to Military Life: The Social Sciences Go to War, 1941-1950. In: SMITH, Merritt R. (org.). Military enterprise and technological change: perspectives on the American experience. Cambridge: MIT Press, 1985; LOWEN, Rebecca S. Creating the Cold War University: the transformation of Stanford. Berkeley: University of California Press, 1997. Big Science and Technology in the Twentieth Century Ao começar pela Primeira Guerra Mundial, estados buscaram a vitória militar por meio das tecnologias de produção em massa desenvolvidas em instalações de engenharia e armaria, bem como laboratórios mantidos por dinheiro estatal para a produção de metralhadoras, tanques, navios, aviões, armas químicas e nucleares, e até de soldados que foram recrutados em larga escala por meio de testes sociais como os de QI para determinar sua capacidade e confiabilidade na batalha (BUCK, 1985; LOWEN, 1997).3 No manejo de recursos naturais, as metáforas industriais começaram a suplantar as de ordem biológica, na medida em que especialistas começavam a trabalhar com oficiais de governo para explorá- los em nome do poder estatal ou econômico, fazendo emergir a agricultura industrial e as indústrias da pesca e da floresta. p As atividades de larga escala industriais e de base científica são apoiadas por uma série crescentemente complexa de instituições e artefatos. Thomas Hughes define os sistemas tecnológicos como constituídos de componentes físicos, recursos naturais e organizações (firmas manufatureiras, companhias utilitárias, bancos de investimento, organizações científicas e de engenharia, além de universidades) que interagem com outros artefatos. Os construtores de sistemas e seus associados trazem os sistemas à tona por meio de processos dificultosos de invenção, de desenvolvimento, de inovação e de transferência tecnológica; por meio de financiamentos, relações públicas, transitando entre obstáculos regulatórios, políticos e de outras ordens. Em outros termos, não podemos olhar para os objetos – uma hidrelétrica ou uma floresta industrializada, por exemplo – isoladamente, mas devemos considerá-los em meio a uma caótica interação de engenharia, ciência, financiamento, governos, consumidores e instituições sociais que lhes dão ímpeto – ou criam 151 obstáculos – para a disseminação de tecnologia, e devemos reconhecer todos os atores envolvidos nessa história. Como lembra David Nye, muitas tecnologias “tornam-se mais rígidas e menos capazes de responder às pressões sociais” uma vez que a propriedade, o controle e as especificações tecnológicas são estabelecidas, e desde que os interesses investidos começam a insistir na modificação e direcionamento do sistema.”4 Nós vemos com frequênccia esse comportamento em institutos, centros de pesquisa, corporações, associações e órgãos reguladores. Esse é o fenômeno do momentum tecnológico,5 por exemplo quando grupos comerciais encontraram dificuldades de se distanciar do governo ao mesmo tempo em que se opõem a regulamentos que podem exigir mudanças. g p 5 Alvin Weinberg, ex-diretor do Oak Ridge National Laboratory e um dos defensores da inovação no design de poder de reatores, considerou, entre outros fatores, o momentum na organização e financiamento da Big Science e na tecnologia na obra Reflections on Big Science (Cambridge: MIT Press, 1968). 6 NOBLEm, David. America by design: science, technology and the rise of corporate capitalism. Cambridge: MIT Press, 1977. 7 Ver ROSENKRANTZ, Barbara. Public health and the state. Cambridge: Harvard University Press, 1972, ou BRANDT, Allan. No magic bullet. New York: Oxford University Press, 1987. 4 NYE, David. Consuming power. Cambridge: MIT Press, 1998. p. 2-3. NYE, David. Consuming power. Cambridge: MIT Press, 1998. p. 2 3. 5 Alvin Weinberg, ex-diretor do Oak Ridge National Laboratory e um dos defensores da inovação no design de poder de reatores, considerou, entre outros fatores, o momentum na organização e financiamento da Big Science e na tecnologia na obra Reflections on Big Science (Cambridge: MIT Press, 1968). 6 NOBLEm, David. America by design: science, technology and the rise of corporate capitalism. Cambridge: MIT Press, 1977. 7 Ver ROSENKRANTZ, Barbara. Public health and the state. Cambridge: Harvard University Press, 1972 ou BRANDT Allan No magic bullet New York: Oxford University Press 1987 Big Science and Technology in the Twentieth Century Mas é, certamente, aparente em laboratórios nacionais ao redor do globo que eles encontram maneiras de manter o financiamento fluindo para seus cofres e para expandir suas atividades para novas direções de pesquisa. Press, 1968). 6 NOBLEm, David. America by design: science, technology and the rise of corporate capitalism. Cambridge: MIT Press, 1977. 7 Ver ROSENKRANTZ, Barbara. Public health and the state. Cambridge: Harvard University Press, A escalada da Big Science: os laboratórios nacionais patrocinados pelo estado Ainda no final do século XIX, várias empresas industriais estabeleceram laboratórios no esforço de amarrar a pesquisa a processos de produção, identificar novos produtos e aumentar a competitividade. Essas empresas incluíam a DuPont, a Westinghouse e outros.6 As empresas petrolíferas reconheceram o papel da geologia e da topografia na sua ascensão ao poder monopolístico. Talvez ainda mais importante, o estado teve um interesse formal e estrutural em ciência e tecnologia devido a sua importância para a saúde pública, por exemplo, no controle da tuberculose, da influenza, de doenças venéreas,7 e de outras epidemias, no fornecimento de água e ar limpos, na economia, particularmente na agricultura, e no setor militar. Esse último setor foi o mais importante para gerar a Big Science do século XX. Nos EUA do final do século XIX essas organizações e burocracias como o Amry Corp of Engineers, a US Geological Survey, o Department of Agriculture, o Forestry Service e o Bureau of Reclamation começaram a direcionar partes significativas do orçamento e constituir 152 portfólios de pesquisa. Na Primeira Guerra Mundial governos financiaram armas químicas e pesquisa aeronáutica, e com a Segunda Guerra Mundial radars, mais armas químicas e biológicas, a bomba atômica que transformou suas instituições de pesquisa e de produção em instituições paradigmáticas para a pesquisa e o desenvolvimento de larga escala no pós-guerra na medicina, na agricultura, na energia e na armaria. g g O governo alemão começou a financiar ciência para fins econômicos a partir dos anos 1870 e 1880 com o surgimento de demandas econômicas, especialmente em setores que demandavam pesquisas intensivas como a engenharia química e elétrica, maquinaria e indústria ótica (por exemplo as corporações Merck e Siemens). Se nos anos 1870 a pesquisa economicamente orientada representava aproximadamente 12% do financiamento científico do império (divididos em 63% para os militares e 25% para a indústria de base), por volta de 1910 ele crescera para aproximadamente 50%. 8 MACRAKIS, Kristie. Surviving the swastika: scientific research in nazi Germany. New York: Oxford University Press, 1993. 9 SPEER, Albert. Inside the Third Reich. New York/Toronto: MacMillan, 1970; ZELLER, Thomas. Driving Germany: the landscape of the German autobahn, 1930-1970. New York/Oxford: Berghahn Books, 2007. 10 KOHLER, Robert. Partners in science: foundations and natural scientists, 1900-1945. Chicago: University of Chicago Press, 1991. A escalada da Big Science: os laboratórios nacionais patrocinados pelo estado No começo do sécuulo XX o governo estabeleceu a Sociedade Kaiser Wilhelm, uma espécie de guarda chuva para muitos institutos, estações experimentais e unidades de pesquisa que também serviu às operações científicas nazistas, mas depois da Segunda Guerra Mundial foi desnazificada e incorporada pela Sociedade Max Planck.8 A máquina de Guerra nazista contribuiu significativamente para a Big Science por meio de projetos de construção de rodovias, em particular a Autobahn, armamentos e estruturas públicas como aquelas usadas para o congresso de Nuremberg.9 g Revistas científicas foram expandidas em quantidade e tamanho, publicando resultados de uma comunidade internacional, como por exemplo a Physical Review e a Zeitschrift fur Physik. Pesquisadores encontravam apoio de benfeitores privados, da indústria e de um crescente número de fundações. A Fundação Rockefeller criou, nos EUA, uma ampla variedade de programas internacionais que beneficiaram a Grã-Bretanha, a Alemanha, a União Soviética e cientistas de outras nações, e foi fundamental na ascensão da biologia molecular na década de 1930.10 No entreguerras esses centros de pesquisa como o Laboratório Cavendish, de Cambridge (Inglaterra), com Ernest Rutherford e John Cockcroft, o Instituto de Física Teórica, em 153 Copenhague (Dinamarca), dirigido por Niels Bohr e financiado pela cervejaria Carlsberg, a Universidade de Gottingen com Max Born e James Franck, Leipzig com Werner Heisenberg, e novos centros soviéticos de pesquisa em Kharkiv (Ucrânia), Leningrado e Moscou atraíram grupos internacionais que desenvolveram a mecânica quântica e da física nuclear. Equipamentos como aceleradores Van de Graaff e as novas gerações de aceleradores de partículas precisaram de aportes financeiros de governos e de fundações privadas, e levvaram à montagem de vastas equipes de investigadores, muitos deles internacionais, e outros conectados com a pesquisa e desenvolvimento para fins de defesa, atrás de laboratórios fechados e protegidos por arame farpado.11 Durante a Grande Depressão, Henry Wallace, o secretário da Agricultura dos EUA, respondeu à preocupação de muitas pessoas, que o avanço tecnológico tinha colocado-os fora do mercado de trabalho, mas conclamado-os para a a fé renovada no progresso. Ele dizia que a ciência e a engenharia investiram muito para escapar das rotinas de trabalho e para aumentar a produção agrícola. 11 GALISON, Peter; HEVLY, Bruce (orgs.) Big Science: the growth of large-scale research. Stanford: Stanford University Press, 1992. Os autores que fazem parte desta obra geralmente focalizam a ciência em grandes instituições. Sobre Big Science e tecnologia numa comparação internacional, ver também THACKRAY, Arnold (org.) Science After ’40. Osiris. 2.ed. v. 7, 1992. A escalada da Big Science: os laboratórios nacionais patrocinados pelo estado Mas para além da produção, ele pediu para que especialistas atentassem para os "estudos imaginativos, não- matemáticas, como a filosofia, a literatura, a metafísica, o drama e a poesia." E continuava: “Desejamos um maior e melhor controle do uso da engenharia e da ciência a fim de que o homem possa ter uma percentagem muito maior de sua energia de sobra para desfrutar das coisas que não são materiais ou econômicas, e gostaria de incluir não só a música, a pintura, a literatura mas em especial a curiosidade do próprio cientista."12 Em vez disso, é claro, os governos optaram por se concentrar em questões militares e em pesquisa e desenvolvimento, que muitas vezes beneficiava apenas as corporações. g f 12 WALLACE, Henry. The Social Advantages and Disadvantages of the Engineering-Scientific Approach to Civilization. Science, v.79, n.2036, p.1-5, 1934. O empreendimento nuclear do Projeto Manhattan Em 2011, os Estados Unidos gastaram US $ 5 trilhões em armas nucleares; enquanto que os outros nove países com armas nucleares gastaram, juntos, US$ 100 bilhões. Os custos extraordinários desses programas – e o tamanho dos processos de mineração, moagem, processamento, separação de isótopos, pesquisa, desenvolvimento e fabricação de armas – estavam claros a partir do Projeto Manhattan, que, em 154 p , 1945, foi o projeto mais caro já concluído pelo governo dos EUA, com despêndio de cerca de US$ 2 bilhões (US$ 30 bilhões em valores atualizados de 2016).13 O projeto da bomba foi a forja nuclear da Big Science. Em dezenas de histórias aprendemos como, no esforço da Guerra Fria e na tentativa de construção da paridade entre as potências, EUA, URSS, França, China e Inglaterra gastaram bilhões de dólares, rublos, francos e libras em pesquisa de mineração e fábricas especiais.14 Esses projetos conduziram institutos de pesquisa e universidades ao grande montante de dinheiro oferecido por esses governos desde então. Os detalhes do Projeto Manhattan, do Distrito de Engenharia de Manhattan do Corpo de Engenheiros do Exército dos EUA - que protegeu a bomba atômica do escrutínio público – são bem conhecidos: como Leo Szilard e Albert Einstein escreveram uma carta ao presidente Roosevelt instando-o a iniciar um programa de bomba devido à preocupação de que os cientistas nazistas já estariam envolvidos no mesmo esforço; a seleção de J. 13 WELLERSTEIN, Alex. The price of the Manhattan Project. Disponível em: «http://blog.nuclearsecrecy.com/2013/05/17/the-price-of-the-manhattan-project» Acesso em: 17 maio 2013. 14 THODES, Richard. The making of the atomic bomb. New York: Simon and Schuster, 1987; HOLLOWAY, David. Stalin and the bomb. New Haven: Yale, 1994. Sobre o projeto nazista ver WALKER, Mark. German national socialism and the quest for nuclear power. Cambridge: Cambridge University Press, 1993. 15 Sobre Oppenheimer ver BIRD, Kai; SHERWIN, Marty. American Prometheus. New York: Knopf, 155 13 WELLERSTEIN, Alex. The price of the Manhattan Project. Disponível em: «http://blog.nuclearsecrecy.com/2013/05/17/the-price-of-the-manhattan-project» Acesso em: 17 maio 2013. 14 THODES, Richard. The making of the atomic bomb. New York: Simon and Schuster, 1987; HOLLOWAY, David. Stalin and the bomb. New Haven: Yale, 1994. Sobre o projeto nazista ver WALKER, Mark. German national socialism and the quest for nuclear power. Cambridge: Cambridge University Press, 1993. 15 Sobre Oppenheimer ver BIRD, Kai; SHERWIN, Marty. American Prometheus. New York: Knopf, 2005. p j WALKER, Mark. German national socialism and the quest for nuclear power. Cambridge: Cambridge University Press, 1993. 15 Sobre Oppenheimer ver BIRD, Kai; SHERWIN, Marty. American Prometheus. New York: Knopf, 2005. y , Oppenheimer ver BIRD, Kai; SHERWIN, Marty. American Prometheus. New York: Knopf, O empreendimento nuclear do Projeto Manhattan Robert Oppenheimer como o diretor científico do projecto e do general Leslie Groves como seu diretor estatal e militar;15 a compartimentação das actividades de investigação dos cientistas para evitar o vazamento de segredos mas, principalmente, ao que parece, para evitar problemas morais acerca do poder de destruição em massa; o Relatório Franck sobre a moralidade do uso de armas em 11 de Junho de 1945, no qual o cientista de Chicago conclamava a nação a não bombardear o Japão mas demonstrar claramente àquele país o estágio vívido dos testes; o Trinity test em Alamogordo, Novo México, de 16 de julho de 1945, que mostrou o sucesso da bomba, e o bombardeamento de Hiroshima e Nagasaki realizado em Alamogordo, Novo México, em 16 de julhoem que Chicago cientista exortou a nação não bombardear os japoneses, mas para encenar uma demonstração vívida para o Japão; Teste Trinity em Alamagordo, Novo México, de 16 de Julho de 1945, que provou o sucesso da bomba; e o bombardeio de Hiroshima e Nagasaki, Japão, em agosto, quando Oppenheimer disse: “Nós pecamos”. Os detalhes do Projeto Manhattan, do Distrito de Engenharia de Manhattan do Corpo de Engenheiros do Exército dos EUA - que protegeu a bomba atômica do escrutínio público – são bem conhecidos: como Leo Szilard e Albert Einstein escreveram uma carta ao presidente Roosevelt instando-o a iniciar um programa de bomba devido à preocupação de que os cientistas nazistas já estariam envolvidos no mesmo esforço; a seleção de J. No final da guerra, em nome da segurança nacional e das forças econômicas, o govenro dos EUA continuou a financiar a investigação 155 científica e o desenvolvimento, com o apoio à comunidade universitária e em outros lugares através de uma variedade de institutos e agências de financiamento. Vannevar Bush, que foi nomeado para o Comitê Consultivo Nacional para a Aeronáutica (NACA), em 1938, e em seguida, para o Comitê Nacional de Pesquisa em Defesa (NDRC), e próximo diretor do Gabinete do Governo dos EUA para a Investigação Científica e o Desenvolvimento, um órgão de coordenação das actividades de 6.000 cientistas que trabalham no esforço de guerra, esteve intimamente ligado ao início do Projeto Manhattan. No final da guerra, apresentou Science, the endless frontier ao presidente Roosevelt, conclamando os EUA a investirem na expansão do apoio governamental à ciência e à criação da National Science Foundation (NSF). 16 Sobre esses grandes projetos ver SCOTT, John. Behind the Urals. Boston: Houghton and Mifflin, 1942/ RASSWEILER, Anne. The generation of power. New York: Oxford University Press, 1989, e McCANNON, John. Red atom. New York: Oxford University Press, 1998. Ver, também, JOSEPHSON, Paul. R. Projects of the century' in Soviet history: large scale technologies from Lenin to Gorbachev. Technology and Culture, v.36, n.3, p.519-559, jul. 1995. 17 Sobre a França ver PRITCHARD, Sara B. Reconstructing the Rhone: the cultural politics of nature and nation in contemporary France, 1945–1997. French Historical Studies, v.27, n.4, p.765-799, 2004, and “From hydroimperialism to hydrocapitalism: ‘French’ hydraulics in France, North Africa, and Beyond”. Social Studies of Science, v.42, n.4, p.591-615, 2012. Sobre os EUA, ver LILIENTHAL, David. TVA--Democracy on the march. New York: Harper Brothers,1944, e REISNER, Marc. Cadillac Desert. New York: Viking, 1986. Sobre hidropoder na Índia e na China, ver PATEL, C. C. Surging Ahead: The Sardar Sarovar Project, Hope of Millions. Harvard International Review, v.15, n.1, p.24-27, 1992; SIEGEL, Katy; MEI, June Y. Yun-Fei Ji's Three Gorges Dam migration. Art Journal, v.69, n.3 p.73-78, 2010; LIU, Changming; MA, Laurence J. C. Interbasin Water Transfer in China. Geographical Review, v.73, n.3, p.253-270, 1983, e XUEMIN, Cheng. China's Hydropower Potential and its Utilization. GeoJournal, v.10, n.2, p.141-149, 1985. O empreendimento nuclear do Projeto Manhattan Ele escreveu: O progresso na guerra contra doenças depende de um fluxo de novos conhecimentos científicos. Novos produtos, novas indústrias e mais empregos exigem adições contínuas ao conhecimento das leis da natureza, e a aplicação desse conhecimento para fins práticos. Da mesma forma, a nossa defesa contra a agressão exige novos conhecimentos para que possamos desenvolver armas novas e melhores. Esse conhecimento novo e essencial só pode ser obtido através da pesquisa científica básica. O progresso na guerra contra doenças depende de um fluxo de novos conhecimentos científicos. Novos produtos, novas indústrias e mais empregos exigem adições contínuas ao conhecimento das leis da natureza, e a aplicação desse conhecimento para fins práticos. Da mesma forma, a nossa defesa contra a agressão exige novos conhecimentos para que possamos desenvolver armas novas e melhores. Esse conhecimento novo e essencial só pode ser obtido através da pesquisa científica básica. A ciência pode ser eficaz para o bem-estar nacional se for parte de um time quer seja na paz ou na guerra. Contudo, sem progresso científico, não é possível alcançar qualquer avanço ou realização no sentido de assegurar nossa saúde, nossa prosperidade, nossa segurança como não do mundo moderno. A NSF (fundada em 1951) forneceu bilhões de dólares para universidades estadunidenses para apoiar a investigação básica e foidos EUA para apoiar a investigação básica, e englobou os Institutos Nacionais de Saúde, o Instituto Nacional do Câncer e muitos outros centros de referência. O Projeto Manhattan foi, portanto, um paradigma para a Big Science (investigação em escala patrocinada pelo estado com vistas ao incremento da produção industrial de armas): em Oak Ridge, Tennessee, onde os cientistas separavam isótopos de urânio com centrífugas e eletroímãs; no Laboratório Metalúrgico, em Chicago, Illinois, onde eles projetaram o primeiro reator do mundo ou uma “pilha atômica” numa quadra de squash para estudar física de neutrons; em Hanford, Washington, com seus reatores de plutônio; no Laboratório Nacional de Los Alamos, Novo México, onde os cientistas projetaram e testaram a bomba; em Nevada, no Atol de Bikini e 156 em outros locais de teste de armas; e por meio de amplo apoio financeiro da Agência de Energia Atêmica para a pesquisa e o desenvolvimento de aplicações pacíficas, como por exemplo, centrais nucleares que fizeram com que os EUA tivesse, sozinho, 113 usinas. A URSS prosseguiu também seu caminho para a Big Science a partir da Revolução Russa. 16 Sobre esses grandes projetos ver SCOTT, John. Behind the Urals. Boston: Houghton and Mifflin, 1942/ RASSWEILER, Anne. The generation of power. New York: Oxford University Press, 1989, e McCANNON, John. Red atom. New York: Oxford University Press, 1998. Ver, também, JOSEPHSON, Paul. R. Projects of the century' in Soviet history: large scale technologies from Lenin to Gorbachev. Technology and Culture, v.36, n.3, p.519-559, jul. 1995. 17 Sobre a França ver PRITCHARD, Sara B. Reconstructing the Rhone: the cultural politics of nature and nation in contemporary France, 1945–1997. French Historical Studies, v.27, n.4, p.765-799, 2004, and “From hydroimperialism to hydrocapitalism: ‘French’ hydraulics in France, North Africa, and Beyond”. Social Studies of Science, v.42, n.4, p.591-615, 2012. Sobre os EUA, ver LILIENTHAL, David. TVA--Democracy on the march. New York: Harper Brothers,1944, e REISNER, Marc. Cadillac Desert. New York: Viking, 1986. Sobre hidropoder na Índia e na China, ver PATEL, C. C. Surging Ahead: The Sardar Sarovar Project, Hope of Millions. Harvard International Review, v.15, n.1, p.24-27, 1992; SIEGEL, Katy; MEI, June Y. Yun-Fei Ji's Three Gorges Dam migration. Art Journal, v.69, n.3 p.73-78, 2010; LIU, Changming; MA, Laurence J. C. Interbasin Water Transfer in China. Geographical Review, v.73, n.3, p.253-270, 1983, e XUEMIN, Cheng. China's Hydropower Potential and its Utilization. GeoJournal, v.10, n.2, p.141-149, 1985. 16 Sobre esses grandes projetos ver SCOTT, John. Behind the Urals. Boston: Houghton and Mifflin, 1942/ RASSWEILER, Anne. The generation of power. New York: Oxford University Press, 1989, e McCANNON, John. Red atom. New York: Oxford University Press, 1998. Ver, também, JOSEPHSON, Paul. R. Projects of the century' in Soviet history: large scale technologies from Lenin to Gorbachev. Technology and Culture, v.36, n.3, p.519-559, jul. 1995. 17 Sobre a França ver PRITCHARD, Sara B. Reconstructing the Rhone: the cultural politics of nature and nation in contemporary France, 1945–1997. French Historical Studies, v.27, n.4, p.765-799, 2004, and “From hydroimperialism to hydrocapitalism: ‘French’ hydraulics in France, North Africa, and Beyond”. Social Studies of Science, v.42, n.4, p.591-615, 2012. Sobre os EUA, ver LILIENTHAL, David. TVA--Democracy on the march. New York: Harper Brothers,1944, e REISNER, Marc. Cadillac Desert. New York: Viking, 1986. Sobre hidropoder na Índia e na China, ver PATEL, C. C. Surging O surgimento da Big Science na URSS Vários fatores levaram ao surgimento da Big Science na URSS. Um deles é de natureza ideológica bolchevique, que legou à centralização de políticas econômicas, sociais, culturais e políticas nas mãos de Moscou. Tecnoloigas de larga escala – sítios de contrução, canais, represas, usinas hidrelétricas, fábricas, etc. – tinham sentido técnico para produção de eletricidade ou para melhorar o transporte, mas também tinha sentido educacional, que era trazer dezenas de milhares de trabalhadores para os objetos econômicas centrais e doutriná-los na história e nos benefícios do Partido Comunista, treinando-os em matemática, leitura e escrita, para portar os símbolos e demonstrar as glórias da lidernaça comunista.16 De modo semelhate, engenheiros franceses e estadunidenses funcionários púbicos, escritores, industriais e outros viram a hidreletricidade como uma forma de produzir energia elétrica, domesticar inundações, facilitar viagens e promover a agricultura, ao mesmo tempo que resguardar interesses locais. A hidroeletricidade tornou-se uma forte instituição do poder do Estado nos anos do pós-guerra, em uma batalha épica contra a natureza, com um rio para ser treinado, conquistado, transformado em lar. Na China e na Índia, de forma semelhante, as barragens foram templos de energia e seus custos foram justificados, mesmo com tantas pessoas transferidas ou removidas de seus locais de habitação e cultura.17 157 Na URSS, já desde 1918, especialistas tiveram vantagens por parte de da burocracia bolchevique, e o governo apoiou a criação de uma série de institutos que refletiam os principais campos de conhecimento. Eles fortaleceram os quadros já existentes na nova física, biologia, química, agronomia, silvicultura, hidrologial, geologia e de outros campos, muitos deles localizados em centros provinciais. Como Graham observa, esse foi um produto da “inovação revolucionária”, qual seja, casar as atitudes soviéticas para com as ciências como elementos fundamentais a serviço das massas e especialmente do estado sem isolar internacionalmente sua ciência, ou seja, entendendo a natureza da organização científica na Europa.18 Eles superaram o isolamento internacional e restabeleceram contatos com o ocidente, publicando novas revistas e ganhando rapidamente status internacionais em vários campos. Alguns dos cientistas que trabalhavam antes da Segunda Guerra Mundial incluem os prêmios Novel Peter Kapitsa, Igor Tamm e Lev Landau, bem como figuras representativas no mundo da bomba atômica como Igor Kurtchátov e Andrei Sakhárov. 18 GRAHAM, Loren. The Formation of Soviet Research Institutes: A Combination of Revolutionary Innovation and International Borrowing. Social Studies of Science, v.5, p.303-329, 1975. 19 BAILES, Kendall. Technology and Society under Lenin and Stalin: Origins of the Soviet Technical Intelligentsia, 1917-1941. Princeton: Princeton University Press, 1978, e GRAHAM, Loren Graham. The Soviet Academy of Sciences and the Communist Party, 1927-1932. Princeton: Princeton University Press, 1967. RAHAM, Loren. The Formation of Soviet Research Institutes: A Combination of Revolutiona ation and International Borrowing. Social Studies of Science, v.5, p.303-329, 1975. O surgimento da Big Science na URSS A tendência à centralização da ciência e à formação de centros de pesquisa dominadas por uma escola ou pesquisador, e as ênfases sobre a ciência aplicada para a indústria às custas da pesquisa básica foram as principais características da stalinização da ciência. A investigação científica foi ligada à industrialização e à coletivização da agricultura. O acompanhamento quase total dos especialistas por oficiais do partido asseguravam que os planos recém-instituídos para a ciência teriam suas metas cumpridas em relação aos programas econômicos mais amplos. O Partido Comunista infiltrava-se em instituições científicas, incluindo a Academia Soviética de Ciências.19 Os cientistas viam um grande aumento dos recursos e um aumento de 50% no número de institutos, com centros regionais de pesquisa agrícola estabelecidos na Ucrânia, no Cáucaso e nos Urais. Por último, as autoridades politizavam a cultura profissional, a realização de campanhas contra o trabalho escravo e a servidão, as perversões pedagógicas. Muitos cientistas foram acusados, presos e mortos por sua falta de vigilância ou alegações de espionagem em nome do ocidente. Mas o estado conseguiu estabelecer a Big Science. O projeto soviético da bomba, levado a cabo pelo director científico Igor Kurtchátov sob supervisão administrativa do chefe de polícia secreta Lavrenty Beria tornou-se o segundo maior do mundo, e talvez tenha sido 158 mais danoso ambientalmente, embora tenha sido semelhante ao dos EUA em termos de uso de rótulos de segurança nacional para evitar que suas informações chegassem às mãos do público, incluindo os residentes em cidades ou empresas militares que foram construídas para esse fim. Dezenas de cidades secretas fechadas a quem não morava nelas, surgiram como suporte para a pesquisa e desenvolvimento nucleares, para o ciclo de combustível e para a produção de armas. Essas cidades de pesquisa em química, física, biologia, armas, enriquecimento de plutônio, fabricação de combustíveis, pesquisa espacial e inteligência militar, denominadas pelo acrônimo russo ZATO (Zakrytye administrativno-territorial'nye Obrazovaniia) foram muitas vezes construídas com trabalho escravo e incluem a Arzamas-16 (para o desenho de bombas), Krasnoiarsk-45 e 26, Cheliabinsk-65 e 70, Zlatoust-20 e Semipalatinsk-121. Na Rússia de 2016 há ao menos 44 ZATOs com uma população total de aproximadamente 1.5 milhões de pessoas, dois terços delas mililares e um quarto delas administradas pela Empresa Russa de Energia Atômica (ROSATOM). ROSATOM embarcou num programa de revitalização para construer, talvez, dezenas de reatores de 1.000 MW antes de 2030, e desenvolver algumas dezenas de usinas nucleares flutuantes. 20 Sobre o comunismo movido a energia atômica, ver JOSEPHSON, Paul R. Nuclear Culture in the USSR. Slavic Review, v.55, n.2, p.297-324, 1996. Uma discussão mais detalhada pode ser lida, no mesmo autor, em Red atom. Pittsburgh: University of Pittsburgh Press, 2005. Para uma avaliação preliminar das novas visões sobre o imaginário nuclear da era Putin, ver o artigo do mesmo autor: _____. Technological Utopianism in the Twenty-First Century: Russia’s Nuclear Future. History and Technology, v.19, n.3, p.279-294, 2003. O surgimento da Big Science na URSS Apesar dos desastres nucleares de Chernobyl e Kichtym e dezenas de outros menores, a utopia atômica perdeu muito pouco seu domínio sobre as mentes dos líderes, cientistas e engenheiros russos Em termos gerais e números absolutos, o programa da ROSATOM é semelhante ao dos soviéticos. No final da era Brejnev, funcionários da indústria vivenciaram a adição de dezenas de milhares de megawatts de capacidade nuclear a cada cinco anos, com oito vasos de pressão e equipamentos associados para reatores de 1.000 MW de água pressurizada produzidos anualmente a uma planta especial construída em princípios fordistas, a Atommash. Em 2016, a ROSATOM é uma organização enorme, distribuída por 350 empresas que empregavam mais de 190.000 pessoas. A criação de um império nuclear centralizado, com tantos funcionários, é uma reminiscência da abordagem soviética. Sergei Kirienko comentou que a ROSATOM é "uma recriação do Minsredmash lendário da URSS, mas em novas condições de mercado." Em outro lugar eu descrevi a coleção de visões e realidades ligadas a um mundo nuclear futuro como "potência atômica comunista." Esse poderoso imaginário, o comunismo sociotécnico alimentado atomicamente, envolveu a redescoberta de visões utópicas soviéticas do mundo socialista extremamente funcionais depois da morte de Stálin, com base na indústria avançada, que produziu grandes quantidades de energia elétrica e libertou os trabalhadores do trabalho manual tedioso. De acordo com essa visão, os 159 cientistas, e especialmente os físicos, em virtude do seu conhecimento sublime - e seu papel no desenvolvimento de armas nucleares - receberam grande respeito dos líderes do partido e do público, e estavam preparados para avançar o estado e a sociedade soviéticos do pós-guerra como concorrentes do ocidente capitalista. Essa visão renasceu sob Vladímir Putin.20 Big Science no Mundo Natural A Big Science prevaleceu no século XX em silvicultura, pesca e agricultura. Além de pesquisa e desenvolvimento patrocinados pelo governo, e, claro, do financiamento do governo através de várias agências e construção de projetos para permitir o controle de inundações, melhorar o transporte, a produção de eletricidade e fornecer mercadorias valiosas, a iniciativa privada desempenhou um papel significativo. Nós vemos a ascensão de poderosas empresas ligadas à extração de matérias primas e produção de energia através de minas, moinhos (têxteis, madeira e fundição), barragens, energia a vapor e energia elétrica e agricultura. Nesse último caso, por exemplo, o Departamento de Agricultura dos EUA em conjunto com universidades públicas e privadas tem desenvolvido novas cultivares, novas práticas e, mais recentemente, organismos geneticamente modificados, que beneficiam o agronegócio e tornam a competição mais difícil para os pequenos agricultores. Nesta seção, discutindo dois casos do mundo da natureza, a floresta e a agricultura (a sojicultura), e brevemente mencionando a prática de geoengenharia, exploro a noção de tecnologia de força bruta para entender como a Big Science caracteriza a história natural do século XX. Eu desenvolvi a ideia da tecnologia de força bruta em Industrialized Nature – sistemas tecnológicos de larga escala que são engajados em projetos de geoengenharia que adquirem impulso institucional. São usinas hidrelétricas, reatores nucleares, rodovias e ferrovias, florestas industriais e agroindústrias, aquicultura e pesca vorazes. As tecnologias de força bruta parecem ter-se desenvolvido autonomamente ou, ao menos, facilitado mais rapidamente o desenvolvimento de outros sistemas de larga escala ligados à exploração de recursos. No caso da infra estrutura de transporte e de produção de energia, 160 o que eu chamo de "corredores de modernização," rodovias, ferrovias e energia deixaram pequenas aldeias de poucas pessoas para trás. linhas de rodovias, ferrovias e energia que deixam pequenas aldeias e pequenas pessoas para trás.21 Por exemplo, como tecnologias patrocinadas pelo estado foram o veículo de colonização da Amazônia brasileira. O mais visível deles foram as rodovias. Um grave seca no Nordeste, em 1970, levou ao Programa de Integração Nacional a construir 15.000 km de estrada centrada na rodovia Transamazônica de Estreita, Piauí com o objetivo de alcançar o Peru a 5.000km. A rodovia passou a cortar 60m de mata no centro da floresta para atravessá-la, muito mais do que o necessário para uma ferrovia. 21 BENNETT, Evan. Highways to Heaven or Roads to Ruin? The Interstate Highway System and the Fate of Starke, Florida. The Florida Historical Quarterly, v.78, n.4, p.451-467, 2000. 22 Para uma discussão inicial sobre a abertura do interior do Brasil e sobre o papel da agricultura e de rodovias ver IPEAN. Contribuicao ao Estudo dos Solos de Altamira. Belém: Instituto des Pesquisa Agropecuaria do Norte, 1967, Circular no.10; IPEAN. Solos da Rodovia Transamazônica: Trecho Itaituba-Rio Branco. Belém: Instituto des Pesquisa Agropecuaria do Norte, 1974; PRESIDÊNCIA DA REPÚBLICA DO BRASIL. Primeiro plano de desenvolvimento: 197274. Brasília: PR, 1971. Ver também OLIVEIRA, Márcio Mauro. As circunstâncias da criação da extensão rural no Brasil. Cadernos de Ciência & Tecnologia, v.16, p.97-134, 1999 e SZMRECSÁNYI, Tamas. Pequena história da agricultura no Brasil. São Paulo: Contexto, 1990. 21 BENNETT, Evan. Highways to Heaven or Roads to Ruin? The Interstate Highway System and the Fate of Starke, Florida. The Florida Historical Quarterly, v.78, n.4, p.451-467, 2000. Big Science no Mundo Natural Planejadores esperavam que 70.000 famílias deixassem as regiões atingidas pela seca e, talvez, 1 milhão de colonos movessem-se para os trópicos, sob a liderança do Instituto Nacional de Colonização e Reforma Agrária (INCRA). O slogan de reassentamento, tendo aestrada como um canal para a Amazônia foi "Pessoas sem terra para uma terra sem pessoas.” A Transamazônica de 5 mil quilômetros vai de João Pessoa, Paraíba para o Peru. No início dos anos 1970, o governo militar forçou a estrada para a florestal como uma solução tecnológica para o desemprego, na esperança de atrair colonos de outras regiões para um espaço de recursos naturais e potencial de trabalho, ao mesmo tempo em que ofereceu uma alternativa à atração potencial de camponeses por insurgentes comunistas. (A Rodovia Interestadual dos EUA foi um projeto de segurança nacional contra as ameaças comunistas por meio do comércio e da mobilidade, inclusive de tropas). Ao invés de promover a reforma agrária e retirar pelo menos uma parcela das terras improdutivas da mão de grandes proprietários de terra, os generais brasileiros construíram uma rodovia que atraiu a pobreza rural para o interior, com poucas oportunidades de serviço e emprego. Da mesma forma que a burocracia e o empresariado estadunidenses, também assumiram a ideia de que uma rodovia poderia automatica e autonomamente atrair bens, serviços e prosperidade.22 Talvez os militares conhecessem um pouco sobre os US Homestead Acts, uma série de leis que datam de 1862 que davam títulos de terra a colonos. As leis dos EUA foram pensadas para 161 apoiar o fortalecimento de fazendeiros independentes, expandir a economia e tirar terras dos nativos americanos. A Belém-Brasília, de 1.800km levou a um crescimento populacional com severos impactos ambientais sobre a floresta. Qualquer sucesso de colonização que envolva milhares de camponeses. A Rodovia Belém- Brasília, de 1.800 quilômetros levou a um aumento populacional responsável por grande impacto sobre a floresta. Todo o sucesso no assentamento de milhares de camponenses sem terra é pouco em comparação com o sucesso do governo em canalizar recursos através da SUDAM para grandes fazendas e empresas com vistas a incrementar a criação de gado na região. 23 Ver FEARNSIDE, Philip M. Human Carrying Capacity of the Brazilian Rainforest. New York: Columbia University Press, 1986; MORAN, Emilio F.. Colonization in the Transamazon and Rondonia. In: SCHMINK, Marianne; WOOD, Charles H. (orgs.) Frontier Expansion in Amazonia. Gainesville: University of Florida Press, 1984; SAWYER, Donald R. Frontier Expansion and Retraction in Brazil. In: SCHMINK, Marianne; WOOD, Charles H. (orgs.) Frontier Expansion in Amazonia. Gainesville: University of Florida Press, 1984. p.188-189; LE BRETON, Binka. Voices from the Amazon. West Hartford: Kumarian Press, 1993; SCHMINK, Marianne; WOOD, Charles. Contested Frontiers in Amazon. New York: Columbia University Press, 1992. p.154-163; BROWDER, John O. Public Policy and Deforestation in the Brazilian Amazon. In: REPETTO, Robert; GILLIS, Malcolm (orgs.) Public Policies and the Misuse of Forest Resources. Cambridge/New York: Cambridge University Press, 1988; MORAN, Emilio F. Following the Amazonian Highways. Indians of the Amazonian Forest. In: DENSLOW, Julie; PADOCH, Christine (orgs.) People of the Tropical Rain Forest. Berkeley/Los Angeles/London: University of California Press, 1988. p.158161; SAWYER, Donald R. Frontier Expansion and Retraction in Brazil. In: SCHMINK, Marianne; WOOD, Charles H. (orgs). Frontier Expansion in Amazonia. Gainesville: University of Florida Press, 1984. p. 183-198. 24 MISA, Tom; BREY, Philip; FEENBERG, Andrew. Modernity and Technology. Cambridge: MIT Press 2003 z y , p Tom; BREY, Philip; FEENBERG, Andrew. Modernity and Technology. Cambridge: MIT 03 25 TROTSKY, Lev. On the ‘Smychka’ Between Town and Country. December 6, 1923. Disponível em: «https://www.marxists.org/archive/trotsky/1923/newcourse/x03.htm» Ver também JOSEPHSON, Paul R. Would Trotsky Wear a Bluetooth? Technological Utopianism Under Socialism. Baltimore: Johns Hopkins University Press, 2009, especialmente o capítulo 1. 26 OSINSKII, Valerian. Amerikanskii Avtomobil ili Rosssiiskaia Telega. Moskva, 1930, p.14-24. Sobre Osinskii e automóveis na URSS, ver BUSCH, Tracy N. Women and Children First? Avtodor's Campaigns and the Limits of Soviet Automobility from 1927 to 1935. The Russian Review, v.70, p.397- 418, 2011: 27 MEANS, Thomas. The Duty of Water in Irrigation. Pacific Rural Press, v.79, n.25, p.491, 2010. Para uma discussão histórica ver WESCOAT JR., J. L. Reconstruction the Duty of Water: A study of Emergent Norms in Socio-hydrology Hydrology and Earth System Sciences v 17 p 4759-4768 2013 Big Science no Mundo Natural A irracionalidade do programa de desenvolvimento refletiu o fato de toda a sua inspiração foi baseada em metáforas militares, em particular a noção de que a Amazônia deveria ser ocupada a qualquer custo, e que rodovias sozinhas fariam o truque.23 Promotores de tecnologias de larga escala empregaram-nos para extrair recursos, para promover a modernização da educação, para aumentar o poder militar da nação, para gerar lucros ao grande negócio, e como símbolos do controle humano sobre a natureza. Em todas essas formas de sistemas de força bruta estão representadas relações dicotômicas entre os seres humanos e o poder ea natureza. O uso da tecnologia para superar a defasagem espacial, temporal, geográfica e política envolvia essas dicotomias como pessoas civilizadas trazendo inevitavelmente o progresso de uma sociedade moderna sobre um mundo incivilizado, através do onhecimento científico universal superior que empurrou o conhecimento local para o lado inadequado.24 Uma das mais importantes dicotomias é entre o mundo urbano e o mundo rural, na qual engenheiros e líderes políticos vêem o mundo urbano como moderno, civilizado e superior à selvageria e precariedade rural – que 162 é preenchida, segundo eles, por pessoas pobres, deseducadas e mal afortunadas, que precisam de tecnologia para superar suas fraquezas. Lev Trotsky, por exemplo, via a revolução marxista como forma de ultrapassar a disparidade de espaço, tempo, visão de mundo e, especialmente, da tecnologia, que ele entendia como o principal motor da sociedade, entre a cidade eo campo e seu campesinato. Ele era especialmente apaixonado pela tecnologia dos EUA.25 Outro entusiasta tecnológico russo também via a tecnologia como uma ferramenta para a modernização. Lênin acreditava que a “eletrificação + poder soviético” iria produzir o comunismo moderno. Outros menos conhecidos, como Valerian Ossinsky, afirmavam que o automóvel – associado a linhas de montagem e produção de massa – era a chave para o futuro da Rússia.26 27 MEANS, Thomas. The Duty of Water in Irrigation. Pacific Rural Press, v.79, n.25, p.491, 2010. Para uma discussão histórica ver WESCOAT JR., J. L. Reconstruction the Duty of Water: A study of Emergent Norms in Socio-hydrology. Hydrology and Earth System Sciences, v.17, p.4759-4768, 2013. Natureza e Indústria Assim como para a natureza, a penetração dos termos industriais e metáforas e práticas nas revistas científicas de biologia, de hidrologia e de outras áreas foi ancorada em abordagens de larga escala. Uma ideia como “o dever da água” mostra que, em essência, o termo não é apenas uma avaliação física do uso da água para irrigação ou para a geração de energia, mas para promover o controle da água por instituições mais amplas.27 O resultado de juntar termos, éthos, e pesquisa científicos foi a industrialização da natureza – a busca por florestas de monocultura e campos agrícolas controlados pelo grande negócio. Consideremos a industrialização da floresta e do setor agrícola. As máquinas e as interpretações maquínicas do século XX penetraram na floresta. Máquinas movidas por motores de combusão interna substituíram as alimentadas por lenha; tratores substituíram arados de boi, cavalos e trenós; moto serras substituíram serrotes e, finalmente, colheitadeiras e cortadoras de árvores que derrubam, desbastam, cortam e empilham madeira de uma só vez apareceram como novos lenhadores ao volante. 163 Quando tudo se limitava a bois e cavalos, machados e serras, era necessário um grande esforço para derrubar uma árvore, movê-la para um córrego e aguardar o degelo da primavera para se ter segurança à jusante para suprir madeira para as fábricas costeiras e mercados de Boston ou Nova Iorque. Mas a mudança tecnológica no poder, transporte, colheita e processamento habilitou latifundiários, silvicultores e fabricantes a encontrar produtos a partir de uma extremidade da floresta a outra. Ao final de um século de revolução tecnológica, pareceria que nem um grama de celulose seria desperdiçado. Se antes madeireiros usavam cavalos e bois para levar madeira para córregos e rios, na metade do século já usavam tratores, caminhões e tratores florestais que podiam ser operados em qualquer época e contavam com ferrovias e rodovias para o transporte de toras para serrarias. Se rios grandes por vezes não tinham muitos obstáculos para o transporte, rios de altitude apresentavam muitos entraves. Nesse caso, o dinamite fazia a diferença, corrigindo a paisagem, mas também eliminando cardumes e corredeiras. Para eles, a solução foram estradas e ferrovias. 28 FITZGERALD, Deborah. Farmers Deskilled: Hybrid Corn and Farmers' Work. Technology and Culture, v.34, n.2, p. 324-343, 1993. Ver também FITZGERALD, D. Beyond Tractors: The History of Technology in American Agriculture. Technology and Culture, v.32, n.1, p. 114-126, 1991. 29 As fonts foram consultadas no site da JICA, disponível em: «http://www.jica.go.jp/brazil/english/office/topics/topics110223.html»; FRECHETTE, Darren L. The Dynamics of Convenience and the Brazilian Soybean Boom. American Journal of Agricultural Economics, v.79, n.4, p. 1108-1118; 1997.GALLOWAY, J. H. Agricultural Reform and the Enlightenment in Late Colonial Brazil. Agricultural History. v.53, n.4, p.763-779, 1979; GRIESSE, Margaret A. Developing Social Responsibility: Biotechnology and the Case of DuPont in Brazil. Journal of Business Ethics, v.73, n.1, p. 103-118, 2007; HYMOWITZ, T.; HARLAN, J. R. The Introduction of Soybean to North America by Samuel Bowen in 1765. Economic Botany, v.37, n.4, p. 371-379, 1983; IRWIN, Scott H.; GOOD, Darrell; MARTINES Filho, João. The Performance of Agricultural Market Advisory Services in Corn and Soybeans. American Journal of Agricultural Economics, v.88, n.1, p. No campo: a agricultura No campo, também, as abordagens de larga escala – tratores e implementos, sementes com herbicidas e pesticidas acomplados, fertilizantes químicos, organismos geneticamente modificados e operações concentradas de alimentação animal – mudarm a forma da fazenda para agroindústria. O pequeno agricultor, o pecuarista e o camponês desapareceram de muitas paisagens sob as pressões da agricultura científica e industrial. Em As Vinhas da Ira (1939), o Prémio Nobel John Steinbeck narra o impacto desconcertante e violento da Grande Depressão, a mudança tecnológica e excesso de oferta de mão de obra de agricultores de Oklahoma e da Califórnia. Como Fitzgerald observa, tecnologia agrícola refere-se ao processo de sistemático cultivo de plantas e criação de animais, incluindo forças econômicas, mecânicas, humanas, científicas e institucionais que apoiam tal atividade. A produção de alimentos (para o gado) e de fibras são, assim como implementos, sementes, agroquímicos e rações, os veículos mais acessíveis para examinar como a tecnologia mudou práticas agrícolas de cultivo do milho, o incremento da criação de suínos, o desenvolvimento de maquinário, o processamento de alimentos. A historiadora nos pede para considerar as mudanças nas práticas de propriedade em relação ao comércio e à indústria, especialmente no que tange a perguntas sobre se as preocupaçõçes e práticas agrícolas são semelhantes àquelas das fábricas e 164 dos trabalhadores na indústria. Fitzgerald observa que a propriedade dos meios de produção por agricultores torna-os mais semelhantes a artesãos do que aos trabalhadores industriais modernos em termos de autonomia, ritmo de trabalho e relações com a comunidade, e sua crescente dependência de sistemas econômicos de mercado mais amplos, preços e decisões legislativas. Ela pontua que o papel do agronegócio no fornecimento de sementes, produtos químicos, equipamentos e animais, bem como no recebimento do produto final transformou o agricultor numa espécie de indústria caseira.28 Mas se a indústria caseira ou a contratação de trabalhadores criam uma dívida de gratidão com o poder do agronegócio, ela enfrenta pressões industriais e os efeitos degradantes da mecanização da criação de animais.ou das commodities internacionais como a soja. ç j A Big Science na agricultura remonta ao século XIX. Os EUA configuraram uma aparato de pesquisa e desenvolvimento na agricultura ainda durante a Guerra Civil (o Morrell Act, de 1862 e a concessão de terras para universidades e faculdades). Nas primeiras décadas, o Departamento de Agricultura dos EUA financiou pesquisas sobre zoonoses, composição química e nutrição, solos e mapeamento, entre outras. 162-181, 2006; MORTON, Douglas C. et al. Cropland Expansion Changes Deforestation Dynamics in the Southern Brazilian Amazon. Proceedings of the National Academy of Sciences of the United States of America, v.103, n.39, p. 14637-14641 2006; MUNN, Alvin. Production and Utilization of the Soybean in the United States. Economic Geography, v.26, n.3, p. 223-234, 1950; PRIMMER, George. United States Soybean Industry. Economic Geography, v.15, n.2, p. 205-211, 1939; PRODÖHL, Ines. A Miracle Bean: How Soy Conquered the West, 1909–1950. Bulletin of the German Historical Institute, v.46, p. 111–129, 2010, e RASK, Kevin. The Social Costs of Ethanol Production in Brazil: 1978-1987. Economic Development and Cultural Change, v.43, n.3, p. 627-649, 1995; WILLIAMS, Gary W.; THOMPSON, Robert L. Brazilian Soybean Policy: The International Effects of Intervention. American Journal of Agricultural Economics, v.66, n.4, p. 488-498, 1984; WITT, Lawrence. Changes in the Agriculture of South Central Brazil. Journal of Farm Economics, v.25, n.3, p. 622-643, 1943. No campo: a agricultura Uma variedade de unidadades do USDA e seus sucessores envolveram-se no desenvolvimento de cuiltivares modernos: o Escritório de Estações experimentais (1888-...), a Secretaria de Indústria Vegetal (1904-...), a Administração de Pesquisa Agropecuária (1942-...) – que, em 1981, foi rebatizada como Serviço de Pesquisa Agrícola. A majoração do orçamento refletiu esse desenvolvimento institucional no segundo pós-guerra, chegando na ordem de US$ 1,1 bilhões destinados ao apoio de 800 projetos de pesquisa, 2.200 cientistas e pós-doutorados e mais de 6.200 empregos ligados à pesquisa em todo o país e no exterior. Forças científicas, políticas e de outras ordens reuniram-se na soja, uma verdaeira tecnologia de força bruta. outros se reuniram na soja, outra tecnologia de força bruta.29 A soja reflete o imperativo industrial como a 165 cultura agrícola desenhada par excellence para a mecanização. Seus grãos são altamente adaptáveis à agricultura mecanizada devido a sua posição na distribuição sazonal do trabalho, sua capacidade como cultura de alto rendimento e à pesquisa visando contínua melhoria tanto da qualidade como da utilidade em aplicações industriais, na alimentação e em casa. Na virada do século XIX, a soja era uma cultura da Manchúria. Em meados da década de 1930, a cultura da soja alastrava-se nos EUA e na década de 1970, no Brasil, impulsionava o agronegócio. A cultura da soja é caracterizada por elevado grau de mecanização, extensivas relações público-privadas quando se fala em investimento em pesquisa e desenvolvimento e importantes consequências para o uso da terra. Além disso, seus produtores desempenham importante papel na política, especialmente no Brasil, onde o lobby do agronegócio tem atuado no enfraquecimento da legislação de relevância ecológica. Big Science no século XX Grandes organizações de ciência e de engenharia adquiriram “momentum tecnológico”. O conceito de momentum tecnológico exposto por Hughe ajuda a compreender a habilidade da indústria nuclear manter-se e se recriar, assegurar financiamentos governamentais e prosseguir em programas ambiciosos por décadas. Ela evolui e se expande, engajando instituições sociais, políticas e financeiras. O número de seus componentes aumenta e apresenta sobreposições. Não se trata de um determinismo tecnológico mas sugere que instituições enormes adquirem tremendo poder e autoridade que, às vezes, é difícil de desarticular. Da mesma forma, como discutiu Alvin Weinberg, diretor do Oak Ridge National Laboratory, grandes empresas de investigação científica aprenderam como expandir projetos nucleares e seus focos de atuação, com o objetivo de manter seus programas com bons financiamentos a partir das promessas de resultados cruciais para a defesa 166 nacional, para a saúde pública, para a inovação industrial, para a medicina e para a agricultura. No século XX, institutos de design, laboratórios nacionais, firmas de negócios e engenharia transformaram globalmente sua pesquisa em instrumentos, técnicas e tecnologias necessárias para levar práticas de manejo e escala de pesquisa a novos patamares. Esses instrumentos e práticas são agora familiares nas grandes fazendas de monocultura que produzem milho híbrido ou soja. Elas são familiares nas sementes que precisam de pesticidas específicos, herbicidas e fertilizantes somente produzidos em laboratórios industriais. Eles existem na floresta industrializada, na irrigação, no controle de enchentes que permitiram entender o “dever da água”, nas incubadoras voltadas ao melhoramento genético anumal, nos laboratórios que desenvolvem variedades híbridas onde mamíferos, pássaros e peixem são fabricados, no concreto pré- fabricado, no redimensionamento de leitos de rios e nos exércitos de trabalhadores que os alimentam. Por fim, uma ideologia de progresso distingue as tecnologias de larga escala do século XX e se sobrepõe à floresta e aos campos. Na floresta, a retórica do progresso alimenta a crença de que com as técnicas modernas de manejo elas estarão disponíveis para gerações futuras para várias finalidades – recreação, serraria, papel ou combustível. Revoluções no corte, no poder de transporte e de penetração no interior da floresta construíram a ideia de fornecimento regular de madeira. De um modo quase determininsta, a tecnologia de força bruta cria seus próprios mercados de consumo para a casa, para o papel e para outros produtos, isto é, alimenta novas demandas de consumo enquanto destrói a natureza. 168 30 SCOTT, James. Seeing Like a State. New Haven: Yale University Press, 1998. Big Science no século XX Os governos promoveram o apoio a essas indústrias nascentes oferecendo várias medidas financieras a elas (subsídios diretos, isenção de taxas, doação de terras justificadas em nome da criação de empregos e de levar a civilização para a floresta selvagem). Em Seeing like a state o antroólogo Jems Scott aborda muitas das questões aqui levantadas, em particular, a forma como o Estado moderno (funcionários do governo, os seus administradores, cientistas e engenheiros) reúne e usa seletivamente o conhecimento abstrato para exercer controle sobre os recursos: as pessoas, a água, as florestas, a agricultura. Segundo ele, os estados descobriram que as abordagens de larga escala são a maneira mais eficiente para mobilizar recursos, organizar estudos, subjugar a natureza, ordenar rios, extrair recursos e organizar as pessoas.. Eles fazem isso através de burocracias, de agências, de bancos, de institutos de pesquisa, de empresas de engenharia e de universidades. Eles limpam, 167 ordenam e uniformizam a natureza por meio da ciência e da tecnologia como uma grade cartesiana sobre o mundo natura. Eles usaram o direito de propriedade e domínio eminente para gerenciar vastas faixas de terra em nome do bem comum. Eles também adotaram a eficiência técnica como fundamento inquestionável na adoção de abordagens de larga escala, comemorada pela importância da mecanização para poupar trabalho e superar gargalos, rejeitnado o conhecimento local como algo válido ou relevante.30 Laboratórios nacionais centralizados, grandes empresas de engenharia e abordagens extensivas de pesquisa são todos parte desse fenômeno do século XX. 168 168
https://openalex.org/W3047152224	https://hal.science/hal-02914915/document	English	null	Kdr genotyping in Aedes aegypti from Brazil on a nation-wide scale from 2017 to 2018	Scientific reports	2,020	cc-by	10,842	Kdr genotyping in Aedes aegypti from Brazil on a nation-wide scale from 2017 to 2018 Monique Melo Costa, Kauara Brito Campos, Luiz Paulo Brito, Emmanuel Roux, Cynara Melo Rodovalho, Diogo Fernandes Bellinato, José Bento Pereira Lima, Ademir Jesus Martins To cite this version: Monique Melo Costa, Kauara Brito Campos, Luiz Paulo Brito, Emmanuel Roux, Cynara Melo Rodovalho, et al.. Kdr genotyping in Aedes aegypti from Brazil on a nation-wide scale from 2017 to 2018. Scientific Reports, 2020, 10, pp.13267. 10.1038/s41598-020-70029-7. hal-02914915 Distributed under a Creative Commons Attribution 4.0 International License OPEN Monique Melo Costa1, Kauara Brito Campos2,3, Luiz Paulo Brito1, Emmanuel Roux4,5, Cynara Melo Rodovalho1, Diogo Fernandes Bellinato1, José Bento Pereira Lima1 & Ademir Jesus Martins1,5,6* Insecticide resistance is currently a threat to the control of Aedes agypti, the main vector of arboviruses in urban centers. Mutations in the voltage gated sodium channel (NaV), known as kdr (knockdown resistance), constitute an important selection mechanism for resistance against pyrethroids. In the present study, we investigated the kdr distribution for the Val1016Ile and Phe1534Cys alterations in Ae. aegypti from 123 Brazilian municipalities, based on SNP genotyping assays in over 5,500 mosquitoes. The alleles NaVS (1016Val+ + 1534Phe+), NaVR1 (1016Val+ + 1534Cyskdr) and NaVR2 (1016Ilekdr + 1534Cyskdr) were consistently observed, whereas kdr alleles have rapidly spread and increased in frequency. NaVS was the less frequent allele, mostly found in Northeastern populations. The highest allelic frequencies were observed for NaVR1, especially in the North, which was fixed in one Amazonian population. The double kdr NaVR2 was more prevalent in the Central-west and South-eastern populations. We introduce the ‘kdr index’, which revealed significant spatial patterns highlighting two to three distinct Brazilian regions. The 410L kdr mutation was additionally evaluated in 25 localities, evidencing that it generally occurs in the NaVR2 allele. This nationwide screening of a genetic mechanism for insecticide resistance is an important indication on how pyrethroid resistance in Ae. aegypti is evolving in Brazil. The number of dengue cases in Brazil totaled over 1.9 million records between 2016 and 2018. During the same period, the recent additional chikungunya and Zika arbovirus epidemics were responsible for around 540 and 240 thousand cases, respectively, according to Ministry of Health official bulletins1. Failure in the control of Aedes aegypti, so far considered the primary vector of the aforementioned arboviruses in Brazil, is considered the main reason for the increase in these records. Despite a series of studies evidencing arbovirus transmission by Brazilian Aedes albopictus populations, this species probably acts as a secondary urban vector, mostly significant in maintaining arbovirus circulation during inter-epidemic seasons and in rural regions2,3. The measures for controlling the density of Aedes mosquitoes largely rely on insecticides. However, their intense use has been, increasingly, selecting resistant populations at a global scale4. This is also true in Brazil, where an extensive insecticide resistance monitoring system has tracked the susceptibility status of Ae. aegypti populations since 19995,6. HAL Id: hal-02914915 https://hal.science/hal-02914915v1 Submitted on 28 May 2021 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 www.nature.com/scientificreports www.nature.com/scientificreports Results Kd Kdr genotyping. The kdr genotypes for 5,341 individuals, distributed in 123 localities, for both the 1016 and 1534 NaV sites were obtained. All evaluated population displayed at least one kdr allele. Six genotypes were substantially observed, all including the S, R1 and R2 (wild-type, kdr in 1534 only and kdr in 1016 + 1534, respectively) haplotypes. The other three genotypes composed by the kdr R3 haplotype (kdr in 1016 site only) accounted for 0.1% of the total samples (Fig. 1 and Table 1). Therefore, the R3 haplotype was not considered in further analyses.h y The most frequent Ae. aegypti genotypes was R1R2, followed by the double kdr homozygote R2R2, with medians of 29.0% and 24.4%, respectively (Table 1). The genotype frequencies per population in their respec- tive geographic region are displayed in Fig. 1. Concerning allelic frequencies, medians were 13.3%, 37.8% and 45.3%, respectively for NaVS, NaVR1 and NaVR2, evidencing the high prevalence of kdr alleles (83.1%) in Brazil. The wild-type allele NaVS was absent from 17 of the 123 evaluated populations, while kdr NaVR1 was present in all localities, ranging from 7.1% (Corumba/MS) to 100% (São Gabriel da Cachoeira/AM) and the kdr NaVR2 ranged from absent (Tucurui/PA, Altamira/PA and São Gabriel da Cachoeira/AM) to 89.3% (Corumba/MS) (Fig. 2). The kdr allelic frequencies were regionalized in the country, as a reflection of their genotypic distribu- tion, as follows: NaVR2 predominated in the Central-West, Southeast and South regions, while NaVR1 was the most frequent allele in the North (Fig. 3). The detailed genotypic and allelic frequencies for each locality are available in the Supplementary Table S1. Kdr index and spatial analyses. The ‘kdr index’ ranged from 1.75 (Jardim do Seridó, RN) to 6.21 (Corumbá, MS) (Fig. 4). The localities presenting the lowest kdr index (first quartile: 1.75 to 3.96) were pre- dominantly located in the Northeast (61.3%), South (19.4%) and North (16.1%) regions. Those beyond the last quartile (5.36–6.21) were distributed in the Southeast (48.4%), Center-West (25.8%), North (12.9%), Northeast (9.7%) and South (6.5%) regions (Supplementary Table S1).h g pp y The spatial distribution kdr index value analysis concerning the geographic coordinates of their respec- tive localities revealed statistically significant spatial patterns, i.e. the existence of clearly identifiable Brazilian regions with different predicted Ae. aegypti resistance status. www.nature.com/scientificreports/ by private companies for mosquito control, as well as against other urban insect vectors, such as Culex, sandflies, anophelines and triatomines6,9,10.hf The main cause of resistance to the pyrethroid knockdown effect is an alteration in the molecular target of this chemical, namely the voltage gated sodium channel (NaV, also commonly referred to as VGSC), caused by one or a few amino acid substitutions that alter the channel conformation, thus avoiding its interaction with pyrethroid molecules11. These substitutions, known as knockdown resistance mutations (kdr) have been linked to pyrethroid and DDT resistance in several arthropod species. The kdr amino acid position is generally convergent through- out distinct taxa, as the NaV gene is very conserved and, therefore, few variations are permissive to maintain its neurophysiological role12. In several species, including Anopheles and Culex mosquitoes, the most classical kdr is a Leu to Phe substitution in the 1014 position (L1014F)13–15. In the Aedes genus, two simultaneous substitutions in the codon 1014 would be necessary, which is unlikely to occur16. Substitutions in the 1016 and 1534 sites have been consistently detected in the NaV of Ae. aegypti populations worldwide. The Phe to Cys substitution in the 1534 site (F1534C) is found in populations from the Americas, Africa and Asia, whilst the wild-type Val is sub- stituted by Ile (V1016I) in the 1016 site in American populations and by Gly (V1016G) in Asian populations17–20. Additional alterations have also been reported, such as the Pro to Ser substitution in the 989 (P989S) and Val to Leu in the 410 position (V410L), respectively, in Asian and Latin American populations4,17,21,22. p p y p p Since the beginning of the 2000s, kdr mutations have been detected in Ae. aegypti populations from Brazil, displaying geographical expansion and increasing frequency trends. A haplotype with kdr mutation F1534C (NaVR1 allele) was present in all but one population among natural populations evaluated from more than 60 localities6, 23. An additional haplotype containing both V1016I and F1534C was absent or detected at very low frequencies in the Brazilian Northeast9,23,24. Although this double kdr allele (NaVR2) presented a high fitness cost in a pesticide-free environment25, its frequency has increased in natural populations and dispersed to localities where it was recently considered absent26. kdr mutations were monitored in Ae. aegypti populations from the state of São Paulo, since they were present at low frequencies, and, although NaVR2 later appeared, it increased faster than NaVR19. www.nature.com/scientificreports/ This can be explained by the fact that NaVR2 confers a higher level of resistance to pyrethroids27. Since the beginning of the 2000s, kdr mutations have been detected in Ae. aegypti populations from Brazil, displaying geographical expansion and increasing frequency trends. A haplotype with kdr mutation F1534C (NaVR1 allele) was present in all but one population among natural populations evaluated from more than 60 localities6, 23. An additional haplotype containing both V1016I and F1534C was absent or detected at very low frequencies in the Brazilian Northeast9,23,24. Although this double kdr allele (NaVR2) presented a high fitness cost in a pesticide-free environment25, its frequency has increased in natural populations and dispersed to localities where it was recently considered absent26. kdr mutations were monitored in Ae. aegypti populations from the state of São Paulo, since they were present at low frequencies, and, although NaVR2 later appeared, it increased faster than NaVR19. This can be explained by the fact that NaVR2 confers a higher level of resistance to pyrethroids27. Additional variations have been reported in the NaV gene of Ae. aegypti populations from Brazil, including the Ile to Met and Val to Leu substitutions in the 1011 (I1011M) and 410 (V410L) positions, respectively. The former is related to a duplication in the NaV gene, always detected in heterozygote individuals28, but with no relevance in pyrethroid resistance27. On the other hand, V410L was found alone or in combination with F1534C, conferring high levels of resistance29, although it is usually found in combination with V1016I and F1534C in Mexican populations21. The present study reports the kdr frequencies in Brazilian Ae. aegypti populations from 123 localities, collected between 2017 and 2018. In order to rank the likely levels of pyrethroid resistance among the evaluated populations, at least in relation to the target site mechanism, an original index called the kdr index was developed. A spatial analysis confirmed the non-randomness of the spatial distribution of the index values and, thus, the existence of clearly identifiable Brazilian regions regarding with pyrethroid resistance levels. h Additional variations have been reported in the NaV gene of Ae. aegypti populations from Brazil, including the Ile to Met and Val to Leu substitutions in the 1011 (I1011M) and 410 (V410L) positions, respectively. The former is related to a duplication in the NaV gene, always detected in heterozygote individuals28, but with no relevance in pyrethroid resistance27. www.nature.com/scientificreports/ On the other hand, V410L was found alone or in combination with F1534C, conferring high levels of resistance29, although it is usually found in combination with V1016I and F1534C in Mexican populations21. The present study reports the kdr frequencies in Brazilian Ae. aegypti populations from 123 localities, collected between 2017 and 2018. In order to rank the likely levels of pyrethroid resistance among the evaluated populations, at least in relation to the target site mechanism, an original index called the kdr index was developed. A spatial analysis confirmed the non-randomness of the spatial distribution of the index values and, thus, the existence of clearly identifiable Brazilian regions regarding with pyrethroid resistance levels. OPEN Organophosphate application has been intensified since the 1980s, substituted by insect growth regulators and pyrethroids two decades later, against larvae and adults, respectively, given the confirmation of resistance to the larvicide temephos7. However, pyrethroid resistance was also confirmed a few years later8. Currently, the organophosphate malathion is employed by governmental campaigns in the entire country as the only alternative to pyrethoids, although the latter class of compounds is still intensively used in households and 1Laboratório de Fisiologia e Controle de Artrópodes Vetores, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, RJ, Brazil. 2Laboratório de Parasitologia Médica e Biologia de Vetores, Faculdade de Medicina, Universidade de Brasília, Brasília, DF, Brazil. 3Coordenação Geral de Vigilância de Aboviroses, Secretaria de Vigilância em Saúde, Ministério da Saúde, Brasília, DF, Brazil. 4ESPACE‑DEV, IRD, Université de Montpellier, Université de La Réunion, Université de la Guyane and Université des Antilles, Montpellier, France. 5Laboratório Misto Internacional “Sentinela”, FIOCRUZ, UnB, IRD, Rio de Janeiro, Brazil. 6Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular (INCT‑EM), Universidade federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil. *email: ademirjr@ioc.fiocruz.br \| https://doi.org/10.1038/s41598-020-70029-7 Scientific Reports \| (2020) 10:13267 www.nature.com/scientificreports/ Results Kd A total of 55 candidate models, related to different (a priori) inter-city “links” structures, were generated (see Supplementary Text S1 for methodological details and intermediary results). Figure 5 displays the most significant model, first evidencing a large scale spatial dif- ferentiation in the Northeast–Southwest direction, separating Northeast (and to a lesser extent, North) localities from the rest of the country (areas A11 and A12, respectively, represented in Fig. 5a and, second, a three-mode differentiation in the same direction, separating: (i) North and Northeast localities; (ii) a strip of cities oriented Scientific Reports \| (2020) 10:13267 \| https://doi.org/10.1038/s41598-020-70029-7 www.nature.com/scientificreports/ Southeast–Northwest and (iii) South localities (areas A21, A22 and A23, respectively, in Fig. 5b. The kdr index 0.0 0.2 0.4 0.6 0.8 1.0 frequency North 0.0 0.2 0.4 0.6 0.8 1.0 Northeast 0.0 0.2 0.4 0.6 0.8 1.0 South 0.0 0.2 0.4 0.6 0.8 1.0 R3/R3 R2/R3 S/R3 R2/R2 R1/R2 S/R2,R1/R3 R1/R1 S/R1 S/S Center-West 0.0 0.2 0.4 0.6 0.8 1.0 frequency Southeast 0.0 0.2 0.4 0.6 0.8 1.0 R3/R3 R2/R3 S/R3 R2/R2 R1/R2 S/R2,R1/R3 R1/R1 S/R1 S/S Brazil Figure 1. Frequency of kdr genotypes in Aedes aegypti from Brazil considering the V1016I and F1534C variations in the voltage gated sodium channel. Populations were distributed according to the Brazilian five macro-geographical regions. Dots represent the genotypic frequency of each population, while the box-plots exhibit the median, quartiles, minimum and maximum values. Genotypes: 1016 + 1534. 0.0 0.2 0.4 0.6 0.8 1.0 frequency North 0.0 0.2 0.4 0.6 0.8 1.0 Northeast 0.0 0.2 0.4 0.6 0.8 1.0 R3/R3 R2/R3 S/R3 R2/R2 R1/R2 S/R2,R1/R3 R1/R1 S/R1 S/S Brazil Northeast frequency 0.0 0.2 0.4 0.6 0.8 1.0 South 0.0 0.2 0.4 0.6 0.8 1.0 R3/R3 R2/R3 S/R3 R2/R2 R1/R2 R2,R1/R3 R1/R1 S/R1 S/S Center-West 0.0 0.2 0.4 0.6 0.8 1.0 frequency Southeast Figure 1. Frequency of kdr genotypes in Aedes aegypti from Brazil considering the V1016I and F1534C variations in the voltage gated sodium channel. Populations were distributed according to the Brazilian five macro-geographical regions. Dots represent the genotypic frequency of each population, while the box-plots exhibit the median, quartiles, minimum and maximum values. Genotypes: 1016 + 1534. 0.0 0.2 0.4 0.6 0.8 1.0 South 0.0 0.2 0.4 0.6 0.8 1.0 R3/R3 R2/R3 S/R3 R2/R2 R1/R2 S/R2,R1/R3 R1/R1 S/R1 S/S Center-West 0.0 0.2 0.4 0.6 0.8 1.0 frequency Southeast Southeast South frequency Figure 1. Results Kd Considering only samples with the six best-represented genotypes, the haplotypic frequencies were calculated as 40.0% (VVC), 38.2% (LIC) and 10.4% (VVF), indicating that 410L was associated with 1016I https://doi.org/10.1038/s41598-020-70029-7 Scientific Reports \| (2020) 10:13267 \| www.nature.com/scientificreports/ PCR results Genotypes Number (frequency)a Median Minb Maxc VV + FF SS 255 (0.048) 0.022 0 0.523 VV + FC SR1 652 (0.122) 0.098 0 0.511 VV + CC R1R1 1,054 (0.197) 0.136 0 1.000 VI + FC SR2, R1R3 524 (0.098) 0.089 0 0.333 VI + CC R1R2 1,548 (0.290) 0.279 0 0.591 II + CC R2R2 1,301 (0.244) 0.222 0 0.810 VI + FF SR3 3 (0.0006) 0.000 0 0.024 II + FC R2R3 2 (0.0004) 0.000 0 0.022 II + FF R3R3 2 (0.0004) 0.000 0 0.023 PCR results Genotypes Number (frequency)a Median Minb Maxc VV + FF SS 255 (0.048) 0.022 0 0.523 VV + FC SR1 652 (0.122) 0.098 0 0.511 VV + CC R1R1 1,054 (0.197) 0.136 0 1.000 VI + FC SR2, R1R3 524 (0.098) 0.089 0 0.333 VI + CC R1R2 1,548 (0.290) 0.279 0 0.591 II + CC R2R2 1,301 (0.244) 0.222 0 0.810 VI + FF SR3 3 (0.0006) 0.000 0 0.024 II + FC R2R3 2 (0.0004) 0.000 0 0.022 II + FF R3R3 2 (0.0004) 0.000 0 0.023 Table 1. Genotypes of 123 Aedes aegypti populations from Brazil, considering the V1016I and F1534C SNPs in the NaV gene. a Number of samples with their respective genotypes, bMinimum and cMaximum observed frequencies. Table 1. Genotypes of 123 Aedes aegypti populations from Brazil, considering the V1016I and F1534C SNPs in the NaV gene. a Number of samples with their respective genotypes, bMinimum and cMaximum observed frequencies. Table 1. Genotypes of 123 Aedes aegypti populations from Brazil, considering the V1016I and F1534C SNPs in the NaV gene. a Number of samples with their respective genotypes, bMinimum and cMaximum observed frequencies. Figure 2. Distribution of the kdr allelic frequencies in Aedes aegypti populations from Brazil considering the V1016I and F1534C variations in the voltage gated sodium channel. Localities are represented by numbers in the legend, exhibited on the map according to their respective geographical coordinates, grouped within the five Brazilian macro-regions. The background map is the “OSM TF Landscape” product (Maps Thunderforest, Data OpenStreetMap contributors, under license CC-BY-SA 2.0). Results Kd Frequency of kdr genotypes in Aedes aegypti from Brazil considering the V1016I and F1534C variations in the voltage gated sodium channel. Populations were distributed according to the Brazilian five macro-geographical regions. Dots represent the genotypic frequency of each population, while the box-plots exhibit the median, quartiles, minimum and maximum values. Genotypes: 1016 + 1534. Figure 1. Frequency of kdr genotypes in Aedes aegypti from Brazil considering the V1016I and F1534C variations in the voltage gated sodium channel. Populations were distributed according to the Brazilian five macro-geographical regions. Dots represent the genotypic frequency of each population, while the box-plots exhibit the median, quartiles, minimum and maximum values. Genotypes: 1016 + 1534. Southeast–Northwest and (iii) South localities (areas A21, A22 and A23, respectively, in Fig. 5b. The kdr index value distributions as a function of the aforementioned areas are displayed in Fig. 5c,d. Southeast–Northwest and (iii) South localities (areas A21, A22 and A23, respectively, in Fig. 5b. The kdr index value distributions as a function of the aforementioned areas are displayed in Fig. 5c,d. Including the V410L SNP. In addition to V1016I and F1534C, the V410L SNP was also evaluated in 25 populations, mostly in the state capitals, totaling 977 genotyped samples for these three sites, which together ren- dered 11 genotypes, amongst which five were poorly represented (Table 2, Fig. 6). The three most prevalent geno- types were VV + VV + CC (27.8%), VL + VI + CC (26.8%) and LL + II + CC (23.9%) (in order, 410 + 1016 + 1534), composed by the kdr haplotypes VVC (kdr in the 1534 site only) and LIC (kdr in 410, 1016 and 1534 sites). Next are the genotypes formed with the wild-type haplotype (VVF): VV + VV + FC (11.1%), VL + VI + FC (6.0%) and VV + VV + FF (2.7%). The remaining five genotypes occurred in frequencies below 1%, which would be formed by alleles such as VIC (kdr in 1016 and 1534), LVC (kdr in 410 and 1534), LVF (kdr in 410 only) and VIF (kdr in 1016 only). Discussion Th l l The molecular nation-wide monitoring of the main pyrethroid resistance marker in 123 Ae. aegypti populations from Brazil, a country comprising 8.5 million km2, where the 26 federal states are infested with this vector, is presented herein. To the best of our knowledge, this is the largest kdr survey performed using samples collected in the time span of one year. The regionalized profile of allelic frequency distribution indicates that this sample size is enough to represent the whole country.h The molecular nation-wide monitoring of the main pyrethroid resistance marker in 123 Ae. aegypti populations from Brazil, a country comprising 8.5 million km2, where the 26 federal states are infested with this vector, is presented herein. To the best of our knowledge, this is the largest kdr survey performed using samples collected in the time span of one year. The regionalized profile of allelic frequency distribution indicates that this sample size is enough to represent the whole country.h The frequency of kdr alleles was very high in Brazil (median of 83.1%), representing an increase of 27.8% since a previous survey was performed, evaluating 29 localities where mosquitoes were collected between 2009 and 201214. The kdr NaVR1 allele (1016Val + 1534Cyskdr) was present in all Brazilian populations evaluated here, and fixed in one of them. This was probably the first kdr allele to spread in Latin America20 and is present in at least two haplotypes of independent phylogenetic origins17. kdr NaVR2 (1016Ilekdr + 1534Cyskdr) confers a higher level of resistance to pyrethroids compared to NaVR117, which may explain why NaVR2 frequency is rapidly increasing compared to the wild-type NaVS and kdr NaVR1 alleles, as observed in Ae. aegypti populations from São Paulo9. It is interesting to note that, out of the 17 populations where the wild-type NaVS was absent, 12 were from the Amazonian region. In many of these localities, chemical control is not only exerted to combat Ae. aegypti, but also targeting other important vectors, such Anopheles mosquitoes, triatomines and phlebotomines, against which pyrethroids are the main choice inside houses and in the peri-domicile area. Therefore, the scenario observed herein may be explained by this constant selection pressure. We also presented the application of a ‘kdr index’ which considers the kdr genotype frequencies weighed by their respective knockdown time resistance ratio (KdT RR95). Results Kd Map was generated with the free and open source software (GNU General Public License) QGIS version 3.12.3 (developd by the Open Source Geospatial Foundation Project, http://qgis.org). Fi 2 Di t ib ti f th kd ll li f i i A d pti l ti f B il id i th Figure 2. Distribution of the kdr allelic frequencies in Aedes aegypti populations from Brazil considering the V1016I and F1534C variations in the voltage gated sodium channel. Localities are represented by numbers in the legend, exhibited on the map according to their respective geographical coordinates, grouped within the five Brazilian macro-regions. The background map is the “OSM TF Landscape” product (Maps Thunderforest, Data OpenStreetMap contributors, under license CC-BY-SA 2.0). Map was generated with the free and open source software (GNU General Public License) QGIS version 3.12.3 (developd by the Open Source Geospatial Foundation Project, http://qgis.org). and 1534C (NaVR2 allele) and that 1534C also occurs alone (NaVR1 allele). Nevertheless, we cannot exclude the possibility that the underrepresented genotypes could be composed by additional haplotypes circulating under low frequencies. The complete dataset displaying the numbers and frequencies for each SNP in all populations is presented in Supplementary Table S2. and 1534C (NaVR2 allele) and that 1534C also occurs alone (NaVR1 allele). Nevertheless, we cannot exclude the possibility that the underrepresented genotypes could be composed by additional haplotypes circulating under low frequencies. The complete dataset displaying the numbers and frequencies for each SNP in all populations is presented in Supplementary Table S2. https://doi.org/10.1038/s41598-020-70029-7 Scientific Reports \| (2020) 10:13267 \| www.nature.com/scientificreports/ Figure 3. Frequency of the kdr alleles in Aedes aegypti from Brazil considering the V1016I and F1534C variations in the voltage gated sodium channel. Plots represent the NaV allelic frequency distribution of each population in the five geographic Brazilian macro-regions and in the country as a whole. The alleles are NaVS (V1016 + F1534), NaVR1 (V1016 + 1534C) and NaVR2 (1016I + 1534C). Circles represent the allelic frequency of each population and bars indicate the median and quartile distribution of the respective allele. Figure 3. Frequency of the kdr alleles in Aedes aegypti from Brazil considering the V1016I and F1534C variations in the voltage gated sodium channel. Plots represent the NaV allelic frequency distribution of each population in the five geographic Brazilian macro-regions and in the country as a whole. Results Kd The alleles are NaVS (V1016 + F1534), NaVR1 (V1016 + 1534C) and NaVR2 (1016I + 1534C). Circles represent the allelic frequency of each population and bars indicate the median and quartile distribution of the respective allele. Discussion Th l l The background map is the “OSM TF Landscape” product (Maps Thunderforest, Data OpenStreetMap contributors, under license CC-BY-SA 2.0). Map was generated with the free and open source software (GNU General Public License) QGIS version 3.12.3 (developd by the Open Source Geospatial Foundation Project, http://qgis.org). Figure 4. kdr index distribution in Aedes aegypti from Brazil. This index represents the kdr genotypic frequency weighted by the deltamethrin knockdown time (KdT RR95) for the respective genotype, as determined elsewhere27. Locality identification is similar to that exhibited in Fig. 2. The background map is the “OSM TF Landscape” product (Maps Thunderforest, Data OpenStreetMap contributors, under license CC-BY-SA 2.0). Map was generated with the free and open source software (GNU General Public License) QGIS version 3.12.3 (developd by the Open Source Geospatial Foundation Project, http://qgis.org). populations in the country. This evidenced that the genetic trend of higher or lower pyrethroid resistant (the kdr index) is not random in Brazil. The populations placed in the A12 or A22 areas, in the models with two or three clusters, respectively, are more likely to be resistant to pyrethroids than the rest of the country, considering the target site mechanism. On the other hand, specific conditions may lead to diverse insecticide resistance pat- terns, including distinct kdr allelic frequencies, as observed in Ae. aegypti populations from five neighborhoods in Merida, Mexico30. New studies including spatial patterns details will further knowledge on the dynamics of insecticide resistance distribution and expansion in natural populations. populations in the country. This evidenced that the genetic trend of higher or lower pyrethroid resistant (the kdr index) is not random in Brazil. The populations placed in the A12 or A22 areas, in the models with two or three clusters, respectively, are more likely to be resistant to pyrethroids than the rest of the country, considering the target site mechanism. On the other hand, specific conditions may lead to diverse insecticide resistance pat- terns, including distinct kdr allelic frequencies, as observed in Ae. aegypti populations from five neighborhoods in Merida, Mexico30. New studies including spatial patterns details will further knowledge on the dynamics of insecticide resistance distribution and expansion in natural populations.h p p p The most frequent genotype in Ae. aegypti from Brazil was R1R2, which was heterozygous for the alleles NaVR1 and NaVR2, in 28.9% of all evaluated samples. Discussion Th l l The lowest kdr indexes were predominately found in populations from the Northeast, where the kdr NaVR2 allele was almost absent from the previous survey24 and is now present in all states in that region, with frequencies ranging from 10.2% (Jardim do Seridó, RN) to 83.8% (Itabaiana, SE). In fact, in a previous study assessing resistance to the pyrethroid deltamethrin amongst the 13 populations with pyrethroid resistance ratios (RR95) below 10, reported that seven were located in the Northeast region. In addition, only two populations in the Northeast were amongst the 24 highly resistant populations (RR95 > 10)6.h p p The study of the spatial distribution of resistance markers can aid in identifying underlying resistance emer- gence processes in surrounding areas. In fact, these determinants can exhibit significant “spatial patterns”, due to their strong dependency to space, climate, land cover and land use, active and passive mosquito mobility between connected cities and regional strategies for vector control, among others. These patterns may display spatial variations at different scales. As a consequence, a spatial distribution of the resistance level results of the mixture of these different multi-scale spatial patterns is observed. The decomposition of the observed level of resistance into various independent and significant spatial patterns can, consequently, not only exhibit spatial clusters of cities that present significantly high (or low) resistance levels, but also highlight spatial patterns typi- cal of phenomena that may, therefore, be considered as potential determinants. In the present study, we tested a series of analyses considering the spatialized kdr index values, revealing two to three well defined clustered Scientific Reports \| (2020) 10:13267 \| https://doi.org/10.1038/s41598-020-70029-7 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 4. kdr index distribution in Aedes aegypti from Brazil. This index represents the kdr genotypic frequency weighted by the deltamethrin knockdown time (KdT RR95) for the respective genotype, as determined elsewhere27. Locality identification is similar to that exhibited in Fig. 2. The background map is the “OSM TF Landscape” product (Maps Thunderforest, Data OpenStreetMap contributors, under license CC-BY-SA 2.0). Map was generated with the free and open source software (GNU General Public License) QGIS version 3.12.3 (developd by the Open Source Geospatial Foundation Project, http://qgis.org). Figure 4. kdr index distribution in Aedes aegypti from Brazil. This index represents the kdr genotypic frequency weighted by the deltamethrin knockdown time (KdT RR95) for the respective genotype, as determined elsewhere27. Locality identification is similar to that exhibited in Fig. 2. Discussion Th l l (c) and (d) display the kdr index value distributions as a function of the identified regions. The background map is the “OSM TF Landscape” product (Maps Thunderforest, Data OpenStreetMap contributors, under license CC-BY-SA 2.0). Map was generated with the free and open source software (GNU General Public License) QGIS version 3.12.3 (developd by the Open Source Geospatial Foundation Project, http://qgis.org). Figure 5. Results of the kdr index value distribution spatial analysis. (a) and (b) indicate the spatial patterns and the corresponding regions identified from the kdr index value spatial distributions in Brazil for Aedes aegypti. These patterns correspond to the most explanatory eigenvectors of a principal coordinates analysis of a given neighbor matrix (see Supplementary Text S1). White and black squares consist of negative and positive eigenvector component values, respectively, and square size is proportional to the absolute value of the vector components. These patterns were selected because they present a significant Moran’s I spatial autocorrelation and explain 51.6% (29.6% and 22.0% for A and B, respectively) of the total variance of the observed kdr index values, by their inclusion as explanatory variables in a multiple linear regression model. (c) and (d) display the kdr index value distributions as a function of the identified regions. The background map is the “OSM TF Landscape” product (Maps Thunderforest, Data OpenStreetMap contributors, under license CC-BY-SA 2.0). Map was generated with the free and open source software (GNU General Public License) QGIS version 3.12.3 (developd by the Open Source Geospatial Foundation Project, http://qgis.org). Table 2. Genotypes of 25 Aedes aegypti populations from Brazil, considering the V410L, V1016I and F1534C SNPs in the NaV gene. a Number of samples with their respective genotypes, bMinimum and cMaximum observed frequencies. Discussion Th l l The second most frequent, at 24.4%, was the double kdr R2R2, homozygous for NaVR2, followed by R1R1 in 19.9%, homozygous for NaVR1. The sum of all “resistant genotypes”27 indicated that 73.2% of all Ae. aegypti from Brazil evaluated herein would be resistant to pyrethroid, at least considering the target-site resistance mechanism. It is worth noting, however, that metabolic resistance might also contribute, alone or in synergism with kdr, to confer pyrethroid resistance in the natural populations of this country6. Although the organophosphate malathion began replacing pyrethroids in official campaigns oriented by the Brazilian MoH in 200931, the selection pressure of pyrethroids is still heavily present in Ae. aegypti due to household aerosol insecticide products9,32,33. Experiments conducted in Merida, Mexico, indicate that 87% of households regularly used pyrethroid-based products against mosquitoes33 and that Ae. aegypti field populations were resistant to commonly household used products. In addition, an increase in kdr 1016Ile was associated with the employment of pyrethroid surface sprays in houses34.i p y py p y kdr mutations, or at least the kdr NaVR2 allele, have a high fitness cost and tend to decrease in insecticide- free environments25. In a very practical example, the first Ae. aegypti lineage infected with Wolbachia released in Rio de Janeiro was actually a result of backcrosses between an original infected Australian lineage and a natural Brazilian population, therefore resulting in a Wolbachia-infected lineage resistant to pyrethroids. However, dur- ing several generations in the laboratory awaiting governmental licenses for release, kdr frequencies decreased and the lineage became susceptible32. As residents were spraying pyrethroid-based aerosols in the study area, Scientific Reports \| (2020) 10:13267 \| https://doi.org/10.1038/s41598-020-70029-7 www.nature.com/scientificreports/ Figure 5. Results of the kdr index value distribution spatial analysis. (a) and (b) indicate the spatial patterns and the corresponding regions identified from the kdr index value spatial distributions in Brazil for Aedes aegypti. These patterns correspond to the most explanatory eigenvectors of a principal coordinates analysis of a given neighbor matrix (see Supplementary Text S1). White and black squares consist of negative and positive eigenvector component values, respectively, and square size is proportional to the absolute value of the vector components. These patterns were selected because they present a significant Moran’s I spatial autocorrelation and explain 51.6% (29.6% and 22.0% for A and B, respectively) of the total variance of the observed kdr index values, by their inclusion as explanatory variables in a multiple linear regression model. Discussion Th l l PCR results (410 + 1016 + 1534) Genotypes Number (frequency)a Median Minb Maxc VV + VV + FF VVF/VVF (SS) 26 (0.027) 0 0 0.250 VV + VV + FC VVF/VVC (SR1) 108 (0.111) 0.089 0 0.356 VV + VV + CC VVC/VVC (R1R1) 272 (0.278) 0.227 0.044 1.000 VL + VI + FC VVF/LIC (SR2), VVC/LIF (R1R3) 59 (0.060) 0.034 0 0.182 VL + VI + CC VVC/LIC (R1R2) 262 (0.268) 0.244 0 0.489 LL + II + CC LIC/LIC (R2R2) 233 (0.239) 0.227 0 0.583 VV + VI + CC VVC/VIC 2 (0.002) 0 0 0.022 VV + VI + FC VVF/VIC, VVC/VIF 4 (0.004) 0 0 0.067 VL + VV + CC VVC/LVC 2 (0.002) 0 0 0.038 LL + VI + FC LVF/LIC, LVC/LIF 6 (0.006) 0 0 0.044 VL + II + CC VIC/LIC 3 (0.003) 0 0 0.047 Table 2. Genotypes of 25 Aedes aegypti populations from Brazil, considering the V410L, V1016I and F1534C SNPs in the NaV gene. a Number of samples with their respective genotypes, bMinimum and cMaximum observed frequencies. Wolbachia dispersion did not occur as expected and the first release failed. Thereafter, a new lineage was again backcrossed with field mosquitoes in order to guarantee similar conditions to the well-adapted field population, which then allowed Wolbachia to expand and stabilize in the study area. The kdr frequency is now monitored in the laboratory lineage and in the target field population in the World Mosquito Program32. Th kd fi d d l f h d h l29 l Wolbachia dispersion did not occur as expected and the first release failed. Thereafter, a new lineage was again backcrossed with field mosquitoes in order to guarantee similar conditions to the well-adapted field population, which then allowed Wolbachia to expand and stabilize in the study area. The kdr frequency is now monitored in the laboratory lineage and in the target field population in the World Mosquito Program32.hi i The kdr SNP V410L was first detected in Ae. aegypti samples from Northern and Southeastern Brazil29, also observed in samples from Mexico since 200221, where it is currently highly disseminated35. The V410L SNP was detected in several of our evaluated samples, with strong association of the mutant variation (410Leu) with 1016Ile (and, therefore, with 1534Cys), i.e. the triple kdr allele. Discussion Th l l In Brazil, the most recent nation-wide Ae. aegypti IR monitoring detected several populations resistant to malathion39. The Brazilian MoH is currently planning the implemen- tation of an alternative compound comprising a mixture of pyrethroids and clothianidin to be sprayed inside houses, in addition to another compound comprising a mixture of pyrethroids and imidacloprid as an ULV for outdoor areas. On the other hand, a deeper discussion should consider environmental issues regarding neoni- cotinoids in non-target organisms40. In meanwhile, new arbovirus case records in 2019 in Brazil consisted of over 1.5 million dengue cases, 130 thousand chikungunya cases and 10 thousand Zika cases1. In Brazil, as well as in several other countries with similar climate and urban landscape conditions, the implementation of new vector control tools is paramount. Discussion Th l l Although these neonicotinoids are slower-acting than pyrethroids and organophosphates, resistant populations to these insecticides seem to not present cross-resistance38, indicating them as promising alternative adulticides. In Brazil, the most recent nation-wide Ae. aegypti IR monitoring detected several populations resistant to malathion39. The Brazilian MoH is currently planning the implemen- tation of an alternative compound comprising a mixture of pyrethroids and clothianidin to be sprayed inside houses, in addition to another compound comprising a mixture of pyrethroids and imidacloprid as an ULV for outdoor areas. On the other hand, a deeper discussion should consider environmental issues regarding neoni- cotinoids in non-target organisms40. In meanwhile, new arbovirus case records in 2019 in Brazil consisted of over 1.5 million dengue cases, 130 thousand chikungunya cases and 10 thousand Zika cases1. In Brazil, as well as in several other countries with similar climate and urban landscape conditions, the implementation of new vector control tools is paramount. also observed in samples from Mexico, where mutations in 410 and 1016 were always associated, or, otherwise, dissociated in very low frequencies35. In order to save consumables in a broad kdr surveillance in Ae. aegypti in Latin American countries, where resources are limited, we recommend that the 1016 site may be genotyped first, and for samples genotyped as 1016 V/V or V/I, a second reaction for the 1534 SNP should be performed. On the other hand, when the samples are detected as 1016 I/I, their genotype are likely to be R2R2, i.e. homozygous for the three kdr mutations. Thus, it would not be necessary to genotype the 410 site at all. For academic purposes however, it would be interesting to track possible fluctuations in the frequency of these rare genotypes over time. l New methods for controlling Ae. aegypti or at least making the species “immune” to arbovirus infections are being tested or have already been implemented as actual control methods36. The use of insecticides however may be maintained, due to their potential of rapidly decreasing the density of a targeted population, unless resistance to the applied compound is present. New products with active neonicotinoid-class ingredients have been recently approved by WHO PQT-VC, namely clothianidin, as an indoor residual spray (IRS), and imidacloprid, as an ultra-low volume (ULV) compound37. Although these neonicotinoids are slower-acting than pyrethroids and organophosphates, resistant populations to these insecticides seem to not present cross-resistance38, indicating them as promising alternative adulticides. Discussion Th l l In a few samples however, 410L was dissociated from these other kdr mutations, as evidenced by individuals genotyped as LL + VI + FC, VL + VV + CC and VL + II + CC (all in the order 410 + 1016 + 1534), which account for only 1.1% of the total. Similar results were https://doi.org/10.1038/s41598-020-70029-7 Scientific Reports \| (2020) 10:13267 \| www.nature.com/scientificreports/ 0.0 0.2 0.4 0.6 0.8 1.0 VL+II+CC LL+VI+FC VL+VV+CC VV+VI+FC VV+VI+CC LL+II+CC VL+VI+CC VL+VI+FC VV+VV+CC VV+VV+FC VV_VV_FF frequency genotypes (410 + 1016 + 1534) Figure 6. Frequency of kdr genotypes in Aedes aegypti from Brazil, considering the 410, 1016 and 1534 sites in the voltage gated sodium channel. Dots represent the allelic frequency of each population and bars indicate the median and quartile distribution of the respective allele. 0.0 0.2 0.4 0.6 0.8 1.0 VL+II+CC LL+VI+FC VL+VV+CC VV+VI+FC VV+VI+CC LL+II+CC VL+VI+CC VL+VI+FC VV+VV+CC VV+VV+FC VV_VV_FF frequency genotypes (410 + 1016 + 1534) Figure 6. Frequency of kdr genotypes in Aedes aegypti from Brazil, considering the 410, 1016 and 1534 sites in the voltage gated sodium channel. Dots represent the allelic frequency of each population and bars indicate the median and quartile distribution of the respective allele. also observed in samples from Mexico, where mutations in 410 and 1016 were always associated, or, otherwise, dissociated in very low frequencies35. In order to save consumables in a broad kdr surveillance in Ae. aegypti in Latin American countries, where resources are limited, we recommend that the 1016 site may be genotyped first, and for samples genotyped as 1016 V/V or V/I, a second reaction for the 1534 SNP should be performed. On the other hand, when the samples are detected as 1016 I/I, their genotype are likely to be R2R2, i.e. homozygous for the three kdr mutations. Thus, it would not be necessary to genotype the 410 site at all. For academic purposes however, it would be interesting to track possible fluctuations in the frequency of these rare genotypes over time. New methods for controlling Ae. aegypti or at least making the species “immune” to arbovirus infections are being tested or have already been implemented as actual control methods36. The use of insecticides however may be maintained, due to their potential of rapidly decreasing the density of a targeted population, unless resistance to the applied compound is present. Discussion Th l l New products with active neonicotinoid-class ingredients have been recently approved by WHO PQT-VC, namely clothianidin, as an indoor residual spray (IRS), and imidacloprid, as an ultra-low volume (ULV) compound37. Although these neonicotinoids are slower-acting than pyrethroids and organophosphates, resistant populations to these insecticides seem to not present cross-resistance38, indicating them as promising alternative adulticides. In Brazil, the most recent nation-wide Ae. aegypti IR monitoring detected several populations resistant to malathion39. The Brazilian MoH is currently planning the implemen- tation of an alternative compound comprising a mixture of pyrethroids and clothianidin to be sprayed inside houses, in addition to another compound comprising a mixture of pyrethroids and imidacloprid as an ULV for outdoor areas. On the other hand, a deeper discussion should consider environmental issues regarding neoni- cotinoids in non-target organisms40. In meanwhile, new arbovirus case records in 2019 in Brazil consisted of over 1.5 million dengue cases, 130 thousand chikungunya cases and 10 thousand Zika cases1. In Brazil, as well as in several other countries with similar climate and urban landscape conditions, the implementation of new vector control tools is paramount. also observed in samples from Mexico, where mutations in 410 and 1016 were always associated, or, otherwise, dissociated in very low frequencies35. In order to save consumables in a broad kdr surveillance in Ae. aegypti in Latin American countries, where resources are limited, we recommend that the 1016 site may be genotyped first, and for samples genotyped as 1016 V/V or V/I, a second reaction for the 1534 SNP should be performed. On the other hand, when the samples are detected as 1016 I/I, their genotype are likely to be R2R2, i.e. homozygous for the three kdr mutations. Thus, it would not be necessary to genotype the 410 site at all. For academic purposes however, it would be interesting to track possible fluctuations in the frequency of these rare genotypes over time. New methods for controlling Ae. aegypti or at least making the species “immune” to arbovirus infections are being tested or have already been implemented as actual control methods36. The use of insecticides however may be maintained, due to their potential of rapidly decreasing the density of a targeted population, unless resistance to the applied compound is present. New products with active neonicotinoid-class ingredients have been recently approved by WHO PQT-VC, namely clothianidin, as an indoor residual spray (IRS), and imidacloprid, as an ultra-low volume (ULV) compound37. Material and methods Collections were performed in the context of the national Brazilian insecticide resistance monitoring program for Ae. aegypti between 2017 and 2018. Field agents installed eggtraps randomly distributed according to the total number of houses in each municipality, as follows: 100, 150, 200 or 300 traps for < 50, 50–200, 200–500 and > 500 thousand houses, respectively. The installation and collection methodology of the eggtraps is described elsewhere39. A presential meeting was organized with field-personnel representatives of each state and a video with step-by-step procedures was launched (https://www.youtube.com/watch?v=2w89kagSOKM) in order to ensure that collections were made as homogeneously as possible. The palettes were sent to the laboratory, where the eggs were stimulated to hatch and larvae were raised up to adults in order to produce an F1 colony of each locality, as previously described21. After 4–5 days in the cages, enough time for copulation, a sampling of this F0 generation of each population, around 45 insects, preferentially males, were removed and saved for kdr genotyp- ing. These mosquitoes were maintained in 80% ethanol solution or cryopreserved prior to DNA extraction. The DNA was extracted from single mosquitoes using the FastDNA Spin (MP Biomedicals) or Nucleo Spin Tissue (Macherey–Nagel) kits, according to their manufacturers’ protocol. The eluted DNA was diluted to 10 ng/µL in extra-pure water and cryopreserved until use. p y p We performed independent genotyping reactions for each kdr site based on a qPCR approach using the Custom TaqMan SNP Genotyping Assay (ThermoFisher) (see Table 3 for the primer and probe sequences for each assay). Reactions consisted of 1X TaqMan Genotyping Master Mix (ThermoFisher), 1X of the respective Custom TaqMan SNP Genotyping Assay, 20 ng of DNA and ultra-pure water q.s. 10 µL, run in a QuantStudio https://doi.org/10.1038/s41598-020-70029-7 Scientific Reports \| (2020) 10:13267 \| www.nature.com/scientificreports/ Table 3. Primer and probe sequences for the SNPs V410L, V1016I and F1534C kdr in Aedes aegypti. a Identification of the customized TaqMan SNP Genotyping Assay (ThermoFischer). NaV site Assay IDa Variation Primers Probes 410 AN2XA9W GTA/TTA For: GTGGCACATGCTCTTCTTCATT Val: VIC-TCGTTCTACCTTGTAAATT-NFQ (Val/Leu) Rev: GGCGACAATGGCCAAGATC Leu: FAM-TTCGTTCTACCTTTTAAATT-NFQ 1016 AHS1DL6 GTA/ATA For: CGTGCTAACCGACAAATTGTTTCC Val: VIC-CCCGCACAGGTACTTA-FAM (Val/Ile) Rev: GACAAAAGCAAGGCTAAGAAAAGGT Ile: FAM-CCGCACAGATACTTA-NFQ 1534 AHWSL61 TTC/TGC For: TCGCGAGACCAACATCTACATG Phe: VIC-AACGACCCGAAGATGA-NFQ (Phe/Cys) Rev: GATGATGACACCGATGAACAGATTC Cys: FAM-ACGACCCGACGATGA-NFQ Table 3. Primer and probe sequences for the SNPs V410L, V1016I and F1534C kdr in Aedes aegypti. a Identification of the customized TaqMan SNP Genotyping Assay (ThermoFischer). Material and methods V/I F/C IIS6 1016 IIIS6 1534 S Haplotypes R1 R2 R3 FF FC CC FF FC CC FF FC CC 1016 VV VI II PCR results 1534 + SS SR1 R1R1 SR3 SR2, R1R3 R1R2 R3R3 R2R3 R2R2 Genotypes Figure 7. Representation of the kdr genotype based on PCR reactions for the 1016 (Val+, Ilekdr) and 1534 (Phe+, Cyskdr) variations in the voltage gated sodium channel of Aedes aegypti. Figure 7. Representation of the kdr genotype based on PCR reactions for the 1016 (Val+, Ilekdr) and 1534 (Phe+, Cyskdr) variations in the voltage gated sodium channel of Aedes aegypti. 6 Flex (Applied Biosystems), under standard conditions: 45 cycles with a DNA denaturation step (95 °C for 15 s) and primer and probe annealing, followed by DNA polymerization (60 °C for 1 min). The genotypes were obtained by the online software Genotype Analysis Module V3.9 (Applied Biosystems, ThermoFischer cloud platform). The kdr sites 1016 (V1016I) and 1534 (F1534C) were evaluated in all populations. For 25 out of the 123 populations, mostly from state capitals, the 410 SNP (V410L) was also assessed. 6 Flex (Applied Biosystems), under standard conditions: 45 cycles with a DNA denaturation step (95 °C for 15 s) and primer and probe annealing, followed by DNA polymerization (60 °C for 1 min). The genotypes were obtained by the online software Genotype Analysis Module V3.9 (Applied Biosystems, ThermoFischer cloud platform). The kdr sites 1016 (V1016I) and 1534 (F1534C) were evaluated in all populations. For 25 out of the 123 populations, mostly from state capitals, the 410 SNP (V410L) was also assessed. y Considering that the evaluated SNPs are linked in the same gene, the genotype of each individual contem- plated the results of each site, 1016 (VI, II) and 1534 (FC, CC), resulting in the total combination of nine possible genotypes, composed by four possible haplotypes, S, R1, R2 and R314,22, as displayed in Fig. 7. For the 25 popula- tions in which the 410 site (VV, VL, LL) was also genotyped, a total of 27 combined genotypes and, consequently, eight haplotypes: VVF, VVC, VIC, VIF, LVF, LVC, LIC and LIF, were possible. Material and methods Considering that the evaluated SNPs are linked in the same gene, the genotype of each individual contem- plated the results of each site, 1016 (VI, II) and 1534 (FC, CC), resulting in the total combination of nine possible genotypes, composed by four possible haplotypes, S, R1, R2 and R314,22, as displayed in Fig. 7. For the 25 popula- tions in which the 410 site (VV, VL, LL) was also genotyped, a total of 27 combined genotypes and, consequently, eight haplotypes: VVF, VVC, VIC, VIF, LVF, LVC, LIC and LIF, were possible. g y We ranked the populations in relation to their predicted level of resistance to pyrethroids, based on the kdr index, consisting of the sum of kdr genotype frequencies weighed with their respective resistance ratios, which were previously obtained in a knockdown time test (KdT RR95, Brito et al.27. The KdT RR95 values for the geno- types were SS (1), SR1 (2.4), SR2 (1.7), R1R1 (4.6), R1R2 (5.4) and R2R2 (6.7). The formula for the kdr index of a population is: We ranked the populations in relation to their predicted level of resistance to pyrethroids, based on the kdr index, consisting of the sum of kdr genotype frequencies weighed with their respective resistance ratios, which were previously obtained in a knockdown time test (KdT RR95, Brito et al.27. The KdT RR95 values for the geno- types were SS (1), SR1 (2.4), SR2 (1.7), R1R1 (4.6), R1R2 (5.4) and R2R2 (6.7). The formula for the kdr index of a population is: Scientific Reports \| (2020) 10:13267 \| https://doi.org/10.1038/s41598-020-70029-7 www.nature.com/scientificreports/ [f(SS) × 1] + [f(SR1) × 2.4] + [f(SR2) × 1.7] + [f(R1R1) × 4.6] + [f(R1R2) × 5.4] + [f(R2R2) × 6.7] + [f(R1R1) × 4.6] + [f(R1R2) × 5.4] + [f(R2R2) × 6.7] In order to test the existence of significant multi-scale spatial patterns (according to Moran’s Index of spatial autocorrelation) regarding the spatial distribution of the kdr frequencies, we considered the aforementioned kdr index and the geographic coordinates of each locality in a series of spatial statistic tests, applying a principal coordinate analysis of neighbor matrices and Moran’s eigenvector maps41, as detailed in Supplementary Text 1. 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Screening Aedes aegypti (Diptera: Culicidae) populations from Pernambuco, Brazil for resistance to temephos, diflubenzuron, and cypermethrin and characterization of potential resistance mechanisms. J. Insect Sci. https://doi.org/10.1093/ jisesa/iez054 (2019). Scientific Reports \| (2020) 10:13267 \| https://doi.org/10.1038/s41598-020-70029-7 Acknowledgements g We would like to thank to the Coordenação Geral de Vigilância de Arboviroses (CGARB, Brazilian Ministry of Health), the state and municipality coordinators and health agents from each respective Health and Environ- mental Secretariats for their key role in the sample collection processes and the teams of technicians from both Laficave/IOC/Fiocruz and Lena/Sucen laboratories for the sample preparations. Special thanks are due to Dr Maria de Lourdes Macoris/Lena/Sucen, who provided part of the samplings and gave important inputs to the discussion of this article. We thank Dr Cristovam Barcellos (ICICT/FIOCRUZ) for advices about possibilities in geographical analyses, and are greateful for the support from the Brazilian Coordination for the Improvement of Higher Education Personnel (CAPES). Funds were obtained from Brazilian Ministry of Health, National Institute of Health (NIH, Grant no. UO1 AI115595), Instituto Nacional de Ciência e Tecnologia em Entomolo- gia Molecular (INCT-EM, Grant no. 465678/2014-9) and Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ, Grants no E-26/201.836/2017 and E-26/202.795/2019). Author contributions A.J.M., K.B.C. and J.B.P.L. conceived the idea and designed the study. C.M.R. and D.F.B. organized the sample collection and funds administration. M.M.C. and L.P.B. were responsible for the DNA extraction, qPCR reactions and analyses. E.R. designed and performed the spatial analyses. A.J.M. compiled the data. All authors wrote, reviewed and discussed and are in accordance with the final version of this manuscript. Additional information Supplementary information is available for this paper at https://doi.org/10.1038/s41598-020-70029-7. Correspondence and requests for materials should be addressed to A.J.M. 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. https://doi.org/10.1038/s41598-020-70029-7 Scientific Reports \| (2020) 10:13267 \| ture.com/scientificreports/ 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/. © Th A th ( ) 2020 www.nature.com/scientificreports/ www.nature.com/scientificreports/ 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) 10:13267 \| https://doi.org/10.1038/s41598-020-70029-7
https://openalex.org/W4380050920	https://zenodo.org/records/8022670/files/ZK_article_102847.pdf	English	null	A new species of Siphlonurus Eaton, 1868 (Ephemeroptera, Siphlonuridae) from Yunnan, China	ZooKeys	2,023	cc-by	5,855	Copyright: © Kun Yang et al. This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International – CC BY 4.0). A new species of Siphlonurus Eaton, 1868 (Ephemeroptera, Siphlonuridae) from Yunnan, China Kun Yang1, Xian-Fu Li1,2,3 , Xiao-Li Tong4, Qing-Hua Cai5 1 Institute of Eastern-Himalaya Biodiversity Research, Dali University, Dali 671000, Yunnan, China 2 Collaborative Innovation Center for Biodiversity and Conservation in the Tree Parallel Rivers Region of China, Dali University, Dali, Yunnan, China 3 Research Center of Ecology and Governance for Er’hai Lake Streams, Dali, Yunnan, China 4 Department of Entomology, College of Plant Protection, South China Agricultural University, Guangzhou 510642, Guangdong, China 5 State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, Hubei China Corresponding author: Xian-Fu Li (lixf@eastern-himalaya.cn) Citation: Yang K, Li X-F, Tong X-L, Cai Q-H (2023) A new species of Siphlonurus Eaton, 1868 (Ephemeroptera, Siphlonuridae) from Yunnan, China. ZooKeys 1166: 121–139. https://doi.org/10.3897/ zookeys.1166.102847 Abstract Siphlonurus dongxi Li & Tong, sp. nov. from Shangri-La City, Yunnan Province, China, is described based on egg, nymph, and winged stages. The new species is closely related to S. davidi (Navás, 1932), and can be distinguished by the colour of the imago, the forking point of MP, the penis, posterolateral spines of tergum IX of imagoes, and first abdominal terga nymph, as well as the structure of the egg. The new species and S. davidi have the same morphological and structural characteristics, such as the long cubital area with many intercalaries, cross veins between C, Sc, RA, and RSa1 surrounded with distinct pigments, the strong curvature of vein CuP in the forewing, the broad expansion of the hindwing, the membranous penis lobes fused without teeth, supporting the proposition of a new species complex, the Siphlonurus davidi group. The structures of the penis and the egg of the new species could help understand the origin and evolution of the genus Siphlonurus. Key words: Hengduan Mountains, Himalaya, Mayfly, Siphlonuridae Research Article ZooKeys 1166: 121–139 (2023) DOI: 10.3897/zookeys.1166.102847 ZooKeys 1166: 121–139 (2023) DOI: 10.3897/zookeys.1166.102847 Introduction ZooBank: https://zoobank. org/6FF7402B-CCC7-4C28-B6D4- E919E2817F7C The genus Siphlonurus Eaton, 1868 (Ephemeroptera, Siphlonuridae) is characterized by many plesiomorphies (Kluge et al. 1995). About 40 Siphlonurus species have been reported from the Nearctic and Palaearctic realms (Kluge 2004). A few species have been mentioned from China, but Kluge (2004) suggested that there was an “unknown group” of Siphlonurus in China. So far, only S. davidi (Navás, 1932) is distributed in China. It was initially described from a single male subimago (Zhou and Peters 2003), while the type specimen was redescribed later (Sartori and Peters 2004). Afterwards, the egg, nymph, and imago of S. davidi were described by Han et al. (2016). Consequently, the lack of research on adults of Siphlonurus might limit the understanding of this genus in China. 121 Kun Yang et al.: Siphlonurus dongxi Li & Tong, sp. nov. Siphlonurus davidi presents some plesiomorphies, indicating a close rela tionship with the ancestor of the Siphlonurus lineage, such as the forking point of MP subequal to that of the fusion point of MA and RS, the cubital area lon ger and with more intercalaries between CuA and the posterior margin of the wing, and the hindwings approximately half the length of the forewings, longer than in other Siphlonurus species (Sartori and Peters 2004; Han et al. 2016). During our recent survey of the mayfly fauna of the Hengduan Mountains area, at the eastern end of the Himalayas, a not yet described species of Siphlonurus similar to S. davidi was found in Shangri-La City, western Yunnan, China, at an altitude of more than 3000 m. Here, we describe this new Siphlonurus species based on imago, subimago, nymph, and egg stages. Materials and methods Siphlonurus nymphs were collected with a D-frame net from the floodplain habitats of the Dugang River in Shangri-La City, northwestern Yunnan, China. Following the guidelines from Li et al. (2022), the habitat photographs were taken using a Huawei Nova 8 mobile phone equipped with a Kase 40–75 mm macro lens. Some specimens were dissected under a stereomicroscope and were mounted on slides with Hoyer’s solution for examination with a digital microscope. Slide-mounted specimens were examined and photographed with a Keyence VHX-S550E digital microscope. For scanning electron microscopy (SEM), eggs were dried, coated with gold, and observed with a VEGA3 SBU SEM (Tescan, Brno, Czech Republic). Measurements were taken using ImageJ image processing software. The final plates were prepared with Adobe Photo shop CC 2018. All examined materials were deposited at the Museum of Biology, Insti tute of Eastern-Himalaya Biodiversity Research, Dali University, Dali, Yunnan, China (MBDU). Results Siphlonurus dongxi Li & Tong, sp. nov. https://zoobank.org/27AA2276-1CF5-4CA9-A6ED-5FCC5EC9F691 Figs 1–17 ZooKeys 1166: 121–139 (2023), DOI: 10.3897/zookeys.1166.102847 Siphlonurus dongxi Li & Tong, sp. nov. Material examined. Holotype: male imago, with final nymphal instar exuvia (in ethanol), China, Yunnan Province, Shangri-La City, Jiantang Town, Dugang river, 27°47′50.4″N, 99°48′43.3″E, 3361 m a.s.l., 12.VI.2022, coll. Xian-Fu Li. Paratypes: 13 nymphs, 30 imagoes and 8 subimagoes reared from nymphs with same data as holotype. 5 nymphs from same location as holotype, but 1.VI.2021, coll. Yi-Hao Fang. Diagnoses. The new species is similar to S. davidi. It can be distinguished from S. davidi by the colour of the imago, the morphological structure of egg, the forking point of MP, the transversal sclerite of the penis with two dorsal elongations, the dorsal elongation of the penis basally expanded, the elongations of the ventral sclerite, the posterolateral spines of tergum IX of imagoes and the first abdominal terga of the nymph, as well as the structure of the egg. ZooKeys 1166: 121–139 (2023), DOI: 10.3897/zookeys.1166.102847 122 Kun Yang et al.: Siphlonurus dongxi Li & Tong, sp. nov. ys 1166: 121–139 (2023) DOI: 10 3897/zookeys 1166 102847 ng et al.: Siphlonurus dongxi Li & Tong, sp. nov. re 1. Male imago of Siphlonurus dongxi Li & Tong, sp. nov. A dorsal view B lateral view C ventra ewing E hindwing F anterior margin of forewing G anterior margin of hindwing. et al.: Siphlonurus dongxi Li & Tong, sp. nov. igure 1. Male imago of Siphlonurus dongxi Li & Tong, sp. nov. A dorsal view B lateral view C ventral D forewing E hindwing F anterior margin of forewing G anterior margin of hindwing. Figure 1. Male imago of Siphlonurus dongxi Li & Tong, sp. nov. A dorsal view B lateral view C ventral view D forewing E hindwing F anterior margin of forewing G anterior margin of hindwing. ZooKeys 1166: 121–139 (2023), DOI: 10.3897/zookeys.1166.102847 123 Kun Yang et al.: Siphlonurus dongxi Li & Tong, sp. nov. Figure 2. Male imago of Siphlonurus dongxi Li & Tong, sp. nov. A foreleg B midleg C hindleg D femur enlarged of foreleg E femur enlarged of midleg F femur enlarged of hindleg G tarsus enlarged of foreleg H tarsus en larged of midleg I cerci J cerci enlarged. Figure 2. Male imago of Siphlonurus dongxi Li & Tong, sp. nov. Siphlonurus dongxi Li & Tong, sp. nov. A foreleg B midleg C hindleg D femur enlarged of foreleg E femur enlarged of midleg F femur enlarged of hindleg G tarsus enlarged of foreleg H tarsus en larged of midleg I cerci J cerci enlarged. Descriptions. Male imago (in ethanol). Body length 18.4–20.5 mm (exclud ing cerci), head width 3.3–3.5 mm, forewing length 17.3–18.6 mm, hindwing length 7.8–8.1 mm, antennae 1.4–1.6 mm. Ratio of hindwing: forewing length about 0.43. Head: compound eyes contiguous (Fig. 1A), each of them spherical, upper portion and lower portion grey, without clear line between them (Fig. 1B). Thorax: generally light yellow and dark brown, mesonotum anterior and legs basal with light yellowish stripes or rings (Figs 1A–C, 2A–C). All legs light yellowish to dark brown, with distinct markings at both ends of femur and tib ia (Fig. 2A–C). Length of femur: tibia: tarsus of foreleg = 1.3: 1.0: 2.4, tarsal ZooKeys 1166: 121–139 (2023), DOI: 10.3897/zookeys.1166.102847 124 Kun Yang et al.: Siphlonurus dongxi Li & Tong, sp. nov. Figure 3. Male imago of Siphlonurus dongxi Li & Tong, sp. nov. A dorsal view of penis B lateral view of penis C ventral view of penis D dorsal view of genitalia E lateral view of genitalia F ventral view of genitalia. Figure 3 Male imago of Siphlonurus dongxi Li & Tong sp nov A dorsal view of penis B lateral view of penis Figure 3. Male imago of Siphlonurus dongxi Li & Tong, sp. nov. A dorsal view of penis B lateral view of penis C ventral view of penis D dorsal view of genitalia E lateral view of genitalia F ventral view of genitalia. segments from basal to apical = 2.2: 2.0: 1.8: 1.4: 1.0; femur: tibia: tarsus of mid leg = 1.4: 1: 1.6, tarsal segments from basal to apical = 3.7: 2.5: 1.8: 1.0: 1.4; fe mur: tibia: tarsus of hindleg = 1.5: 1.0: 1.6, tarsal segments from basal to apical = 4.3: 2.8: 1.9: 1.0: 1.8. Inner margin of foreleg tarsus densely covered with circular bulges (Fig. 2A, G), and that of midleg and hindleg with stout setae (Fig. 2B, C, H). Outer margin of foreleg femur and tibia relatively densely covered with stout setae (Fig. 2A–F). Similar to other Siphlonurus species, all legs end with one blunt and one hooked claw. Forewings (Fig. Siphlonurus dongxi Li & Tong, sp. nov. 1D) generally hyaline except outer 1/3 of C and Sc, Sc and RA fields, base of forewing distinctly pigmented with black inlaid with yellow. Cross veins between C, Sc, RA, and RSa1 surrounded with distinct pigments, with dark spots. The forking point of MP is more proximal from that of fusion point of MA and RS. Anterior margin of forewing with small setae (Fig. 1F). Base of hindwing (Fig. 1E) distinctly pigmented with black inlaid with yellow, an additional large dark patch in the middle of Sc and RA cells, RA, and RS cells. Distal half of hindwing washed with black, making this area semi transparent, areas near centre and near margin of hindwing darker than others. Ratio of width: length about 0.79. Anterior margin without setae (Fig. 1G). segments from basal to apical = 2.2: 2.0: 1.8: 1.4: 1.0; femur: tibia: tarsus of mid leg = 1.4: 1: 1.6, tarsal segments from basal to apical = 3.7: 2.5: 1.8: 1.0: 1.4; fe mur: tibia: tarsus of hindleg = 1.5: 1.0: 1.6, tarsal segments from basal to apical = 4.3: 2.8: 1.9: 1.0: 1.8. Inner margin of foreleg tarsus densely covered with circular bulges (Fig. 2A, G), and that of midleg and hindleg with stout setae (Fig. 2B, C, H). Outer margin of foreleg femur and tibia relatively densely covered with stout setae (Fig. 2A–F). Similar to other Siphlonurus species, all legs end with one blunt and one hooked claw. Forewings (Fig. 1D) generally hyaline except outer 1/3 of C and Sc, Sc and RA fields, base of forewing distinctly pigmented with black inlaid with yellow. Cross veins between C, Sc, RA, and RSa1 surrounded with distinct pigments, with dark spots. The forking point of MP is more proximal from that of fusion point of MA and RS. Anterior margin of forewing with small setae (Fig. 1F). Base of hindwing (Fig. 1E) distinctly pigmented with black inlaid with yellow, an additional large dark patch in the middle of Sc and RA cells, RA, and RS cells. Distal half of hindwing washed with black, making this area semi transparent, areas near centre and near margin of hindwing darker than others. Ratio of width: length about 0.79. Anterior margin without setae (Fig. 1G). ZooKeys 1166: 121–139 (2023), DOI: 10.3897/zookeys.1166.102847 125 Kun Yang et al.: Siphlonurus dongxi Li & Tong, sp. nov. Figure 4. Male subimago of Siphlonurus dongxi Li & Tong, sp. Siphlonurus dongxi Li & Tong, sp. nov. nov. A dorsal view B lateral view C ventral view D forewing E hindwing F forceps of genitalia. Kun Yang et al.: Siphlonurus dongxi Li & Tong, sp. nov. Figure 4. Male subimago of Siphlonurus dongxi Li & Tong, sp. nov. A dorsal view B lateral view C ventral view D forewing E hindwing F forceps of genitalia. ZooKeys 1166: 121–139 (2023), DOI: 10.3897/zookeys.1166.102847 126 126 Kun Yang et al.: Siphlonurus dongxi Li & Tong, sp. nov. Figure 5. Male subimago of Siphlonurus dongxi Li & Tong, sp. nov. A foreleg B midleg C hindleg D tarsus enlarged of foreleg E cerci F cerci enlarged. Figure 5. Male subimago of Siphlonurus dongxi Li & Tong, sp. nov. A foreleg B midleg C hindleg D tarsus enlarged of foreleg E cerci F cerci enlarged. Abdomen (Fig. 1A–C) light yellow and dark brown, dorsal (Fig. 1A) and lateral sides (Fig. 1B) of terga II–X each with “W” shaped dark stripes; the ventral side (Fig. 1C) of terga II–VIII each with two dark symmetric spots and dark stripes, part and all of terga VIII–X distinct pigmented. Posterolateral spines of tergum IX well developed (Fig. 3D–F). Middle filament lost, terminal filament vestigial. Cerci lengths 29.5 mm, densely covered with long setae (Fig. 2I, J). Genitalia. Penis relatively long (Fig. 3A–C), brown with dark brown markings, visible in distinct views (Fig. 3D–F); transversal sclerite with two elongations (Fig. 3A, indicated by red arrow) and two semicircular white plates shown in dorsal and lateral views (Fig. 3A, B); dorsal elongation of penis arched without spines, basally expanded (Fig. 3A, indicated by black arrow) apical half slim, penis lobe white fused without spines; elongations of ventral sclerite long, gradually widen from basal to subapical; styliger (Fig. 3D–F) 4-segmented, dark brown with stout setae, basal segment shortest but broadest, second segment about twice the length of third and apical ones together, the latter two subequal in length, each slightly longer than basal one. Styliger plate (Fig. 3F) slightly notched in middle. Male subimago (in ethanol) (Fig. 4A–G) similar to male imago except for following characters: thorax and terga VIII–X overall lighter than the male imago (Fig. 4A–C); forewing (Fig. 4E) and hindwing (Fig. 4F) subhyaline; ZooKeys 1166: 121–139 (2023), DOI: 10.3897/zookeys.1166.102847 127 Kun Yang et al.: Siphlonurus dongxi Li & Tong, sp. nov. Figure 6. Male subimago of Siphlonurus dongxi Li & Tong, sp. nov. Siphlonurus dongxi Li & Tong, sp. nov. A dorsal view of penis B lateral view of penis C ventral view of penis. Figure 6. Male subimago of Siphlonurus dongxi Li & Tong, sp. nov. A dorsal view of penis B lateral view of penis C ventral view of penis. forewing without intercalaries at MP1 to iMP, with tiny setae on outer and hind margins; length of femur: tibia: tarsus of foreleg (Fig. 5A) = 1.4:1.0: 2.0, tarsal segments from basal to apical = 1.8: 1.6: 1.3: 1.0: 1.0; femur: tibia: tarsus of midleg (Fig. 5B) = 1.5: 1.0: 1.7, tarsal segments from basal to apical = 3.2: 2.1: 1.4: 1.0: 1.4; femur: tibia: tarsus of hindleg (Fig. 5C) = 1.5: 1.0: 1.5, tarsal segments from basal to apical = 3.9: 2.3: 1.6: 1.0: 1.8; inner of tarsus of fore leg with circular bulges and setae (Fig. 5D). The sclerite structure of penis incomplete (Fig. 6A–C). Styliger plate only shallowly curved, posterior margin waved; styliger with relative densely thick setae (Fig. 4G). Forewing length 17.0–17.5 mm, hindwing length 7.8–8.1 mm, cerci length 15.4–16.5 mm (Fig. 5E, F). Female imago (in ethanol). Colour pattern similar to male; body length 19.6– 22.2 mm, head width 3.1–3.4 mm, caudal filaments 20.7–24.4 mm, forewing 18.5–21.3 mm, hindwing 9.0–9.4 mm (Fig. 7A–F). Length of femur: tibia: tar sus of foreleg = 1.3: 1.0: 1.8, tarsal segments from basal to apical = 3.0: 2.3: 1.7: 1: 1.3; femur: tibia: tarsus of midleg = 1.5: 1.0: 1.6, tarsal segments from basal to apical = 3.2: 2.3: 1.6: 1: 1.4; femur: tibia: tarsus of hindleg = 1.5: 1.0: 1.6, tarsal segments from basal to apical = 3.8: 2.8: 1.7: 1.0: 1.6. Subgenital plate produced to 1/3 length of sternum VIII (Fig. 7B). Compared with male, forewing of female imago with sparse cross veins between C and Sc; all of Sc and RS fields, and outer part of RA and RS cells subhyaline (Fig. 7D). Inner margins of tarsus of foreleg, midleg and hindleg densely covered with spines (Fig. 8A–C, I). Female subimago (in alcohol). Similar to male subimago except the tarsal segments of foreleg with more spines and usual sexual differences (Figs. 8D–I). ZooKeys 1166: 121–139 (2023), DOI: 10.3897/zookeys.1166.102847 ZooKeys 1166: 121–139 (2023), DOI: 10.3897/zookeys.1166.102847 Siphlonurus dongxi Li & Tong, sp. nov. ZooKeys 1166: 121–139 (2023), DOI: 10.3897/zookeys.1166.102847 130 Kun Yang et al.: Siphlonurus dongxi Li & Tong, sp. nov. Winged stages of Siphlonurus dongxi Li & Tong, sp. nov. (living) are shown in Fig. 9A–D. Eggs. Oval with irregular flat areas (Fig. 10A), length of 224–240 um, and width of 168–175 um. Chorion without obvious reticulation, with micropyle with or without reticulation (Fig. 10A–D). No accessory attachment structure apparent, but the egg surface has convex rough structures (Fig. 10A, B). The eggs were stuck tightly together, and the mass remained intact when placed in the water or ethanol. Persistent mouthparts of winged stages. The new species presents per sistent mouthparts in winged stages; in ventral view of head, the labial and maxillary are present and clearly visible (Fig. 11A–D, indicated by white arrow). Final nymphal instar (in ethanol) light yellow with red and dark markings (Fig. 12A, B), body length 14.1–16.7 mm (excluding cerci); head width 2.6– 2.8 mm, cerci lengths 6.7–7.7 mm, median filament 5.6–6.8 mm, antennae 1.8–2.1 mm. Morphology and structure of the head (Fig. 12A, B) and mouth parts (Fig. 13A–H) of the new species similar to that of S. davidi. All legs simi lar (Fig. 14A), surface with short thick sparse setae, the apex of femora, tibiae, and tarsi with black spots or rings; femora broad, median marking black band; midleg with one clear patellar–tibial suture and hindleg with two ones on the tibiae (Fig. 14A, indicated by red arrow). Length of femur: tibia: tarsus of fore leg = 1.8: 1.0: 1.3, femur: tibia: tarsus of midleg = 1.9: 1.0: 1.3, and femur: tibia: tarsus of hindleg = 1.8: 1.0: 1.2. Claws simple, without teeth (Fig. 14A). Abdo men with each tergite with one pair of parallel stripes near median line and tra cheae with distinct pigmentation (Fig. 12A), posterolateral spines present on terga II–IX, surface with short, thick, sparse setae (Fig. 15A). Abdominal ster num (Fig. 14C) light yellow with dark spots and oblique dashes similar to that of winged stages; surface with short thick sparse setae (Fig. 14D); posterior margin of sternum IX of male and female concave (Fig. 15B, C). Middle instars light yellow without pigmentation (Fig. 14B). Caudal filaments with dark bands at the top of each segment, each segment with whorls of short, thick setae apically and long, hair-like setae laterally (Fig. 15D). Siphlonurus dongxi Li & Tong, sp. nov. Length of femur: tibia: tarsus of foreleg = 1.4: 1.0: 1.6, tarsal segments from basal to apical = 3.1: 2.1: 1.5: 1: 1.6; femur: tibia: tarsus of midleg = 1.4: 1.0: 1.5, tarsal segments from basal to apical = 4.1: 2.6: 1.7: 1: 2.3; femur: tibia: tarsus of hindleg = 1.4: 1.0: 1.4, tarsal segments from basal to apical = 4.0: 2.5: 1.7: 1.0: 1.9. Inner margins of tarsus of foreleg, midleg and hindleg densely covered with spines (Fig. 8D–F, J). Forewing length 17.7–20.2 mm, hindwing length 8.9–9.1 mm, cerci length 13.2–14.2 mm. ZooKeys 1166: 121–139 (2023), DOI: 10.3897/zookeys.1166.102847 128 Kun Yang et al.: Siphlonurus dongxi Li & Tong, sp. nov. 6: 121–139 (2023), DOI: 10.3897/zookeys.1166.102847 l.: Siphlonurus dongxi Li & Tong, sp. nov. Female imago of Siphlonurus dongxi Li & Tong, sp. nov. A dorsal view B lateral view C ven g E hindwing F posterior part of abdomen (ventral view). Figure 7. Female imago of Siphlonurus dongxi Li & Tong, sp. nov. A dorsal view B lateral view C ventra D forewing E hindwing F posterior part of abdomen (ventral view). Figure 7. Female imago of Siphlonurus dongxi Li & Tong, sp. nov. A dorsal view B lateral view C ventral view D forewing E hindwing F posterior part of abdomen (ventral view). ZooKeys 1166: 121–139 (2023), DOI: 10.3897/zookeys.1166.102847 129 Kun Yang et al.: Siphlonurus dongxi Li & Tong, sp. nov. Figure 8. Female imago and subimago of Siphlonurus dongxi Li & Tong, sp. nov. A foreleg of imago B midleg of imago C hindleg of imago D foreleg of subimago E midleg of subimago F hindleg of subimago G forewing H hindwing I tarsus enlarged of imago J tarsus enlarged of subimago. Figure 8. Female imago and subimago of Siphlonurus dongxi Li & Tong, sp. nov. A foreleg of imago B midleg of imago C hindleg of imago D foreleg of subimago E midleg of subimago F hindleg of subimago G forewing H hindwing I tarsus enlarged of imago J tarsus enlarged of subimago. Figure 8. Female imago and subimago of Siphlonurus dongxi Li & Tong, sp. nov. A foreleg of imago B midleg of imago C hindleg of imago D foreleg of subimago E midleg of subimago F hindleg of subimago G forewing H hindwing I tarsus enlarged of imago J tarsus enlarged of subimago. ZooKeys 1166: 121–139 (2023), DOI: 10.3897/zookeys.1166.102847 ZooKeys 1166: 121–139 (2023), DOI: 10.3897/zookeys.1166.102847 Distribution. China (Yunnan). Ecology. Nymphs of this new species prefer to live in pools or slow current areas with aquatic plants close to the bank in clear, high-altitude, wide streams (Fig. 17A). The last instar nymphs molted at noon and stayed on the grass for a relatively long time (Fig. 17B). In the laboratory, the larvae showed a behaviour of tearing water grass and collecting sediment (Fig. 17C). The emergence time is consistent with that observed in the field, the mature larvae crawled out of the water, the subimago stopped for a short time to drain a little water from the end of the abdomen before taking off (Fig. 17D). The subimago stage persisted until the third night while the observed lifespan of imagoes was about 4 days. Siphlonurus dongxi Li & Tong, sp. nov. A male subimago B male imago C female subimago D female imago. Siphlonurus dongxi Li & Tong, sp. nov. Gills double on segments I and II; dorsal lamella of gill I (Fig. 16A) triangle, small, anterior rim relatively short, posterior margin straight. Dorsal lamella of gill II (Fig. 16C) leaf-shaped, anterior rim relatively short, posterior margin round, apically pointed. Ventral lamellae of gills I (Fig. 16B) and II (Fig. 16D) heart-shaped and their posteri or margin slightly notched. Gills III (Fig. 5E), single, leaf-shaped, anterior rim relatively long, posterior margin round, apically slightly pointed. Gills IV–VII (Fig. 16F–J) single, oval, apically round, anterior rim relatively long; posterior margin round. Anterior rims of each gill with very short stout setae (Fig. 16J). Gill size gradually increased from gill I to gill III and decreasing from gill III to gill VII. Etymology. The specific epithet dongxi, is named after the Chinese ab breviation of the Institute of Eastern-Himalaya Biodiversity Research, Dali University (https://www.eastern-himalaya.com.cn/contents/16/923.html). We hope that the Institute of Eastern-Himalaya Biodiversity Research can become an important platform for biodiversity research in the world. At the same time, we hope to cooperate with scientists from all countries through the International Centre of Biodiversity and Primate Conservation (http:// www.icbpc.org/index.html). ZooKeys 1166: 121–139 (2023), DOI: 10.3897/zookeys.1166.102847 131 Kun Yang et al.: Siphlonurus dongxi Li & Tong, sp. nov. Figure 9. Winged stages of Siphlonurus dongxi Li & Tong, sp. nov (living) A male subimago B male imago C female subimago D female imago. Figure 9. Winged stages of Siphlonurus dongxi Li & Tong, sp. nov (living) A male subimago B male imago C female subimago D female imago. Figure 10. Egg of Siphlonurus dongxi Li & Tong, sp. nov. (SEM image) A shape and exochorionic surface of egg B–D micropyles enlarged. Scale bars: 50 μm (A); 10 um (B–D). Figure 10. Egg of Siphlonurus dongxi Li & Tong, sp. nov. (SEM image) A shape and exochorionic surface of egg B–D micropyles enlarged. Scale bars: 50 μm (A); 10 um (B–D). ZooKeys 1166: 121–139 (2023), DOI: 10.3897/zookeys.1166.102847 132 Kun Yang et al.: Siphlonurus dongxi Li & Tong, sp. nov. Figure 11. Persistent mouthparts of Siphlonurus dongxi Li & Tong, sp. nov. A male subimago B male imago C female subimago D female imago. Figure 11. Persistent mouthparts of Siphlonurus dongxi Li & Tong, sp. nov. A male subimago B male imago C female subimago D female imago. Figure 11. Persistent mouthparts of Siphlonurus dongxi Li & Tong, sp. nov. Discussion The identification key of three Asian Siphlonurus species, S. binotatus Eaton, 1892 (Gose 1979: fig. 59), S. davidi (Han et al. 2016: figs 3–6), S. palaearcti cus (Tshernova, 1930) (Kluge 1982: fig. 3) with coloured wings was provided ZooKeys 1166: 121–139 (2023), DOI: 10.3897/zookeys.1166.102847 133 Kun Yang et al.: Siphlonurus dongxi Li & Tong, sp. nov. Figure 12. Last nymphal instar, dorsal habitus of Siphlonurus dongxi Li & Tong, sp. nov. A male (just after molt) B female. Kun Yang et al.: Siphlonurus dongxi Li & Tong, sp. nov. Figure 12. Last nymphal instar, dorsal habitus of Siphlonurus dongxi Li & Tong, sp. nov. A male (just after molt) B female. Figure 12. Last nymphal instar, dorsal habitus of Siphlonurus dongxi Li & Tong, sp. nov. A male (just after molt) B female. by Sartori and Peters (2004) and Han et al. (2016). However, S. yoshinoensis Gose, 1979, with colourful wings (Takayanagi 2021: figs 1–3), was ignored. In Asia, five species with colourful wings have been identified. Among them, the imagoes of S. binotatus and S. palaearcticus have ventral penis lobe with teeth, which can be differentiated from the other three species; S. davidi and S. dongxi Li & Tong, sp. nov. feature a fused penis lobe, while S. yoshinoensis has a penis lobe with a deep median incision. by Sartori and Peters (2004) and Han et al. (2016). However, S. yoshinoensis Gose, 1979, with colourful wings (Takayanagi 2021: figs 1–3), was ignored. In Asia, five species with colourful wings have been identified. Among them, the imagoes of S. binotatus and S. palaearcticus have ventral penis lobe with teeth, which can be differentiated from the other three species; S. davidi and S. dongxi Li & Tong, sp. nov. feature a fused penis lobe, while S. yoshinoensis has a penis lobe with a deep median incision. Siphlonurus dongxi Li & Tong, sp. nov. is closely related to S. davidi, whose adults share the markings of the wings, the strong curvature of vein CuP, the broad expansion of the hind wing, the longer cubital area in the forewing, and the membranous penis lobes fused without teeth. Siphlonurus dongxi Li & Tong, sp. nov. and S. davidi differ from all other described Siphlonurus species in these characters (Sartori and Peters 2004; Han et al. 2016), which offer support that a new species complex, the Siphlonurus davidi group, should be distinguished. ZooKeys 1166: 121–139 (2023), DOI: 10.3897/zookeys.1166.102847 Discussion ZooKeys 1166: 121–139 (2023), DOI: 10.3897/zookeys.1166.102847 134 Kun Yang et al.: Siphlonurus dongxi Li & Tong, sp. nov. Figure 13. Nymphal characters of Siphlonurus dongxi Li & Tong, sp. nov. A antenna B labrum (ventral view on left, dorsal view on right) C left mandible D right mandible E left maxilla F right maxilla G hypopharynx H labium. Figure 13. Nymphal characters of Siphlonurus dongxi Li & Tong, sp. nov. A antenna B labrum (ventral view on left, dorsal view on right) C left mandible D right mandible E left maxilla F right maxilla G hypopharynx H labium. Figure 14. Nymphal characters of Siphlonurus dongxi Li & Tong, sp. nov. A legs (dorsal view), from top to bot tom foreleg, midleg and hindleg B abdomen of the middle instar (ventral view) C abdomen of the last instar (ventral view) D enlarged of abdomen (ventral view). Figure 14. Nymphal characters of Siphlonurus dongxi Li & Tong, sp. nov. A legs (dorsal view), from top to bot tom foreleg, midleg and hindleg B abdomen of the middle instar (ventral view) C abdomen of the last instar (ventral view) D enlarged of abdomen (ventral view). ZooKeys 1166: 121–139 (2023), DOI: 10.3897/zookeys.1166.102847 135 Kun Yang et al.: Siphlonurus dongxi Li & Tong, sp. nov. Figure 15. Nymphal characters Siphlonurus dongxi Li & Tong, sp. nov. A posterior part of abdomen (dorsal view) B posterior part of abdomen of male (ventral view) C posterior part of abdomen of female (ventral view) D enlarged of caudal filaments. Figure 15. Nymphal characters Siphlonurus dongxi Li & Tong, sp. nov. A posterior part of abdomen (dorsal view) B posterior part of abdomen of male (ventral view) C posterior part of abdomen of female (ventral view) D enlarged of caudal filaments. Figure 16. Gills of Siphlonurus dongxi Li & Tong, sp. nov. A dorsal lamella of gill I B ventral lamella of gill I C dor sal lamella of gill II D ventral lamella of gill E gill III F gill IV G gill V H gill VI I gill VII J anterior enlarged of gill VII. Figure 16. Gills of Siphlonurus dongxi Li & Tong, sp. nov. ZooKeys 1166: 121–139 (2023), DOI: 10.3897/zookeys.1166.102847 ZooKeys 1166: 121–139 (2023), DOI: 10.3897/zookeys.1166.102847 Discussion 137 Kun Yang et al.: Siphlonurus dongxi Li & Tong, sp. nov. These numerous and significant differences between S. dongxi Li & Tong, sp. nov. and S. davidi suggest that the existence of a species bridging the gap between them is possible. Obviously, the new species shows characteristics that fall somewhere be tween S. davidi and other ones. The discovery of this new species bridges the gap between S. davidi and other Siphlonurus species, and could help reveal the origin and evolution of the genus Siphlonurus. Funding The Second Tibetan Plateau Scientific Expedition and Research Program (2019QZKK0402) and the National Natural Science Foundation of China (31960255). Conflict of interest No conflict of interest was declared. Author contributions Kun Yang: Writing - Original Draft; Xian-Fu Li: Conceptualization, Methodology, Writing - Review & Editing; Xiao-Li Tong: Writing - Review & Editing; Qing-Hua Cai: Project admin istration, Funding acquisition. Data availability All of the data that support the findings of this study are available in the main text or Supplementary Information. Author ORCIDs Xian-Fu Li https://orcid.org/0000-0002-1258-1573 Discussion A dorsal lamella of gill I B ventral lamella of gill I C dor sal lamella of gill II D ventral lamella of gill E gill III F gill IV G gill V H gill VI I gill VII J anterior enlarged of gill VII. Figure 16. Gills of Siphlonurus dongxi Li & Tong, sp. nov. A dorsal lamella of gill I B ventral lamella sal lamella of gill II D ventral lamella of gill E gill III F gill IV G gill V H gill VI I gill VII J anterior enlar ZooKeys 1166: 121–139 (2023), DOI: 10.3897/zookeys.1166.102847 136 Kun Yang et al.: Siphlonurus dongxi Li & Tong, sp. nov. There are significant differences within the Siphlonurus davidi group, such as the following characters: 1. Imagoes of S. davidi are reddish brown, while S. dongxi Li & Tong, sp. nov. are light yellow and dark brown. 2. Forking point of MP in forewing of S. davidi is subequal to the fusion point of MA and RS, then MP2 bends backwards strongly near to CuA. This con dition is common in Ephemeridae and Potamanthidae, and is similar to Siphlonurus chinensis (Ulmer, 1920; Han et al. 2016), but it is not found in S. dongxi Li & Tong, sp. nov.. 3. Penis of S. davidi has only membranous lobe, but the penis lobe of S. dongxi Li & Tong, sp. nov. has long elongations of ventral sclerite. 4. Posterolateral spines of tergum IX of S. dongxi Li & Tong, sp. nov. imagoes are well developed. While, the ones of S. davidi are poorly developed. 5. All abdominal terga of S. davidi nymph have distinct posterolateral spines, while the spines of S. dongxi Li & Tong, sp. nov. are only on segments 2–9. 6. The egg exochorionic surface of S. dongxi Li & Tong, sp. nov. has micro pyle, but in S. davidi the egg exochorionic surface is without micropyle (Han et al. 2016: fig. 7). Figure 17. Habitat of Siphlonurus dongxi Li & Tong, sp. nov. A nymphal habitat in Dugang River B female subimago in the field (living) C nymphal habitat in laboratory (living) D male subimago in laboratory (living). Figure 17. Habitat of Siphlonurus dongxi Li & Tong, sp. nov. A nymphal habitat in Dugang River B female subimago in the field (living) C nymphal habitat in laboratory (living) D male subimago in laboratory (living). Acknowledgements This work was supported by the Second Tibetan Plateau Scientific Expedition and Research Program (2019QZKK0402) and the National Natural Science Foundation of China (31960255). Thanks are due to Yi-Hao Fang (Dali University) for help with fieldwork. We are grateful to Michel Sartori (Museum of Zoology, Lausanne, Switzerland), an anonymous reviewer, and Davide Fornacca (Dali University) for their useful suggestions and corrections that improved the quality of the manuscript. Ethical statement No ethical statement was reported. 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https://openalex.org/W4307354976	https://www.researchsquare.com/article/rs-2183097/latest.pdf	English	null	Clustering Honey Samples with Unsupervised Machine Learning Methods using FTIR Data	Research Square (Research Square)	2,022	cc-by	5,045	Clustering Honey Samples with Unsupervised Machine Learning Methods using FTIR Data Fatih Mehmet Avcu (  fatih.avcu@inonu.edu.tr ) Inonu University Fatih Mehmet Avcu (  fatih.avcu@inonu.edu.tr ) Inonu University Research Article Keywords: FTIR, Clustering, HCA, Machine Learning, Deep Learning Posted Date: October 26th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-2183097/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License DOI: https://doi.org/10.21203/rs.3.rs-2183097/v1 Page 1/19 Abstract Honey is a food item that people consume because of its taste and positive effects on health. The importance of honey is increasing day by day because of the difficulties in production, the threat of the bee population due to environmental conditions and climate changes, and the increasing population. In this work, data obtained from Fourier transform infrared (FTIR) spectra of honey samples were used for clustering of honey data. First of all, the number of clusters was determined by applying elbow method to the spectrum data obtained from the samples. After this process, the data was divided into 5 clusters. The data were reduced to 2 dimensions with principal components analysis (PCA), clusters of samples were determined by applying Hierarchical clustering (HCA). 20% of the data whose clusters were determined were randomly selected to be used as test data. The rest of the data was used as training data in Deep Learning. After the training, the test data was checked and the accuracy was found to be 96.15%. The proposed method gives reliable results in clustering of honey samples with the advantages of being fast, cheap and not requiring preprocess procedure. 1 Introduction Honey is a nutritious and high-energy food produced by bees from the liquid called nectar obtained from flowers. Honey, which has been known for centuries and is one of the oldest foods, has never lost its importance for human nutrition and health. The first sweetener that humanity noticed is honey. Finding a picture of a person collecting honey, which was determined to have been drawn eight thousand years ago in the Pineapple Cave of Spain[1], shows that honey has had an economic value since ancient times. While most honey consists of carbohydrates (such as glucose, fructose, and sucrose), water, small amounts of other substances such as proteins, ash, amino acids, enzymes, vitamins, and phenolic acids. It is known that each of these components, which are found in small amounts, has characteristic nutritional value or medicinal properties. Although the main components of honey (carbohydrates and water) are almost the same in all honey samples, the chemical composition and physical properties of honey differ according to the plant species from which bees take nectar. In this way, the geographical origin of the honey or whether foreign additives have been added can be understood. The main purpose of cluster analysis is to find natural classification from datasets of complex structures to separate objects into homogeneous and inhomogeneous clusters. When examining unknown structures in nature, cases with natural classifications may need to be reconsidered. Cluster analysis can be used to deal with situations whose natural classification is determined according to a small number of variables, by increasing the number of variables, and to examine whether the previous classification has changed. For example, conditions such as the positive contributions of the developing technology in data collection, the development of modern measurement tools, and the ability to collect data on new variables from the units necessitate the control of traditional information. Therefore, the classification of variables and units should be reconsidered with multivariate statistical methods. Page 2/19 Page 2/19 FTIR is a chemical analytical method in which the intensity of the infrared beam passing through the sample is measured against the wave number of the beam with the mathematical Fourier transform method. It is used not only in the identification of microbial cells, but also in the structural analysis of macromolecules (such as naturalness and quantity). Application areas of FTIR spectroscopy are common due to the broad spectrum available. 1 Introduction Obtaining fast results has popularized the use of traditional FTIR. FTIR spectroscopy is a direct and reversible method. This spectroscopic method[2], which gives results in a short time with a small amount of sample, is used in the analysis of solid, liquid and gas samples. Machine learning was founded in the 1960s. Contrary to the popular belief, its mathematical background is not new. For deep neural networks, it can be said that classical neural networks are a special case of multi-layer and multi-neuron[3]. The most important feature of deep learning networks, which are designed in different models according to the usage area, is that there is no need for a separate study to extract the features suitable for the problem. In layers with deep structure, features are formed by learning of the network. Deep learning networks, which can decide on their own what information to learn instead of using the information presented to them, therefore produce more successful results than classical methods[4–7]. Deep learning consists of an advanced neural network with many hidden layers. Deep learning is used in various applications such as image recognition[8, 9], computer vision[10, 11], text classification[12, 13], multiple classification[14, 15] and regression problems[16, 17]. Since honey has an economic value, many studies have been done on it[18–23. The aim of this study is to propose a machine learning combined with FTIR spectroscopy to cluster honey samples. After clustering, deep learning was used and it was seen that an accurate clustering was made at the rate of 96.15%. In this study, we validated the feasibility of the machine learning algorithm combined with FTIR spectroscopy. 2.1 FTIR Measurements In this study, FTIR spectrometer with KBr emitter and DLaTGS detector was used. For the measurements to be made with infrared spectrophotometer, 5 ml samples were prepared using vials. In order to dilute the honey samples with very high viscosity, 5 ml carbon tetrachloride was added to 5 ml honey sample volumes, which did not give an absorbance peak between the wavelengths of the honey sample in infrared spectrophotometer. Measurements were made in the middle IR region (wavelength range of 600 to 4,000 cm${-1}$). For each sample, 3 separate measurements were made with the FTIR device and the averages were taken and evaluated. The device was cleaned after each measurement. The results obtained from the measurements were recorded in Csv format without any preprocessing. The spectra obtained from the measurements made with the FTIR device of 65 samples are shown in Fig. 1. In this study, FTIR spectrometer with KBr emitter and DLaTGS detector w to be made with infrared spectrophotometer, 5 ml samples were prepare honey samples with very high viscosity, 5 ml carbon tetrachloride was a volumes, which did not give an absorbance peak between the waveleng infrared spectrophotometer. Measurements were made in the middle IR to 4,000 cm${-1}$). For each sample, 3 separate measurements were ma averages were taken and evaluated. The device was cleaned after each obtained from the measurements were recorded in Csv format without a obtained from the measurements made with the FTIR device of 65 sam 1.1 Related Work S. Segato et al. have proposed a multivariate machine learning model to understand how the physicochemical properties of honey change with heat[24]. In their study, they used the Support Vector Model. They said that heating the honey as a pre-treatment, especially increasing it above 39 degrees, creates a significant change in the internal structure of honey. Liu et al. used the Leave One Out Cross Validation test after obtaining the sequencing of honey by DNA extraction[25]. With this method, they determined the geographical origin of honey with a rate of 99%. Chien et al. classified the pre-treated honey spectra with multiLayer perceptron (MLP), support-vector machines (SVM) and principal component analysis (PCA), and showed that SVM and PCA gave better results than MLP[26]. A. Noviyanto and W. H. Abdulla performed a classification using near-IR spectra of honey samples. They used support vector classifier (SVC) and k-nearest neighbors (kNN) in supervised learning algorithms for classification. They reported the classification success as 90% for closed clusters and 88% for open Page 3/19 Page 3/19 clusters [27]. M. A. Al-Awadhi and R. R. Deshmukh used machine learning to find the botanical origin of honey. In their method, they used Linear Discrimination Analysis (LDA) to extract features and reduce the number of dimensions, and then used SVM and KNN algorithms for classification. They stated the accuracy of the proposed model as 95.13%[28]. S. Hu et al. applied supervised learning techniques such asprobabilistic neural network (PNN), convolutional neural network (CNN) and SVM to Raman spectroscopy to determine the contribution of maltose sugar in honey[29]. B.L.Batista et al., conducted a study based on SVM multilayer perceptron and random forests algorithms[30]. They obtained results with 65%, 83% and 79% accuracy, respectively. They stated that they found the geographical origin of a honey quickly with the method they proposed. O. Anjos et al. conducted a neural network-based study to determine the botanical origin of honey based on moisture content, electrical conductivity, water activity, ash content, pH, and free acidity[31]. They stated that this method, in which they used the color and electrical properties of the samples, found the botanical origin correctly with a 5% margin of error. While deep learning is used on large datasets, it has recently been found to be a good tool for classification examples containing small datasets[32, 33]. 2 Material And Methods This study was carried out on honey samples from Turkey with different botanical and geographical origins. Before analysis, samples were stored at room temperature in a dark place. The samples were naturally crystallized on the surface of a thin liquid layer. After the honey samples were scanned with the FTIR-ATR spectrometer, the obtained spectra were used for PCA and HCA. The data set obtained as a result of the clustering process was reclassified by deep learning. For calculations, Open source Tensorflow[34], Keras[35] and Scikit-learn[36] libraries are used. 2.2 Chemometric Methods Cluster analysis, one of the multivariate statistical techniques, is used to classify ungrouped and unknown data according to their similarity. Cluster analysis is similar to discriminant analysis in that it Page 4/19 aims to collect similar samples in the same groups, and with factor analysis because it aims to collect similar variables in the same groups, and it also has data reduction features. aims to collect similar samples in the same groups, and with factor analysis because it aims to collect similar variables in the same groups, and it also has data reduction features. As it is known, PCA is an unsupervised learning method. The basic idea of PCA is to transform the original features into a new feature array in order of importance via a set of orthogonal vectors[37]. It is often used to obtain a lower dimensional graphical representation that describes the maximum variation in a data set. The first component of PCA takes into account the most possible variability in the data, while the second component takes into account as much of the remaining variability as possible. Another unsupervised learning algorithm is HCA[38].The purpose of HCA is to collect similar samples in the same groups or clusters, to identify these clusters and to predict which group the new samples belong to, based on the similarity of the samples in terms of all variables. Clustering is based on similarities (proximity) or dissimilarity (distance) of two samples. In hierarchical clustering methods, clusters are merged sequentially and once one group is combined with another, they are not separated again in subsequent steps. 3 Result And Discussion The number of clusters can be found with a line drawn parallel to the x-axis from the location of the sharp rise in the dendogram. The number of points where this line intersects the dendrogram gives the number of clusters. Each point where the red line in Fig. 3 intersects represents a cluster. The number of 5 clusters found supports the number k in the Fig. 2. In machine learning processes, all features of the data must be brought to the same size. These differences in initial properties can cause problems for many machine learning models. Variables measured at different scales in the dataset do not contribute equally to the fitting of the model and the learning function, thus creating a bias in the result. For example, since clustering algorithms are distance- based, having a large value on a feature in our data will make it the dominant feature. For eliminating this situation, the data are scaled by feature standardization or Z-score normalization methods. We used the "StandardScaler" function in the "Sklearn.Preprocessing" library in Python for this process. The StandardScaler operation adjusts each data column so that its mean is 0 and its standard deviation is 1. In machine learning processes, all features of the data must be brought to the same size. These differences in initial properties can cause problems for many machine learning models. Variables measured at different scales in the dataset do not contribute equally to the fitting of the model and the learning function, thus creating a bias in the result. For example, since clustering algorithms are distance- based, having a large value on a feature in our data will make it the dominant feature. For eliminating this situation, the data are scaled by feature standardization or Z-score normalization methods. We used the "StandardScaler" function in the "Sklearn.Preprocessing" library in Python for this process. The StandardScaler operation adjusts each data column so that its mean is 0 and its standard deviation is 1. For the HCA method, score vectors obtained from PCA were used. Python scripting language was used for PCA and HCA analysis. In this study, in which agglomerative clustering is used, hierarchical clustering is performed with Euclidean distance calculations, starting with the most similar samples. This process is done with the AgglomerativeClustering function from the Sklearn.cluster library. Other hyper parameters of HCA are euclidean and ward parameters for affinity and linkage, respectively. 3 Result And Discussion There are many methods to determine the number of clusters in a data set. The oldest and most used one these methods is the elbow method[39]. In the Elbow method, the number of clusters (k) is changed between 1 and 10. For each k value, the WCSS (Within Cluster Sum of Squares) value is calculated as in the Eq. 1. Eq. 1 WCSS = ∑cn ck∑dm diincidistance (di, Ck)2 C, d are cluster centroids and data point each cluster respectively. C, d are cluster centroids and data point each cluster respectively. WCSS is the sum of the square of the distance between each point and the center point in a cluster. The WCSS value and the k value are passed to a chart. As the number of clusters increases, the WCSS value will begin to decrease. When k = 1, the WCSS value is the largest. Examining Fig. 2, we can see that the graph changes rapidly at one point, thus forming an elbow shape. From this point on, the graph starts to move almost parallel to the x-axis. The k value corresponding to this point is the optimal k value or the optimal number of clusters. In Fig. 2, the number of clusters is clearly seen as 5. Another method of determining the number of clusters is the dendrogram method. The ward method and Euclidean distance were used in the calculations for the dendrogram drawing. L. Ferreira and D. Hitchcock stated in their study that Ward method is the best among other methods[40]. The Ward method is also called the least variance method[41]. In short, the Ward method performs the merging process based on the variances. The merging process starts with the clusters with the least variability. The method is calculated as in Eq. 2. Eq. 2. SSE = ∑K i=1 ∑ni j=1 (yij − −yi) ′ −(yij − −y) Page 5/19 Page 5/19 is the jth object in the ith cluster and is the number of objects in the i th cluster. yij ni There are many distance methods for intercluster calculations. Euclidean distance is one of them. M.Sha et al. reported that Euclidean distance gave the best results in their studies based on raman spectroscopy[42]. Looking at Fig. 3, it will be seen that the graph starts with 65 separate clusters for 65 honey samples. 3 Result And Discussion As is known, the lower the loss function, the better the structure is optimized. Figure 8 shows the success of education and losses. The Loss function of the model is very close to 0.1. It is also seen that the accuracy is above 96%. Considering both the loss and the accuracy, we can say that the training is successful. One of the methods used to find the model accuracy is the complexity matrix. The complexity matrix formed after testing the algorithm with 13 test data not used for training is given in the Fig. 9. The rows of the matrix show the true classes and the columns of the matrix show the predicted classes. Looking at Fig. 9, it is seen that the trained model correctly predicted the test data. 4 Conclusion In this study, a suitable method is proposed for clustering honey samples obtained from different regions without any chemical separation process. The method is based on FTIR data that fingerprints a honey. In this study, the number of clusters was confirmed in 2 different ways. Each frequency point of the FTIR data was evaluated as features and the number of features was reduced to 2 dimensions with PCA. This 2D data was clustered with HCA. The obtained clustering data was reclassified by deep learning. It has been observed that the model we propose can accurately predict the test data at a rate of 96.15%. As a result; successful implementation of the model demonstrated that chemometric techniques applied on FTIR data quickly and accurately clustered honey samples without the need for sample preparation. 3 Result And Discussion As you can see in Fig. 4, Cluster 3 samples, which are very different from other samples, should not be considered as outliers. Outlier sample is expressed as samples that cannot be included in any cluster as a result of cluster analysis. As you can see in Fig. 4, Cluster 3 samples, which are very different from other samples, should not be considered as outliers. Outlier sample is expressed as samples that cannot be included in any cluster as a result of cluster analysis. Looking at the HCA (Fig. 4) and K-Means (Fig. 5) clustering results, it is seen that there are transitions between cluster 1 and cluster 5 and between cluster 2 and cluster 5. Likewise, clustering results from HCA (Fig. 4) and Gaussian Mixture (Fig. 6) show a more intense transition between cluster 1 and cluster 5, and cluster 2 and cluster 5. After the clustering process was done, the classification process was carried out with deep learning to test the accuracy of the clustering. For the classification process, 20% of the data set was randomly selected for the test and the remaining part was used as training data. Process, the train_test_split command from the sklearn.model_selection library is used. The structure of the deep learning model consists of the input, the hidden and the output layer. Each parameter for the hidden layer and epoch number was tried 30 times and the average is shown in Fig. 7. The model created within the scope of this study consists of a total of 3 layers. The rectified linear Unit Page 6/19 Page 6/19 (relu) function is used as the activation function of the input and hidden layers. The output value of the Relu function is between zero and .The reason why the Relu activation function is often used is that inputs greater than 0 have a fixed derivative value. The sigmoid function is used for the output layer activation. The sigmoid function is a continuous and differentiable function. It is often preferred because it is non-linear. It takes a value in the range of 0 to 1 depending on the input value. Since we have 5 classes, the output layer is set to 5. +∞ The most commonly used Adaptive Moment Estimation (adam) function was used as the optimizer[43]. Deep Learning is initiated with the number of epochs 150. Binary_crossentropy was used as the loss function in the structure. Acknowledgments The authors would like to thank Gökhan Durmaz, Department of Food Engineering, Inonu University and Tamer Arslan, Department of Food Processing, Darende Vocational School, Turgut Özal University, for support FTIR spectra. Declarations Data Availability Statement Data are available on request from the authors. Data supporting the findings of this study can be obtained from the relevant author upon reasonable request. Declaration of Competing Interest The author declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Page 7/19 Author Contribution Author Contribution F. M. Avcu developed the software, drew the graphics, and wrote the manuscript. No funding was received for this study. References 1. Nayik, G. et al. 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Page 10/19 Figures Figure 1 The FTIR-ATR spectra of honey samples The FTIR-ATR spectra of honey samples The FTIR-ATR spectra of honey samples The FTIR-ATR spectra of honey samples Page 11/19 Figure 2 Identification of The Elbow point Figure 2 Identification of The Elbow point Page 12/19 Page 12/19 Figure 3 Determine of Cluster Numbers with Dendrogram Figure 3 Determine of Cluster Numbers with Dendrogram Page 13/19 Page 13/19 Page 13/19 Figure 4 Hi hi l Cl t i Figure 4 Hierarchical Clustering Page 14/19 Figure 5 Figure 5 KMeans Clustering Page 15/19 Figure 6 Gaussian Mixture Clustering Figure 6 Figure 6 Figure 6 Gaussian Mixture Clustering Gaussian Mixture Clustering Page 16/19 Figure 7 Figure 7 Optimization of Epocs and Hidden Layer Optimization of Epocs and Hidden Layer Optimization of Epocs and Hidden Layer Page 17/19 Figure 8 Deep Learning Loss and Accuracy Figure 8 Deep Learning- Loss and Accuracy Page 18/19 Figure 9 Figure 9 Figure 9 Confusion Matrix Page 19/19 Page 19/19
https://openalex.org/W2164382771	https://nhess.copernicus.org/articles/13/1015/2013/nhess-13-1015-2013.pdf	English	null	Characteristics of rainfall triggering of debris flows in the Chenyulan watershed, Taiwan	Natural hazards and earth system sciences	2,013	cc-by	8,368	y Atmosphe M Characteristics of rainfall triggering of debris ﬂows in the Chenyulan watershed, Taiwan Te J. C. Chen1, C. D. Jan2, and W. S. Huang2 1Dept. of Environmental and Hazards-Resistant Design, Huafan University, New Taipei, Taiwan 2Dept. of Hydraulic and Ocean Engineering, National Cheng Kung University, Tainan, Taiwan Te J. C. Chen1, C. D. Jan2, and W. S. Huang2 1Dept. of Environmental and Hazards-Resistant Design, Huafan University, New Taipei, Taiwan 2Dept. of Hydraulic and Ocean Engineering, National Cheng Kung University, Tainan, Taiwan Correspondence to: J. C. Chen (jinnchyi@cc.hfu.edu.tw) Received: 12 June 2012 – Published in Nat. Hazards Earth Syst. Sci. Discuss.: – Revised: 4 February 2013 – Accepted: 21 March 2013 – Published: 19 April 2013 Received: 12 June 2012 – Published in Nat. Hazards Earth Syst. Sci. Discuss.: – Revised: 4 February 2013 – Accepted: 21 March 2013 – Published: 19 April 2013 Climate of the Past pen Access Earth System Dynamics Open Access Geoscientific Instrumentation Methods and Data Systems Open Access Clague, 1994; Van Steijn, 1996; Jomelli et al., 2007; Pelﬁni and Santilli, 2008; Floris et al., 2010). This paper examines the variation in the characteristics of rainfall events triggering debris ﬂows and the effects on the number of debris ﬂows in the Chenyulan watershed, central Taiwan. Chen et al. (2012) found that the number of extreme rainfall events within the watershed showed an increasing trend, which was associated with the greater number and magnitude of debris ﬂows dur- ing the last decade. Landslides and debris ﬂows caused by severe rainfall events affect the volume of loose debris within a watershed, which may change the critical rainfall threshold for the initiation of debris ﬂow in subsequent rainfall events. Therefore, understanding the variation in rainfall character- istics and its inﬂuence on debris ﬂow initiation is important for debris ﬂow warning and hazard mitigation. Abstract. This paper reports the variation in rainfall charac- teristics associated with debris ﬂows in the Chenyulan water- shed, central Taiwan, between 1963 and 2009. The maximum hourly rainfall Im, the maximum 24 h rainfall Rd, and the rainfall index RI (deﬁned as the product RdIm) were analysed for each rainfall event that triggered a debris ﬂow within the watershed. The corresponding number of debris ﬂows initi- ated by each rainfall event (N) was also investigated via im- age analysis and/or ﬁeld investigation. The relationship be- tween N and RI was analysed. Higher RI of a rainfall event would trigger a larger number of debris ﬂows. y Atmosphe M Characteristics of rainfall triggering of debris ﬂows in the Chenyulan watershed, Taiwan This paper also discusses the effects of the Chi-Chi earthquake (CCE) on this relationship and on debris ﬂow initiation. The results showed that the critical RI for debris ﬂow initiation had sig- niﬁcant variations and was signiﬁcantly lower in the years immediately following the CCE of 1999, but appeared to re- vert to the pre-earthquake condition about ﬁve years later. Under the same extreme rainfall event of RI = 365 cm2 h−1, the value of N in the CCE-affected period could be six times larger than that in the non-CCE-affected periods. Data Systems s Geoscientific Model Development Open Access Hydrology and Earth System Sciences Open Access Ocean Science Open Acce Debris ﬂow, a highly hazardous hydrological process, oc- curs frequently in the Chenyulan watershed. Some signiﬁcant debris ﬂow events in the watershed were previously studied and documented (Lin and Jeng, 2000; Chang et al., 2001; Cheng et al., 2005; Jan and Chen, 2005; Chen et al., 2011). The major Chi-Chi earthquake of 1999 (7.6 on the moment magnitude scale, 7.3 on the Richter scale) caused numerous landslides in the Chenyulan watershed. Previous investiga- tors examined some phenomena of debris ﬂows following the Chi-Chi earthquake, such as the effect of the earthquake on the characteristics of debris ﬂows (Lin et al., 2003; Liu et al., 2008), the effects of strong ground motion on the susceptibil- ity of debris ﬂows (Liu et al., 2009), the variation in rainfall conditions required to trigger debris ﬂows after the Chi-Chi earthquake (Chen, 2011), and recent changes in the number of rainfall events related to debris ﬂow occurrence (Chen et al., 2012). 1 Introduction Over the last few decades, global-warming-induced climate change has manifested in a number of ways including ex- treme rainfall events (for example, Katz and Brown, 1992; Fauchereau et al., 2003; Fowler et al., 2005; Sillmann and Roeckner, 2008). As a consequence, higher frequencies and larger magnitudes of rainfall-induced landslides and/or de- bris ﬂows have been reported worldwide (Eybergen and Ime- son, 1989; Rickenmann and Zimmermann, 1993; Evans and Nat. Hazards Earth Syst. Sci., 13, 1015–1023, 2013 www.nat-hazards-earth-syst-sci.net/13/1015/2013/ doi:10.5194/nhess-13-1015-2013 © Author(s) 2013. CC Attribution 3.0 License. Nat. Hazards Earth Syst. Sci., 13, 1015–1023, 2013 www.nat-hazards-earth-syst-sci.net/13/1015/2013/ doi:10.5194/nhess-13-1015-2013 © Author(s) 2013. CC Attribution 3.0 License. Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. J. C. Chen et al.: Characteristics of rainfall triggering of debris ﬂows 1016 1016 J. C. Chen et al.: Characteristics of rainfall triggering of debris ﬂows Table 1. Debris ﬂow events and related rainfall characteristics in the Chenyulan watershed between 1963 and 2009 (Modiﬁed from Chen et al., 2012). ble 1. Debris ﬂow events and related rainfall characteristics in the Chenyulan watershed between 1963 and 2009 (Modiﬁed from Chen 2012). Published by Copernicus Publications on behalf of the European Geosciences Union. Year Date of the rainfall event Name of rainfall event Number of debris ﬂows N Maximum hourly rainfall, Im (mm h−1) Maximum 24 h rainfall, Rd (mm) Rainfall duration, T (h) Rainfall index, RI (cm2 h−1) 1963 10–12 Sep Typhoon Gloria 1∗ 36.1 668.1 82.0 241.2 1985 23–25 Aug Typhoon Nelson 2∗ 47.2 418.8 50.0 197.7 1986 21–22 Aug Typhoon Wayne 2∗ 50.8 329.8 18.0 167.5 1992 30–31 Aug Typhoon Polly 1∗ 41.3 479.5 41.0 198.0 1996 31 July–1 Aug Typhoon Herb 37 71.6 1181.6 35.0 846.0 1998 7–8 June Rainstorm 3 28.1 227.8 40.0 64.0 1998 4–5 Aug Typhoon Otto 4 64.6 311.7 19.0 201.4 1998 15–16 Oct Typhoon Zeb 2 24.6 251.0 31.0 61.7 1999 27–28 May Rainstorm 2 24.3 254.3 32.0 61.8 2000 01 Apr Rainstorm 2 20.0 75.1 8.0 15.0 2000 25 Apr Rainstorm 1 8.4 30.6 9.0 2.6 2000 28–29 Apr Rainstorm 1 7.9 78.2 14.0 6.2 2000 2 May Rainstorm 1 8.1 30.6 5.0 2.5 2000 12–14 June Rainstorm 4 18.0 228.1 47.0 41.1 2000 18 July Rainstorm 3 12.7 30.0 3.0 3.8 2000 22 July Rainstorm 3 16.3 20.7 2.0 3.4 2000 5 Aug Rainstorm 4 11.6 38.8 10.0 4.5 2000 22–23 Aug Typhoon Bilis 2 20.6 234.5 24.0 48.3 2001 5 June Rainstorm 1 7.5 27.0 8.0 2.0 2001 14–15 June Rainstorm 3 18.4 200.1 24.0 36.8 2001 29–30 July Typhoon Toraji 78 78.5 587.6 18.0 461.3 2001 10 Aug Rainstorm 3 22.4 22.4 1.0 5.0 2001 17 Sep Typhoon Nari 4 35.7 252.5 23.0 90.1 2002 31 May Rainstorm 4 14.4 53.0 7.0 7.6 2002 3–4 July Rainstorm 2 13.3 117.9 19.0 15.7 2002 12 Aug Rainstorm 1 17.1 26.5 3.0 4.5 2004 2–3 July Typhoon Mindulle 17 54.0 681.4 38.0 368.0 2004 23–25 Aug Typhoon Aere 2 35.0 385.4 47.0 134.9 2005 4–5 Aug Typhoon Matsa 1 42.3 411.9 34.0 174.2 2005 31 Aug–1 Sep Rainstorm 1 44.3 495.0 27.0 219.3 2006 8–11 June Rainstorm 10 77.5 682.8 78.0 529.2 2006 13–15 July Typhoon Bilis 2 29.9 371.7 56.0 111.1 2007 17–20 Aug Typhoon Sepat 1 31.6 328.4 66.0 103.8 2007 6–7 Oct Typhoon Krosa 1 54.3 669.4 35.0 363.5 2008 17–18 July Typhoon Kalmaegi 3 67.2 515.7 18.0 346.6 2008 12–15 Sep Typhoon Sinlaku 2 35.0 612.4 66.0 214.3 2009 6–11 Aug Typhoon Morakot 41 85.5 1192.6 110.0 1019.7 Note: ∗: Debris ﬂow data obtained only from scientiﬁc papers/reports before 1996; no aerial photographs and satellite images were available to identify debris ﬂows in the whole watershed before Typhoon Herb. Published by Copernicus Publications on behalf of the European Geosciences Union. N = total number of individual debris ﬂow triggered by each rainfall event in the Chenyulan watershed; Im = maximum hourly rainfall in each rainfall event; Rd = maximum 24 h rainfall amount in each rainfall event; RI = rainfall index, RI = RdIm. Table 1. Debris ﬂow events and related rainfall characteristics in the Chenyulan watershed between 1963 and 2009 (Modiﬁed from Chen et al., 2012). Note: ∗: Debris ﬂow data obtained only from scientiﬁc papers/reports before 1996; no aerial photographs and satellite images were available to identify debris ﬂows in the whole watershed before Typhoon Herb. N = total number of individual debris ﬂow triggered by each rainfall event in the Chenyulan watershed; Im = maximum hourly rainfall in each rainfall event; Rd = maximum 24 h rainfall amount in each rainfall event; RI = rainfall index, RI = RdIm. Extreme rainfall causes numerous landslides and debris ﬂows in mountainous watersheds, which generally deposit large amounts of loose debris in gullies and on slopes (Dong et al., 2009; Chen et al., 2012). The supply of loose debris plays an important role in the occurrence of future debris ﬂows during subsequent rainfall events (Jakob et al., 2005). Both the critical rainfall threshold for debris ﬂow initiation and the number of debris ﬂows caused by a rainfall event may differ before and after an extreme rainfall event or a major earthquake. This study analysed the characteristics of rain- fall related to debris ﬂow occurrence between 1963 and 2009 in the Chenyulan watershed, in order to investigate (1) the variation in the regional rainfall conditions related to debris ﬂow occurrences, and (2) the empirical relationship between rainfall characteristics and the corresponding number of de- bris ﬂows. 2.1 Debris ﬂows in the Chenyulan watershed The watershed of Chenyulan stream, located in Nantou County, central Taiwan (Fig. 1), has an area of 449 km2, main stream length of 42 km, average stream-bed gradi- ent of 4◦, and elevations ranging from 310 to 3952 m. The annual regional rainfall in the watershed is between 2000 and 5000 mm, with an average of approximately 3500 mm. Nat. Hazards Earth Syst. Sci., 13, 1015–1023, 2013 www.nat-hazards-earth-syst-sci.net/13/1015/2013/ www.nat-hazards-earth-syst-sci.net/13/1015/2013/ 1017 J. C. Chen et al.: Characteristics of rainfall triggering of debris ﬂows 1 Fig. 1. Locations of debris ﬂows and meteorological stations in the Chenyulan watershed (Modiﬁed from Chen et al., 2012). 1 Fig. 1. Locations of debris ﬂows and meteorological stations in the Chenyulan watershed (Modiﬁed from Chen et al., 2012). 16 most 2000 mm, which initiated 37 debris ﬂows, leading to 27 deaths and 14 people being reported missing in the wa- tershed (Jan and Chen, 2005). The Chi-Chi earthquake of 21 September 1999 was the largest earthquake in Taiwan for a hundred years (Shin and Teng, 2001), and caused numer- ous landslides in the Chenyulan watershed. The extreme rain- fall event caused by Typhoon Toraji from 29–30 July 2001 had a maximum hourly rainfall of 78.5 mm and 24 h rain- fall of 587.6 mm; this event caused more than 30 debris ﬂows in the watershed, resulting in more than 100 people being reported dead or missing, and widespread damage to houses, roads and bridges (Chen et al., 2012). The heavy rainfall brought by Typhoon Morakot in August 2009 had (Modified from Chen et al., 2012). 3 4 Approximately 80 % of the annual rainfall occurs in the rainy season between May and October, especially during typhoons, which generally occur three or four times annu- ally. Owing to steep topography, loose soils, young (3 million years) and weak (due to ongoing orogenesis) geological for- mations, heavy rainfall and active earthquakes, many debris ﬂows were triggered by more than 30 rainfall events between 1963 and 2009 in the watershed, as shown in Table 1. The most signiﬁcant debris ﬂow events were those caused by heavy rainfall brought by Typhoon Herb in 1996, Typhoon Toraji in 2001 and Typhoon Morakot in 2009. During the two day period from 31 July to 1 August 1996, Typhoon Herb brought an unexpectedly high cumulative rainfall of al- www.nat-hazards-earth-syst-sci.net/13/1015/2013/ Nat. Hazards Earth Syst. Sci., 13, 1015–1023, 2013 2.3 Regional rainfall characteristics versus debris ﬂows To investigate the variation in rainfall characteristics in the Chenyulan watershed, long-term rainfall records were ob- tained from three meteorological stations (Sun Moon Lake, Yushan and Alisan stations, as shown in Fig. 1). These data were used to estimate the regional rainfall characteristics for the whole Chenyulan watershed, via the reciprocal-distance- squared (RDS) method (Chow et al., 1988). The estimated point using this method was taken at the centroid of the watershed area. The rainfall characteristics estimated by the RDS method may not actually reﬂect the rainfall character- istics at speciﬁc locations when local rainfall varied signiﬁ- cantly owing to abrupt changes in elevation, but it is a sim- ple method to directly compute the regional average rainfall characteristics for a watershed. Moreover, the regional aver- age rainfall estimated using the RDS method can easily rep- resent the variation trend for regional rainfall characteristics throughout the Chenyulan watershed (Chen et al., 2012). The present paper calculated regional hourly rainfall, cumulative rainfall and rainfall duration, etc., of each rainfall event as- sociated with debris ﬂow occurrence, in order to study the relationship between rainfall characteristics and debris ﬂow occurrence. Identifying debris ﬂows, it was decided that each of the soil slip, ﬂow path and deposits should be identiﬁable from the images – that is, the source, transportation, and deposi- tion zones should be identiﬁable from aerial photographs or satellite images. These features on aerial photographs were previously used by Liu et al. (2008) to identify debris ﬂows. A debris ﬂow is a rapid movement of debris materials along the ﬂow path. Some important features of a debris ﬂow in- clude traces of channelised stream ﬂow, a large erosion ca- pacity (such as sharp bank erosion, fallen tree trunks), the transport of large boulders, the formation of levees, uneven and poorly sorted deposits, and scarring high on streamsides and deposits high in trees (Costa, 1984). These features pro- Parameters such as peak hourly rainfall, daily rainfall, maximum daily rainfall, cumulative rainfall, average rain- fall intensity and rainfall duration have been used by pre- vious researchers to investigate the occurrence of debris ﬂows. The choice of rainfall parameters reﬂects different re- search objectives. For example, the empirical relationship between average rainfall intensity and rainfall duration was used to assess the potential for debris ﬂow occurrence in a rainfall event (Caine, 1980; Keefer et al., 1987; Chen, 2011). J. C. Chen et al.: Characteristics of rainfall triggering of debris ﬂows J. C. Chen et al.: Characteristics of rainfall triggering of debris ﬂows 1018 a maximum hourly rainfall of 123 mm and 48 h rainfall of 2361 mm (measured at Alishan rainfall station), and caused numerous debris ﬂows that buried more than 20 houses, es- pecially in Shenmu, Tongfu and Xinyi villages (Chen et al., 2011). vide evidence of debris ﬂow, and can be used to identify de- bris ﬂow in ﬁeld investigation. In this study, only gullies with obvious patterns of debris ﬂow were identiﬁed as debris ﬂow events. Table 1 shows 37 rainfall events, including 18 rainstorms and 19 typhoon-induced heavy rainfall events, that triggered debris ﬂows in the Chenyulan watershed during the study pe- riod; and the number of debris ﬂow events (N) for each rain- fall event throughout the watershed. Table 1 shows that most rainfall events triggered four or fewer debris ﬂows, but that some particularly severe events, such as those associated with heavy rainstorms in 2006, and with Typhoons Herb (1996), Toraji (2001), Mindulle (2004) and Morakot (2009), in- duced ten or more debris ﬂows. The maximum number of de- bris ﬂows was 78, which were triggered by Typhoon Toraji, approximately two years after the Chi-Chi earthquake; Ty- phoons Herb and Morakot triggered 37 and 41 debris ﬂows, respectively, within the watershed. 2.2 Identiﬁcation of debris ﬂow events Naturally occurring high-discharge ﬂows of water and sed- iment in open channels vary over a wide and continuous spectrum of sediment concentration and particle-size distri- bution (Pierson, 2005). Water ﬂoods normally transport rel- atively small quantities of mostly ﬁne sediment. At the other end of the spectrum, high-discharge debris ﬂows may trans- port more sediment than water. The term “debris ﬂood” or “mud ﬂood” is often applied to ﬂows intermediate between these two extremes (Pierson, 2005). For hazard management purposes, only a debris ﬂow that occurs in a watershed with an area more than 3.0 ha and of main upstream slope more than 10◦is ofﬁcially recognised as a debris ﬂow in Taiwan; otherwise, the ﬂow would be treated as a sediment-laden ﬂow, a debris ﬂood or a mud ﬂood. Therefore, the present study also adopts this classiﬁcation when counting the num- ber of debris ﬂows triggered during a rainfall event. Debris ﬂow data prior to 1996 was obtained from the scientiﬁc lit- erature (such as Yu and Chen, 1987; Chiang and Lin, 1991; Chang et al., 2001). No aerial photographs and satellite im- ages were available to identify debris ﬂows in the whole wa- tershed before 1996. After 1996, according to their speciﬁc features, debris ﬂows were identiﬁed through interpretation of aerial photographs, satellite images or/and ﬁeld investiga- tions. The identiﬁcation of debris ﬂows involved two basic stages. In the ﬁrst stage, the possible locations of debris ﬂow were identiﬁed from media reports (local newspapers, TV news), related documents and papers (Lin and Jeng, 2000; Cheng et al., 2005; Jan and Chen, 2005; Chen et al., 2009, 2012; Chen, 2011), and interpretation of images, such as aerial photographs and satellite images (SPOT images before 2004; FORMOSAT2 images after 2004) to assess whether the event was classiﬁed as a debris ﬂow. The second stage was to conduct a ﬁeld investigation to conﬁrm whether the event identiﬁed in the ﬁrst stage qualiﬁed as a debris ﬂow. 2.3 Regional rainfall characteristics versus debris ﬂows Rainfall data on a daily scale, such as maximum 24 h rainfall, daily rainfall, or 3 day rainfall, have been used to analyse the inﬂuence of rainfall change on debris ﬂow www.nat-hazards-earth-syst-sci.net/13/1015/2013/ Nat. Hazards Earth Syst. Sci., 13, 1015–1023, 2013 J. C. Chen et al.: Characteristics of rainfall triggering of debris ﬂows 1960 1 1 1960 1965 1970 1975 1980 1985 1990 19 0 1019 2010 J. C. 2.3 Regional rainfall characteristics versus debris ﬂows Chen et al.: Characteristics of rainfall triggering of debris ﬂows 1019 1960 1965 1970 1975 1980 1985 1990 1995 2000 2005 2010 Year 0 200 400 600 800 1000 1200 1400 Rd (mm/h) TH TT HR TM EQ TMi : Rainfall events triggering 10 or more debris flows TH : Typhoon Herb (July 31Aug 01, 1996) CCE : Chi-Chi earthquake (Sep 21, 1999) TT : Typhoon Toraji (July 2930, 2001) TMi: Typhoon Mindulle (July 0203, 2004) HR : Heavy rainstorm (June 0811, 2006) TM : Typhoon Morakot ( Aug 0611, 2009) : Rainfall events triggering 9 or less debris flows Critical rainfall condition (Rd = 580 mm/h) for triggering 10 or more debris flows in a rainfall event (a) d R index 1960 1965 1970 1975 1980 1985 1990 1995 2000 2005 2010 Year 0 20 40 60 80 100 Im (mm/h) TH TT HR TM EQ TMi : Rainfall events triggering 10 or more debris flows TH : Typhoon Herb (July 31Aug 01, 1996) CCE : Chi-Chi earthquake (Sep 21, 1999) TT : Typhoon Toraji (July 2930, 2001) TMi: Typhoon Mindulle (July 0203, 2004) HR : Heavy rainstorm (June 0811, 2006) TM : Typhoon Morakot ( Aug 0611, 2009) : Rainfall events triggering 9 or less debris flows Critical rainfall condition (Im = 54 mm/h) for triggering 10 or more debris flows in a rainfall event (b) m I index : Rainfall events triggering 10 or more debris flows TH : Typhoon Herb (July 31Aug 01, 1996) CCE : Chi-Chi earthquake (Sep 21, 1999) : Rainfall events triggering 9 or less debris flows Year 1 (a) d R index 2 1960 1965 1970 1975 1980 1985 1990 1995 2000 2005 2010 Year 0 20 40 60 80 100 Im (mm/h) TH TT HR TM EQ TMi : Rainfall events triggering 10 or more debris flows TH : Typhoon Herb (July 31Aug 01, 1996) CCE : Chi-Chi earthquake (Sep 21, 1999) TT : Typhoon Toraji (July 2930, 2001) TMi: Typhoon Mindulle (July 0203, 2004) HR : Heavy rainstorm (June 0811, 2006) TM : Typhoon Morakot ( Aug 0611, 2009) : Rainfall events triggering 9 or less debris flows Critical rainfall condition (Im = 54 mm/h) for triggering 10 or more debris flows in a rainfall event 3 (b) m I index 4 1960 1965 1970 1975 1980 1985 1990 1995 2000 2005 2010 Year 0 200 400 600 800 1000 1200 RI (cm2/h) : Rainfall events triggering 10 or more debris flows Critical rainfall condition (RI = 365 cm2/h) for triggering 10 or more debris flows in a rainfall event TH : Typhoon Herb (July 31Aug 01, 1996) CCE : Chi-Chi earthquake (Sep 21, 1999) TT : Typhoon Toraji (July 2930, 2001) TMi: Typhoon Mindulle (July 0203, 2004) HR : Heavy rainstorm (June 0811, 2006) TM : Typhoon Morakot ( Aug 0611, 2009) TH TT HR TM CCE TMi : Rainfall events triggering 9 or less debris flows 5 (c) RI index 6 Figure 2: Variations in three rainfall parameters contributing to debri 7 and 2009 in the Chenyulan watershed The Chi-Chi earthquake sign 8 1960 1965 1970 1975 1980 1985 1990 1995 2000 2005 2010 Year 0 (a) d R index 1960 1965 1970 1975 1980 1985 1990 1995 2000 2005 2010 Year 0 20 40 60 80 100 Im (mm/h) TH TT HR TM EQ TMi : Rainfall events triggering 10 or more debris flows TH : Typhoon Herb (July 31Aug 01, 1996) CCE : Chi-Chi earthquake (Sep 21, 1999) TT : Typhoon Toraji (July 2930, 2001) TMi: Typhoon Mindulle (July 0203, 2004) HR : Heavy rainstorm (June 0811, 2006) TM : Typhoon Morakot ( Aug 0611, 2009) : Rainfall events triggering 9 or less debris flows Critical rainfall condition (Im = 54 mm/h) for triggering 10 or more debris flows in a rainfall event (b) m I index 1960 1965 1970 1975 1980 1985 1990 1995 2000 2005 2010 Year 0 200 400 600 800 1000 1200 RI (cm2/h) : Rainfall events triggering 10 or more debris flows Critical rainfall condition (RI = 365 cm2/h) for triggering 10 or more debris flows in a rainfall event TH : Typhoon Herb (July 31Aug 01, 1996) CCE : Chi-Chi earthquake (Sep 21, 1999) TT : Typhoon Toraji (July 2930, 2001) TMi: Typhoon Mindulle (July 0203, 2004) HR : Heavy rainstorm (June 0811, 2006) TM : Typhoon Morakot ( Aug 0611, 2009) TH TT HR TM CCE TMi : Rainfall events triggering 9 or less debris flows (c) RI index Figure 2: Variations in three rainfall parameters contributing to debris flows between 1963 and 2009 in the Chenyulan watershed. 2.3 Regional rainfall characteristics versus debris ﬂows The Chi-Chi earthquake significantly lowered the rainfall threshold for debris flow occurrence during approximately the subsequent 5 years Fig. 2. Variations in three rainfall parameters contributing to debris ﬂows between 1963 and 2009 in the Chenyulan watershed. The Chi-Chi earthquake signiﬁcantly lowered the rainfall threshold for debris ﬂow occurrence, during approximately the subsequent 5 yr. Im (mm/h) mm/h ws in : Rainfall events triggering 10 or more debris flows TH : Typhoon Herb (July 31Aug 01, 1996) : Rainfall events triggering 9 or less debris flows (c) RI index 6 Figure 2: Variations in three rainfall parameters contributing to debris 7 d 2009 i th Ch l t h d Th Chi Chi th k i 8 Figure 2: Variations in three rainfall parameters contributing to debris flows between 1963 and 2009 in the Chenyulan watershed. The Chi-Chi earthquake significantly lowered the i f ll h h ld f d b i fl d i i l h b 5 Fig. 2. Variations in three rainfall parameters contributing to debris ﬂows between 1963 and 2009 in the Chenyulan watershed. The Chi-Chi earthquake signiﬁcantly lowered the rainfall threshold for debris ﬂow occurrence, during approximately the subsequent 5 yr. s flow occurrence, during approximately the s 17 rainstorm in 2006, and Typhoon Morakot (2009), all had the extreme characteristics, of Rd > 580 mm (this thresh- old was used by Chen et al. (2012) to analyse the vari- ation of extreme rainfall events in the Chenyulan water- shed) and Im > 54 mm h−1, especially Typhoon Morakot, with Rd = 1192.6 mm and Im = 85.5 mm h−1. Each of these ﬁve extreme rainfall events caused 10 or more debris ﬂows. However, as shown in Table 1 and Fig. 2a and b, only ﬁve of the eight Rd > 580 mm or Im > 54 mm h−1 extreme rain- fall events caused 10 or more debris ﬂows. Therefore, it is 400 600 800 1000 1200 RI (cm2/h) Critical rainfall condition (RI = 365 cm2/h) for triggering 10 or more debris flows in a rainfall event yp ( y ) HR : Heavy rainstorm (June 0811, 2006) TM : Typhoon Morakot ( Aug 0611, 2009) TH TT HR TM CCE TMi rainfall threshold for deb 9 10 activity (Rebetez et al., 1997; Zhuang et al., 2011; Chen et al., 2012). 2.3 Regional rainfall characteristics versus debris ﬂows Extreme rainfall refers to events of relatively high rainfall intensity and/or high cumulative rainfall. De- bris ﬂows caused by a rainfall event generally occurred within the period of the maximum 24 h rainfall, and were closely related to the maximum hourly rainfall Im (Lin and Jeng, 2000; Chen et al., 2011, 2012). The corresponding Im and Rd for debris ﬂow events in the Chenyulan water- shed between 1963 and 2009 (see Table 1) shows that Ty- phoons Herb (1996), Toraji (2001), Mindulle (2004), heavy Nat. Hazards Earth Syst. Sci., 13, 1015–1023, 2013 0 www.nat-hazards-earth-syst-sci.net/13/1015/2013/ 3 Inﬂuence of rainfall variation on debris ﬂows occurrence Both severe rainfall and earth- quakes would change the condition of RI, in terms of both the magnitude and number of debris ﬂow events. The data for the Chenyulan watershed suggest that the Chi-Chi earthquake was the most signiﬁcant inﬂuence on the rainfall threshold for debris ﬂow occurrence. ﬂow occurrence in the period between CCE and TMi was only one-thirtieth of that (60 cm2 h−1) before the CCE (be- tween TH and CCE). After about 5 years, the loose sedi- ment generally became consolidated and re-orientated over time, less soil and rock remained deposited in streams, and the shear strength of the soil gradually increased (Fan et al., 2003; Chen, 2011). The critical RI gradually recovered from 2 cm2 h−1 to 100 cm2 h−1. The variation of critical RI is related to debris supply within the watershed. Dong et al. (2009) developed a susceptibility index (SI) that ac- counted for debris supply to estimate the susceptibility to debris ﬂow. They reported that the SI increased following the CCE and after TT (before the occurrence of debris ﬂow), because the volume of debris contributed by shallow land- slides increased; and that SI decreased when the volume of debris was reduced by debris outﬂow during TT and TMi. A higher SI may lead to a smaller critical RI; conversely, a lower SI may require a larger critical RI to trigger debris ﬂow. These results conﬁrm that the RI for debris ﬂow occur- rence decreased in the early periods after the CCE and that the RI showed an increasing tendency between TT and TMi, as shown in Fig. 3. Our results are consistent with those ob- tained by Dong et al. (2009). Both severe rainfall and earth- quakes would change the condition of RI, in terms of both the magnitude and number of debris ﬂow events. The data for the Chenyulan watershed suggest that the Chi-Chi earthquake was the most signiﬁcant inﬂuence on the rainfall threshold for debris ﬂow occurrence. As shown in Fig. 3, the critical RIs for debris ﬂow occur- rence were approximately 165 cm2/h before Typhoon Herb (TH) in 1996; 60 cm2 h−1 between TH and the Chi-Chi earth- quake (CCE) in 1999; 2 cm2 h−1 between CCE and Typhoon Mindulle (TMi) in 2004; and 100 cm2 h−1 between TMi and Typhoon Morakot (TM) in 2009. 0 www.nat-hazards-earth-syst-sci.net/13/1015/2013/ et al.: Characteristics of rainfall triggering of debris ﬂows 1960 1965 1970 1975 1980 1985 1990 1995 2000 2005 2010 Year 1 10 100 1000 10000 RI (cm2/h) : Rainfall events triggering 10 or more debris flows RI =165 RI =365 cm2/h TH : Typhoon Herb (July 31Aug 01, 1996) CCE : Chi-Chi earthquake (Sep 21, 1999) TT : Typhoon Toraji (July 2930, 2001) TMi: Typhoon Mindulle (July 0203, 2004) HR : Heavy rainstorm (June 0811, 2006) TM : Typhoon Morakot ( Aug 0611, 2009) TH TT HR TM CCE TMi RI = 60 : Rainfall events triggering 9 or less debris flows RI =2 RI =100 3: The variations in rainfall index RI for all rainfall events triggering deb 1963 and 2009 in the Chenyulan watershed. The critical RI for deb nce in the years following the Chi-Chi earthquake is much smaller than thos hquake and five years after the earthquake. Fig. 3. The variations in rainfall index RI for all rainfall events trig- gering debris ﬂows between 1963 and 2009 in the Chenyulan wa- tershed. The critical RI for debris ﬂow occurrence in the years fol- lowing the Chi-Chi earthquake is much smaller than those before the earthquake and ﬁve years after the earthquake. J. C. Chen et al.: Characteristics of rainfall triggering of debris ﬂows 1 1020 inappropriate to apply a single rainfall parameter such as Rd or Im as a critical condition for the occurrence of multiple debris ﬂows. The occurrence of debris ﬂow is related to not only accumulated rainfall, but also rainfall intensity. A trig- gering index RI of a rainfall event, deﬁned as the product of Rd and Im (i.e., RI = RdIm), could be used as a critical condi- tion for the occurrence of multiple debris ﬂows. As shown in Fig. 2c, for RI > 365 cm2 h−1, all ﬁve of the extreme rainfall events caused multiple debris ﬂows, with N ≥10. The rain- fall data used in this study is limited to hourly rainfall data because the minute-scale rainfall data, such as 5 or 10 min rainfall data was not available. Other rainfall indices, such as total storm rainfall, or rainfall duration, have less correlation with the number of debris ﬂow events when compared to the RI index. 2 3 Inﬂuence of rainfall variation on debris ﬂows occurrence gg g 1963 and 2009 in the Chenyulan watershed. The critical RI for deb ce in the years following the Chi-Chi earthquake is much smaller than thos quake and five years after the earthquake. Fig. 3. The variations in rainfall index RI for all rainfall events trig- gering debris ﬂows between 1963 and 2009 in the Chenyulan wa- tershed. The critical RI for debris ﬂow occurrence in the years fol- lowing the Chi-Chi earthquake is much smaller than those before the earthquake and ﬁve years after the earthquake. Figures 2a–c show parameters for rainfall events that initi- ated debris ﬂows between 1936 and 2009. There are eight rainfall events with maximum 24 h rainfall Rd greater than 580 mm, of which seven occurred after 1996; rainfall events with maximum hourly rainfall Im greater than 54 mm or rain- fall index RI larger than 365 cm2 h−1 all occurred after 1996. This implies that rainfall events became more severe after 1996. occu 6 the e 7 8 9 10 ﬂow occurrence in the period between CCE and TMi was only one-thirtieth of that (60 cm2 h−1) before the CCE (be- tween TH and CCE). After about 5 years, the loose sedi- ment generally became consolidated and re-orientated over time, less soil and rock remained deposited in streams, and the shear strength of the soil gradually increased (Fan et al., 2003; Chen, 2011). The critical RI gradually recovered from 2 cm2 h−1 to 100 cm2 h−1. The variation of critical RI is related to debris supply within the watershed. Dong et al. (2009) developed a susceptibility index (SI) that ac- counted for debris supply to estimate the susceptibility to debris ﬂow. They reported that the SI increased following the CCE and after TT (before the occurrence of debris ﬂow), because the volume of debris contributed by shallow land- slides increased; and that SI decreased when the volume of debris was reduced by debris outﬂow during TT and TMi. A higher SI may lead to a smaller critical RI; conversely, a lower SI may require a larger critical RI to trigger debris ﬂow. These results conﬁrm that the RI for debris ﬂow occur- rence decreased in the early periods after the CCE and that the RI showed an increasing tendency between TT and TMi, as shown in Fig. 3. Our results are consistent with those ob- tained by Dong et al. (2009). 3 Inﬂuence of rainfall variation on debris ﬂows occurrence yp Between 1963 and 1996, the most severe rainfall event was TH, and the condition for debris ﬂow occurrence was RI > 165 cm2 h−1. TH brought severe rainfall and caused nu- merous landslides and debris ﬂows in the Chenyulan water- shed. Subsequently, abundant loose debris deposits remained on slopes or in gullies to serve as source material for later rainfall-induced debris ﬂows. Since this loose sediment was generally of lower soil strength, less rainfall (with RI ap- proximately equal or greater than 60 cm2 h−1) was needed to trigger debris ﬂows in subsequent years, between TH and CCE. The CCE in 1999 was the largest earthquake in Taiwan for a century, and caused numerous landslides in the Chenyu- lan watershed. Since a large amount of loose sediment with lower soil strength was deposited on streambeds or hillsides after the earthquake, much lower pore water pressure or rainfall was required to initiate the movement of this sed- iment (Lin et al., 2003; Chen et al., 2007; Chen and Jan, 2008). Therefore, the critical RI for debris ﬂow occurrence dropped sharply in the subsequent early years, to 2 cm2 h−1. That is to say that the critical RI (2 cm2 h−1) for debris Nat. Hazards Earth Syst. Sci., 13, 1015–1023, 2013 5 Conclusions d d d d Debris ﬂows and their corresponding rainfall events were studied in the Chenyulan watershed, central Taiwan, between 1963 and 2009. A rainfall index RI, deﬁned as the product of the maximum 24 h rainfall Rd and the maximum hourly rain- fall Im (i.e., RI = RdIm), was used to analyse rainfall condi- tions critical for debris ﬂow occurrence. Since the occurrence of debris ﬂow depends not only on the accumulated rainfall, but also on the rainfall intensity, the triggering index RI of a rainfall event is introduced herein to indicate either high accumulated rainfall or high rainfall intensity could trigger debris ﬂows. The results show that there were ﬁve extreme rainfall events with RI > 365 cm2 h−1 during the study pe- riod (1963–2009), each of which caused 10 or more debris ﬂows in the watershed. These ﬁve extreme rainfall events were associated with Typhoon Herb (TH) in 1996, Typhoon Toraji (TT) in 2001, Typhoon Mindulle (TMi) in 2004, heavy rainstorm (HR) in 2006 and Typhoon Morakot (TM) in 2009. RI (cm2/h) ponding rainfall index RI for two groups. The first group refers to the p cantly affected by the Chi-Chi earthquake (2000–2004; solid line), while the se represents the periods not significantly affected by the Chi-Chi earthquake (da Fig. 4. Relationships between the number of debris ﬂows N in a rainfall event and corresponding rainfall index RI for two groups. The ﬁrst group refers to the period signiﬁcantly affected by the Chi- Chi earthquake (2000–2004; solid line), while the second group rep- resents the periods not signiﬁcantly affected by the Chi-Chi earth- quake (dashed line). Nat. Hazards Earth Syst. Sci., 13, 1015–1023, 2013 www.nat-hazards-earth-syst-sci.net/13/1015/2013/ www.nat-hazards-earth-syst-sci.net/13/1015/2013/ J. C. Chen et al.: Characteristics of rainfall triggering of d 1 10 100 1000 RI (cm2/h) 1 10 100 N : Before TH : Between TH and CCE : Between CCE and TMi : Between TMi and TM TH TT TM HR TMi TH : Typhoon Herb (July 31Aug 01, 1996) CCE : Chi-Chi earthquake (Sep 21, 1999) TT : Typhoon Toraji (July 2930, 2001) TMi: Typhoon Mindulle (July 0203, 2004) HR : Heavy rainstorm (June 0811, 2006) TM : Typhoon Morakot ( Aug 0611, 2009) e 4: Relationships between the number of debris flows N in a rainfall even sponding rainfall index RI for two groups. The first group refers to the p icantly affected by the Chi-Chi earthquake (2000–2004; solid line), while the se p represents the periods not significantly affected by the Chi-Chi earthquake (d Fig. 4. Relationships between the number of debris ﬂows N in a rainfall event and corresponding rainfall index RI for two groups. The ﬁrst group refers to the period signiﬁcantly affected by the Chi- Chi earthquake (2000–2004; solid line), while the second group rep- resents the periods not signiﬁcantly affected by the Chi-Chi earth- quake (dashed line). J. C. Chen et al.: Characteristics of rainfall triggering of debris ﬂows 1021 J. C. Chen et al.: Characteristics of rainfall triggering of 1 10 100 1000 RI (cm2/h) 1 10 100 N : Before TH : Between TH and CCE : Between CCE and TMi : Between TMi and TM TH TT TM HR TMi TH : Typhoon Herb (July 31Aug 01, 1996) CCE : Chi-Chi earthquake (Sep 21, 1999) TT : Typhoon Toraji (July 2930, 2001) TMi: Typhoon Mindulle (July 0203, 2004) HR : Heavy rainstorm (June 0811, 2006) TM : Typhoon Morakot ( Aug 0611, 2009) (2) show that the number of debris ﬂow events increases with the increase of rainfall index. N is larger during the period affected by CCE than the periods unaffected by CCE. According to Eqs. (1) and (2), for a rainfall event of RI = 60 cm2 h−1, the number N of debris ﬂows in the CCE-affected period could potentially be double that of the non-CCE periods. However, for an extreme rainfall event of RI = 365 cm2 h−1, the value of N in the CCE-affected period could be about six times larger than in the non-CCE periods. 4 Relationship between rainfall characteristics and the number of debris ﬂows log(N) = 0.290 + 0.003(RI −2) (1) for the period between 2000 and 2004 (1) The study also presented two empirical relationships between the number of debris ﬂows N in a rainfall event and their corresponding rainfall index RI for the CCE-affected period (between 2000 and 2004) and the non-CCE-affected periods (1963–1999 and 2004–2009). Higher RI of a (2) log(N) = 0.121 + 0.0015(RI −60) for other periods (2) Here, the coefﬁcients of determination for Eqs. (1) and (2) are r2 = 0.78 and 0.75, respectively. Equations (1) and www.nat-hazards-earth-syst-sci.net/13/1015/2013/ 4 Relationship between rainfall characteristics and the number of debris ﬂows We allocated the debris ﬂows to two groups: one represent- ing the period between CCE and TMi (2000–2004), in which the rainfall condition required for debris ﬂow occurrence is signiﬁcantly affected by CCE (shown in Fig. 4 as solid cir- cles); the other representing the periods that were unaffected by the CCE, i.e., before TH, between TH and CCE, and be- tween TMi and TM (shown in Fig. 4 as open triangles, cir- cles and rectangles, respectively). Comparison within the pe- riods without CCE effect shows that the RIs between TH and CCE (open circles in Fig. 4) are smaller, and the correspond- ing number of debris ﬂows N is slightly greater compared to other periods (before TH, and between TMi and TM). This result may be attributed to abundant loose debris remaining on slopes or in gullies in the early periods after TH; these deposits were more susceptible to the effects of rainfall, and served as source material for subsequent debris ﬂows. The relationships for the number of debris ﬂows (N) associated with a rainfall event of rainfall index (RI) in the two groups are shown in Fig. 4, and represented by Eqs. (1) and (2). 9 The Chi-Chi earthquake (CCE) occurred in 1999, and caused severe landslides in the Chenyulan watershed. The ex- treme rainfall events and the severe earthquake were shown to affect the critical condition for the occurrence of debris ﬂows. The critical RIs for occurrence of debris ﬂows be- tween 1963 and 2009 could be classiﬁed into four categories: the periods before TH, between TH and CCE, between CCE and TMi, and between TMi and TM; and had critical RIs of approximately 165, 60, 2 and 100 cm2 h−1, respectively. It is shown that: (1) TH caused numerous landslides and de- bris ﬂows in the watershed, which reduced the critical rain- fall threshold for debris ﬂow in subsequent years; (2) CCE signiﬁcantly lowered the critical rainfall threshold for debris ﬂow occurrence in the subsequent ﬁve years – after CCE, the critical RI dropped sharply to approximately 2 cm2 h−1, which was one-thirtieth of that before the CCE (critical RI = 60 cm2 h−1). The results also show that, approximately ﬁve years after the CCE, the critical RI gradually recovered from 2 cm2 h−1 to 100 cm2 h−1 (the critical RI between TMi and TM). References Fowler, H. J., Ekstr¨om, M., Kilsby, C. G., and Jones, P. D.: New estimates of future changes in extreme rainfall across the UK us- ing regional climate model integrations, 1. Assessment of control climate, J. Hydrol., 300, 212–233, 2005. Caine, N.: The rainfall intensity-duration control of shallow land- slides and debris ﬂows, Geograﬁska annaler, 62A (1/2), 23–27, 1980. 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https://openalex.org/W2997147441	https://revistas.ucm.es/index.php/POSO/article/download/57444/4564456552400	English	null	Russia-Spain Cooperation on Counter-Terrorism: New Challenges or Old Problems?	Política y sociedad	2,019	cc-by	9,679	Recibido: 03-10-2017 / Aceptado: 25-09-2019 Abstract. Against the backdrop of globalization, international terrorism is becoming a complex threat, which can be countered only by the united efforts of all countries. The level of cooperation of states does not fully correspond to the scale of the world terror challenge. Cooperation in the fight against terrorism at the bilateral level opens up more opportunities because it is based on mutual trust between States, with greater effectiveness, as well as practical impact and benefits, due to the knowledge of the situation and the ability to respond quickly. The inefficiency of the global counter- terrorism system can be compensated by the mechanism of bilateral cooperation in the sphere of counter-terrorism, despite all the existing problems such as: sanctions, human rights, the need to promptly equate national laws and legislation in line with international laws, etc. The choice of the Russian Federation and Spain as a case study for this research is not accidental, since the interaction of both countries allows us to trace the development of mechanisms and instruments to counter terrorism on a bilateral basis. As a result of the comparative analysis, it is established that while Russian-Spanish relations began to deteriorate in 2014 as a result of the sanctions policy, the bilateral contexts of cooperation in the field of countering extremism have become comparable in two parameters: the overall level of the threat of terrorism in Russia and Spain, as well as the types of terrorist challenges on their territory. Keywords: bilateral cooperation; terrorism; counter-terrorism strategy; Russia; Spain; UN; international relations; globalization; contractual legal framework; sanctions. [es] Cooperación ruso-española contra el terrorismo: ¿nuevos desafíos o problemas viejos? MISCELÁNEA Política y Sociedad ISSN: 1130-8001 ISSN-e:1988-3129 http://dx.doi.org/10.5209/poso.57444 Russia-Spain Cooperation on Counter-Terrorism: New Challenges or Old Problems? Russia-Spain Cooperation on Counter-Terrorism: New Challenges or Old Problems? Svetlana Bokeriya 1 1 People’s Friendship University of Ruisia, RUDN (Rusia). E-mail: petrovsvet@yandex.ru [es] Cooperación ruso-española contra el terrorismo: ¿nuevos desafíos o problemas viejos? Resumen. El nivel de cooperación entre las naciones por ahora no concuerda con la escala del problema que abarca el terrorismo internacional. Sin embargo, la interacción en la lucha contra el terrorismo a nivel bilateral abre nuevas posibilidades en virtud de que se basa en la mutua confianza entre las naciones. Tiene un gran resultado y también es eficaz. Se caracteriza por tener conocimiento de la situación en el lugar y contar con la posibilidad de reaccionar rapidamente en conjunto. La inefectividad del sistema global de contraterrorismo puede ser compensada por el mecanismo de cooperación bilateral en el campo de la contraposición al terrorismo, a pesar de todos los problemas existentes: sanciones, derechos humanos, la necesidad de poner operativamente la legislación nacional en correspondencia con los actos territoriales, etc. La elección de la FR y de España como case-status para llevar a cabo este estudio no es casual, ya que el contenido de la interacción entre estos países permite velar por el desarrollo de los mecanismos y los instrumentos de contraposición al 737 Polít. Soc. (Madr.) 56(3) 2019: 737-756 738 Bokeriya, S. Polít. Soc. (Madr.) 56(3) 2019: 737-756 terrorismo en una base bilateral. Como resultado de un análisis comparativo, se estableció que al mismo tiempo que las relaciones ruso-españolas comenzaron a empeorar en el año 2014, a causa de la política de sanciones, los contextos recíprocos de interacción en el campo de contraposición al extremismo llegaron a compararse en dos índices: según el nivel general de amenaza de terrorismo en Rusia y en España, y según los tipos de desafíos terroristas en sus territorios. Palabras clave: cooperación bilateral; terrorismo; estrategia contra el terrorismo; Rusia; España; ONU; relaciones internacionales; globalización; marco contractual legal; sanciones. Cómo citar: Bokeriya, S. (2019): “Russia-Spain Cooperation on Counter-Terrorism: New Challenges or Old Problems?”, Política y Sociedad, 56(3), pp. 737-756. Sumario. 1. Introduction. 2. Conceptual Approaches of the EU to International Security and Terrorism. 3. UN Activity in Counter-Terrorism. 4. Russia-Spain Cooperation to Counter-Terrorism: Challenges and Legal Approaches. 5. Conclusion. 6. Bibliography. Sumario. 1. Introduction. 2. Conceptual Approaches of the EU to International Security and Terrorism. 3. UN Activity in Counter-Terrorism. 4. Russia-Spain Cooperation to Counter-Terrorism: Challenges and Legal Approaches. 5. Conclusion. 6. Bibliography. Acknowledgements. [es] Cooperación ruso-española contra el terrorismo: ¿nuevos desafíos o problemas viejos? This paper was financially supported by the Ministry of Education and Science of the Russian Federation on the program to improve the competitiveness of Peoples’ Friendship University of Russia (RUDN University) among the world’s leading research and education centers in the 2016-2020. 1. Introduction A distinctive feature of the modern period of development of the world community was the aggravation of global problems, challenges and threats at the turn of the twentieth and twenty-first centuries, requiring a collective and coordinated response. These problems have become factors that determine not only the key trends in the development of international relations, but also the main directions of world politics, as well as the international agenda. Against the background of fundamental and dynamic changes that affect the interests of each State, the formation of new political blocs, as well as the economic crisis and globalization processes, blurring the boundaries between the center and the periphery, foreign and domestic policies of States, the problems of protecting a global society, each individual nation and person from complex interdependent challenges and threats are becoming more acute. New challenges and threats, such as international terrorism, drug trafficking, global poverty, organized crime, regional conflicts and illegal migration, are global in scale and complex, quickly transforming and changing, requiring rapid response and preventive measures. The situation is aggravated by the fact that these threats can come from both non-state actors and States. All this, on the one hand, destabilizes the international system, on the other hand, activates and intensifies international and regional cooperation between States, which should become a key motive and an effective way to confront complex challenges and threats. Terrorism statistics shows a temporal growth. According to the latest international report Global Terrorism Index 2018, analyzing the impact of terrorism for 163 countries and which covers 99.7 per cent of the world’s population, every region in the world recorded a higher average impact of terrorism in 2017 than in 2002. The increase in the impact of terrorism was greatest in the Middle East and North Africa, followed by sub-Saharan Africa. In Western Bokeriya, S. Polít. Soc. (Madr.) 56(3) 2019: 737-756 739 Europe, deaths fell by 52 per cent, from 168 in 2016 to 81 in 2017. From January until October 2018, fewer than ten deaths were recorded in the region. Europe, deaths fell by 52 per cent, from 168 in 2016 to 81 in 2017. From January until October 2018, fewer than ten deaths were recorded in the region. Despite the fall in deaths, the number of incidents rose in Western Europe. Increased counter-terrorism spending and security measures have reduced the lethality of attacks. 1. Introduction In 2017, just under 95 per cent of total deaths from terrorism occurred in countries involved in at least one violent conflict. When countries in conflict are combined with countries with high levels of political terror, the number climbs to 99 per cent. Terrorist attacks in conflict-affected countries are also more lethal, killing 2.4 people per attack on average, compared to 0.84 people in non- conflict countries in 2017 (Global Terrorism Index, 2018). ( ) This article seeks to provide an analysis of the international terrorism impact on global security with particular emphasis to Russia-Spain bilateral interaction as a part of counter-terrorism efforts on a multinational level. The article is divided into 6 sections, including introduction, conclusion, and bibliography. The first section features conceptual approaches to international security and terrorism of the European Union. The second section addresses and analyses the UN counter- terrorism strategy that requires updating with due regard to the current circumstances. The third section represents an analysis of bilateral cooperation between Russia and Spain with regard to counter-terrorism efforts in order to illustrate the influence of terrorist attacks on national policies, and the cooperation dynamics, and to estimate the established mechanism of bilateral cooperation with regard to the struggle against this threat. 2. Conceptual Approaches of the EU to International Security and Terrorism Cooperation within the EU in the fight against terrorism before the terrorist attack of September 11, 2001 was not an official part of the institutional architecture of the European Union. The "European Union counter-Terrorism strategy", adopted after the London bombing on 7 July 2005, was not aligned with comprehensive strategies (in particular, internal and external security of EU countries) and sub-strategies aimed at countering the financing of terrorism, radicalization and recruitment. Later in 2008, the Council adopted the EU strategy against radicalization and recruitment of terrorists. g Since 2013, the issues of radicalization and the fight against terrorists became permanent items in the agenda of the EU Council and the Council of Europe. The provisions and main tasks for internal and external counter-terrorism activities were developed. p In 2015, EU leaders declared the need to expand cooperation with third world countries in the area of security and the fight against terrorism. Furthermore, the "European Security Agenda" was then initiated, which focused on the exchange of information between States and the prevention of radicalization, created after the attacks in Brussels in March 2016. The concept of "Security Agenda", highlighted the important transition from protecting of a country’s internal security to protecting the collective security of the EU as a whole (European Commission, 2016: 2, 9). ( p , , ) One of the difficulties in developing conceptual frameworks for anti-terrorism in the EU has been the large number of participants (the EU, the EU Council and the European Commission) involvement in this process, as well as duplication and the intersection of tasks, which significantly complicates and slows down the formation of structural and integrated approach. This, in turn, plays into the hands of terrorists, since the system of counteraction against radicalization and terrorism cannot respond to quickly transforming challenges and threats. p q y g g In March 2017, the Council adopted a directive on counter-terrorism, which provides for responsibility for such actions as: conducting training or travelling for terrorist purposes, organizing or facilitating such a trip, as well as providing or collecting funds directed to terrorist groups or their activities. However, there is a critical point of view based on the fact that this directive, due to its broad interpretation and general nature, can exacerbate the problem of human rights violations: criminalize public protests and other peaceful actions, suppress freedom of expression, including opposition political views. 2. Conceptual Approaches of the EU to International Security and Terrorism In the context of reviewing the conceptual aspects of counter terrorism, special attention should be paid to the analysis of challenges, key trends and gaps in the counter-terrorism approaches of the EU, of which Spain is a member. The EU's antiterrorist activities rightfully play a key role in shaping the international legal framework of the European security system in the sphere of countering the threat of terrorism, because creating a security zone, freedom and justice is one of the main objectives of the Union. The following events had a significant impact on the development of the EU's counter-terrorism program: 1) the September 11, 2001 terrorist attacks in the United States. Ten days after these events, at an extraordinary meeting, the European Council declared the fight against terrorism a priority for the EU; 2) the Madrid bombing in 2004 and London in 2005; 3) the Syrian war and the emergence of ISIS; 4) the attacks in Nice and Berlin in 2016 and the crimes involving lone terrorists (The European Union's Policies on Counter-Terrorism, 2017). A common feature of these attacks was the connection with Islamic terrorism, which is why it has become a key focus of the anti-terrorist struggle in the EU. Over the past decade, there has been an increase in the number of terrorist acts of right-wing and jihadist extremism against the backdrop of a decline in separatist and left-wing extremist protests. The trend in recent years has been the increase in terrorist attacks by lone terrorists, as well as active propaganda activities through the internet, with the aim of recruiting new members and 740 Bokeriya, S. Polít. Soc. (Madr.) 56(3) 2019: 737-756 organizing terrorist acts. According to Deputy Secretary of the Security Council of the Russian Federation Yuri Kokov, "today there are about 30 thousand extremist and terrorist sites. The era of technological and digital terrorism is coming, which in terms of the scale of the consequences in the near future can be comparable with weapons of mass destruction" (Tarasenko, 2019). p ( ) However, with the earlier use of internet, members of the Islamic state group urged their supporters to move to Syria and Iraq to commit terrorist attacks, now they are offered to commit Jihad in Western countries, where they are. The fact that this appeal was accepted, the terrorist attacks in Norway, the Netherlands, France and Spain in 2019, among other things, is a confirmation. 2. Conceptual Approaches of the EU to International Security and Terrorism There are concerns that the directive will be applied at the national level to discriminate against vulnerable and marginalized ethnic and religious communities (Aoláin, 2018). Bokeriya, S. Polít. Soc. (Madr.) 56(3) 2019: 737-756 741 Also in 2017, a resolution was adopted to amend the code of Schengen territory and to strengthen the verification of relevant databases at external borders. It requires member States to systematically review relevant databases for all persons crossing external borders. In April 2017, the Council signed a directive on the control of the acquisition and possession of weapons, containing measures to strengthen the control of firearms and prevent its use. Analyzing the development of counter-terrorism architecture of the EU has shown that the main incentive for the elaboration of legal acts regulating the fight against terrorism were the terrorist acts, committed at short intervals in different European countries. It can be said that such an approach involves taking measures after the fact, and not for the future. In addition, threats are not updated on a regular basis within the EU. In particular, Europol and the EU Intelligence and Situation Center are engaged in threat assessment on a situational basis, not comprehensively and not on an ongoing basis. The emphasis in anti-terrorist strategies on jihadism leads to underestimation of threats from other extremist movements, for example, far-right radicals. In parallel with the adoption and application of a large number of strategies, legal mechanisms and tactics in the field of anti-terrorism, the problem of human rights protection is becoming increasingly acute, which at first glance is not obvious. However, in the broad legal field of counter-terrorism, there is a cumulative effect of the joint action of various norms and measures affecting freedom of movement, expression of opinion, the right to hold rallies and speeches of peaceful citizens of a state (Aoláin, 2018). p ( ) Human rights can be violated even at the level of data collection, as confidentiality and data protection are not sufficiently regulated. Among the authorities of various States, guided by both national and international legal acts, there is no clear understanding of the rights of persons who hold confidential data. In the case of a terrorist threat, data collection often covers a wide range of people (family members, representatives of religious communities and public associations). 2. Conceptual Approaches of the EU to International Security and Terrorism The result of the information exchange for a person who possesses classified or secret information, may include surveillance (often unknown to the subject), consequences associated with further employment, restrictions on border crossings, as well as control or blocking access to financial resources for individuals and families. Thus, the EU's counter-terrorism system is multilateral, but not yet completed and requires improvement in terms of ensuring and developing human rights, updating the list of threats, collecting and evaluating data, as well as a clearer delineation of tasks within the authorized structures. However, a common understanding of the need to apply a consolidated approach to solving the problem of terrorism and extremism should be a key motive for cooperation between the EU and Russia, which shares this approach. 3. UN Activity in Counter-Terrorism The bilateral Russian-Spanish cooperation cannot be entirely analyzed without the best practices elaborated by the most powerful international organization in the Bokeriya, S. Polít. Soc. (Madr.) 56(3) 2019: 737-756 742 field of countering international terrorism— the UNO. The main UN counter- terrorism bodies are a group of organizations working under the aegis of the General Assembly (UN GA), as well as the Security Council (UN SC). However, counter-terrorism tasks given in the mandates of these organizations sometimes coincide, despite their different specialization, which hinders coherence of the UN activities generally in this field and requires optimization of resources and reformation of the UN hierarchical system. y The UN Security Council established the Counter Terrorism Committee after the 9/11 terrorist attack and approved resolution 1373 (2001), devoted to measures in fighting against terrorism. The UN Security Council adopts resolutions and is responsible for the Counter-Terrorism Strategy, as well as the UN General Assembly that consolidates and coordinates the UN Global Counter-Terrorism Strategy. This Strategy was adopted on 8 September 2006. Russia and Spain contributed to its adoption by consensus. The Global Counter-Terrorism Strategy in the form of a resolution and the Plan of Action (A/RES/60/288) are composed of four pillars, which are addressing the conditions conducive to the spread of terrorism; measures to prevent and combat terrorism; measures to build states’ capacity and to strengthen the role of the United Nations system in that regard; measures to ensure respect for human rights and the rule of law. The Resolution proposes certain measures to prevent and eliminate conditions contributing to the spread of terrorist activities. In particular, to examine the question of creating national systems of assistance that would promote the needs of victims of terrorism and their families and facilitate the normalization of their lives; to establish an international center for counter-terrorism; to use the experience of similar international, regional and sub-regional organizations with the aim to exchange the cutting-edge experience in the field of strengthening potential in countering terrorism and many other. The Strategy is being implemented via the UN agencies —the UN Secretariat, the UN Security Council and observers—, 38 bodies in total (UN Office of Counter-Terrorism, 2019a). , ( , ) Moreover, the UN Secretary-General proposed to institutionalize the Counter- Terrorism Implementation Task Force within the Secretariat in order to ensure overall coordination in the United Nations system's counter-terrorism efforts. 3. UN Activity in Counter-Terrorism y There have been adopted 26 UN resolutions (13 of them were adopted by the GA and the same quantity by the SC) devoted to the counter-terrorism subject and their number increases annually, which attests to the fact that high global attention is given to this problem: Resolution 70/291, which is the UN Global Counter- Terrorism Strategy; Resolution 71/38 highlights the measures to prevent terrorists from acquiring weapons of mass destruction; Resolution 71/67 on Nuclear Disarmament Verification; Resolution 71/151 highlighted the measures to eliminate international terrorism. In addition, the following resolutions of the UN Security Council are to be included in this list: 2309 (2016) devoted to the terrorist threats to civil aviation; 2331 (2016) —on combating human trafficking and terrorism, as well as 2341 (2017)— on protecting critical infrastructure against terrorist attacks (UN Office of Counter-Terrorism, 2019b). ( ) It is noteworthy that resolution 71/151 adopted by the UN General Assembly has included recommendations to all member states to accelerate the process of solving the unsettled tasks associated with development of a draft Comprehensive Bokeriya, S. Polít. Soc. (Madr.) 56(3) 2019: 737-756 743 Convention on International Terrorism, whose elaboration started in December 1999 after adopting Resolution 54/110 by the UN General Assembly. However, its preparation is complicated by the lack of consensus among the experts on certain questions. At the moment, the project exists only in the form of a working paper. Convention on International Terrorism, whose elaboration started in December 1999 after adopting Resolution 54/110 by the UN General Assembly. However, its preparation is complicated by the lack of consensus among the experts on certain questions. At the moment, the project exists only in the form of a working paper. q p j y g p p The first key institutional reform initiated by the Secretary-General was the establishment of the United Nations Office of Counter-Terrorism in June, 2017. The Office is aimed at cooperation with Security Council bodies and member states, strengthening current and developing new partnerships through regular attendance at counter-terrorism-related meetings, enhancing coordination across the 38 Counter-Terrorism Implementation Task Force entities to provide the balanced implementation of the 4 pillars of the UN Global Counter-Terrorism Strategy. At the request of member states the UN Office of Counter-Terrorism via its UN Counter-Terrorism Centre is already implementing over 40 counter- terrorism capacity-building projects. 3. UN Activity in Counter-Terrorism For instance, the initiative for assisting young people to become resilient to radicalization in Bangladesh and Pakistan (United Nations Office in the Russian Federation, 2018). , ) The Secretary-General signed the new United Nations Global Counter- Terrorism Coordination Compact in February 2018, which changes the Counter- Terrorism Implementation Task Force coordination mechanism. Despite of the first steps in the UN counter-terrorism system reforming and having a wide range of relevant tools in the UN framework, why isn’t the international security system functioning regardless? Nevertheless, the existing international regulatory framework is not sufficiently efficient. The reason is that conventions define and regulate only separate aspects for combating terrorism. That means they are of a sectoral nature, which, in its turn, makes it impossible to solve the problem comprehensively. Additionally, the UN’s line of work can be characterized by piecemeal approaches and by the tendency to create special-purpose committees and plans of action following on from the result of certain incidents, and not by creation of a coordinated program. Furthermore, there is a duplication of tasks within the framework of different agencies that indicates the necessity of optimization and reformation of the UN system in order to increase the efficiency of its activity. Besides, there is no a unified definition of the terms international terrorism, terrorist attack and terrorism, that fact hinders the creation of international treaty obligations framework and gives the states an opportunity to manipulate the terms for their purposes. For example, the term terrorism has multidisciplinary nature, connecting such interpretations as computer attacks, politically-motivated murders, war crimes, etc. There is no global agreement on the definition of terrorism under the UN. The European Union documents present no universal definition for the term as well. During the High-level Conference in Counter-terrorism, held in June 2018 in New York, António Guterres, the United Nations Secretary-General explained that the “terrorism threat continued to evolve and the international community needed to adapt and learn lessons from what worked and what did not. There is a need to strengthen the capacities of counter-terrorism structures and institutions and build the resilience and cohesion of societies at national and regional levels” (Report of the United Nations High-level Conference in Counter-terrorism, 2018). He also noted the major goals for combating terrorism: strengthening international counter- Bokeriya, S. Polít. Soc. 3. UN Activity in Counter-Terrorism (Madr.) 56(3) 2019: 737-756 744 terrorism cooperation, a renewed and sustained focus on preventing terrorism, underlining the importance of fully respecting human rights while tackling terrorism, reinforcing the need to make a strategic investment in young people to counter terrorism and prevent violent extremism, shining a light on the tragic human cost of terrorism, and strengthening the role of the United Nations to assist member states in tackling terrorism. According to Report of the United Nations High-level Conference in Counter-terrorism 2018, the goals show the concerted approaches to the problems of counter-terrorism, which should be solved via an “all-of-government”, “all-of-society” and “all-of-UN” mechanisms. g y In the framework of common efforts, Spain and Russia are actively involved in developing the conceptual framework for combating international terrorism under the auspices of the UN. Thus, For example, Spain is participating in the "Special Counter-Terrorism Strategy Group", particularly active in the Victim Support Group of Terrorism, which is a significant aspect of the post-conflict settlement. Spain has contributed to the adoption of measures and resolutions of importance in the fight against terrorism. These include the adoption of July 2015 Madrid Guidelines on the control of the movement of foreign terrorist fighters and UN Security Council resolution 2322 of December 2016 on international judicial and police cooperation in matters of terrorism. p p In 2017, Spain played a key role in the UN General Assembly resolution (A/C.3/72/L.24), which declared 21st of August the International Day of Remembrance of the victims of terrorism. Spain has one of the most progressive systems of support for victims of terrorism (Argomaniz, 2015). y pp ( g ) During Spain’s presidency of the UN Security Council, Spain introduced Resolution 2331 on human trafficking in conflict situations and contributed to the approval of the UNGA Resolution (A/RES/71/322) on "Strengthening and promoting effective measures and international cooperation in organ donation and transplantation to prevent and combat human trafficking". p p g In 2017, an Anti-Terrorism Bureau was set up to strengthen the UN's counter- terrorism structure, headed by Russian diplomat Vladimir Voronkov. In 2018, he signed the UN Global coordination Pact on combating terrorism (the “Global compact") along with 36 divisions of the Organization, Interpol and the World Customs Organization. Its goal is to improve coordination and coherence in the field of anti-terror and to provide support in this direction to member states. 4. Russia-Spain Cooperation to Counter-Terrorism: Challenges and Legal Approaches Internationalization of global terrorism and changeable combination of scenarios of terrorist attacks, expansion of terrorist networks arise significance of bilateral interaction against terrorism. The mechanism of Russia-Spain efforts in terrorism prevention is of big interest due to old joint dialogue, due to transnational activity of terrorist groups in both countries and identity of political views regarding modern challenges and threats. Since 1990s this mechanism is moved to combating terrorism direction, so as Russia and Spain have a congruent understanding of Bokeriya, S. Polít. Soc. (Madr.) 56(3) 2019: 737-756 745 terrorist attack and necessity to adhere principles and norms of the UN. Both countries ratified 13 universal anti-terrorism protocols and conventions. p The National Security Strategy of Spain 2013 underlines that Russia is a large neighbor and a key strategic actor of the EU. After terroristic acts in Barcelona and Cambrils in 2017 Spanish Ambassador to Russia Ignacio Ybanez Rubio said: “Unfortunately one has to state that Spain’s experience of fighting terrorism is very great and long, but it also shows that terrorism can be fought successfully. We hope that we shall continue this successful experience of fighting terrorism, and other countries will help us including Russia”. So Spain hopes to continue the joint fighting against terrorism with Russia, as it has a wide experience in this sphere and it has made a significant contribution to counter-terrorism in the Middle East. In addition, after independence referendum held in Catalonia in 2017, which was not recognized by the government of Spain, as well as by no other country in the world. Russia was one of the first countries that supported the territorial integrity of Spain, the efforts of the Spanish government, stating that all internal problems of Spain should be solved within the framework of the Spanish constitution and legislation. p g Despite of the mutual support between Russia and Spain, in the light of freezing of political dialogue between Russia and the West, there is a real risk that the common distrust can have a negative result in combating global terrorism. But nevertheless, the counter-terrorism issue is a core motif for Russia-West cooperation. There are objective reasons for that. “Terrorism will continue to be a threat for the world, Europe, our citizens, our security and our way of life for the foreseeable future. In 2017 while jihadist-inspired attacks increased, so did our preventive actions— with at least 11 attacks foiled. 4. Russia-Spain Cooperation to Counter-Terrorism: Challenges and Legal Approaches This is the strongest testament to the necessity of working together to defeat terrorism. Europol’s annual report leaves no doubt that the problem affects all of us equally, and we can only address it with collective action, unity of purpose, and above all trust. A genuine and effective Security Union in Europe is the surest way to tighten the screws on terrorists until they no longer have space to commit their atrocities”, said Dimitris Avramopoulos, European Commissioner for Migration, Home Affairs and Citizenship (Europol, 2018). According to EU Terrorism Situation and Trend Report 2018, the latest trends in international terrorism in Europe are that internet played a significant role in radicalization of some criminal groups, during attacks terrorists preferred for improvised explosive devices, terrorist strategies have further diversified and the threat has increased (EU Terrorism Situation and Trend Report, 2018). ( p ) The events in Ukraine and the EU sanctions adopted in 2014 have a negative influence on bilateral relations between Russia and Spain, both from an economic point of view and in terms of cooperation in the area of countering terrorism. Despite the complicated financial and economic situation in the world, the total volume of trade between Russia and Spain in 2013 was 11 billion US dollars, while trade and investment markers grew by 3%. As a result of the EU’s sanctions policy towards Russia, trade has decreased from 2013 to 5 billion US dollars by 2017, and the number of Russian tourists visiting Spain from 1.5 million to 1.1 million people a year. In 2018, Russia’s Ambassador to Spain, Yu.P. Korchagin, cited a sum of 700 million euros, “which Spain lost as a result of anti-Russian sanctions, as well as counter measures taken by the Russian Federation”. However, in 2018, there were some positive trends and prospects for the growth of trade (Anikeeva, 2019). 746 Bokeriya, S. Polít. Soc. (Madr.) 56(3) 2019: 737-756 Disunity, lack of coordination of actions becomes another threat to each country, as well as the whole world. These factors are beneficial for terrorists and their destructive plans. Therefore, the problem of overcoming any obstacles to bilateral and multilateral mutual measures is extremely relevant now (Shpakovskaya et al, 2017). Spain has never been a full supporter of the EU sanctions against Russia. 4. Russia-Spain Cooperation to Counter-Terrorism: Challenges and Legal Approaches According to Josep Borrell Fontelles, the Spanish Minister of Foreign Affairs: “Spain is one of the countries that suffered the most from the Russian response to European sanctions. We are more than anyone willing to normalize relations and are working in this direction” (El País, 2018). g g ( ) Despite the deterioration of bilateral Russian-Spanish relations as a result of the sanctions policy since 2014, there is a mutual interest of the two countries in the joint fight against terrorism, as well as the convergence in the aspect of counter extremism, due to the comparable level and type of terrorist threats. , p yp Terrorism threats in Russia and Spain vary in scale, type and ways of radicalization. After the cold war and starting in the 1990s, Russia has been more exposed to terrorist attacks than Spain (see figure 1) because of terrorist attacks in the North Caucasus. During the same period, there were fewer terrorist attacks in Spain. Since 2012, Russia has ranked higher in the Global terrorism index than Spain (see table 1). In the 2012 Index, Russia was one of the top ten countries in the world in terms of terrorist activity, ranking 9th, while in 2014- 11th, and in 2015 -23rd, while Spain was ranked 45th position in 2012, 69th in 2014, and 65th in 2015. However, the gap began to decline. In the Index for 2018, Russia was ranked 34th and Spain 50th. Diagram 1. Dynamics of terrorist attacks in the Russian Federation and in Spain (1990-2017) Diagram 1. Dynamics of terrorist attacks in the Russian Federation and in Spain (1990-2017) Source: Global Terrorism Database. Source: Global Terrorism Database. Bokeriya, S. Polít. Soc. (Madr.) 56(3) 2019: 737-756 747 As for the diagram 1, it shows dependence of the number of terrorist acts on a particular period of time (17 years) (see diagram 1). It can be seen that since 1992, there has been observed a starting expansion of terrorist activity in both countries. One more peak of terrorist activity has been noticed in 2000-2001 and in 2010, which can be related to the financial crisis. A decline in terrorist activity has been marked in Russia and Spain from 2004 till 2007. A long period of stabilization has been in Spain from 2007 to 2017, which might be connected with the country’s refusal to take part in the Syrian and the Iraqi campaigns. 4. Russia-Spain Cooperation to Counter-Terrorism: Challenges and Legal Approaches The greatest number of terrorist attacks in Russia was in 2010. As shown in diagram 1 the dynamics of terrorist attacks in Spain systematically reduces while in Russia it develops in chaotic way. y y p y An insignificant difference in the scale and scope of threats can be seen in the lists of terrorist organizations of the EU and the Russian Federation. The official lists of terrorist organizations of the Russian Federation and the European Union are dominated by radical Islamist groups. Thus, the Russian list of foreign terrorist organizations, as of March 14, 2019 noted 30 organizations, most of which are Islamist (Federal Security Service of the Russian Federation, 2019), while in the list of the Council of the European Union as of January 8, 2019 there are only 21 terrorist organizations (EUR-Lex, 2019) with a similar predominance of radical Islamist organizations. g ISIS is a serious threat to both Russia and Spain. For the Russian Federation, the danger posed by this international Islamist terrorist organization is not limited only to conflicts in the North Caucasus (Stepanova, 2017). ISIS has initiated small radical cells outside the region and recruited individuals in different parts of the country. ISIS initiated small radical cells outside this region and recruited individuals in different parts of the country. This type of threat resembles the phenomenon problem of individual terrorists and network agents prevalent in Europe and the United States. p Russia's practical experience in combating terrorism was based on the anti- insurgency campaign in the North Caucasus, as well as a militarized strategy for participating in the Middle East settlement, while in Spain this experience included both political (granting broad autonomy to the region) and a strong security force (the creation of anti-terrorist armed groups and the activities of special services) in the fight against Basque separatism (Mihajlin, 2016). With regard to participation in the stabilization of the situation in the Middle East, Spain has long been neutral, refraining from military operations in favor of assistance in the training of soldiers. g y p g However, the Russian Federation began to pay more attention to the causes and conditions of terrorism, as well as preventive measures, while Spain in the new counter-terrorism strategy focused on the processes of de-radicalization, paying great attention to former members of ISIS and Al-Qaeda, who are returning from conflict zones, prisons, where petty criminals become radical. 4. Russia-Spain Cooperation to Counter-Terrorism: Challenges and Legal Approaches Among common initiatives realized between Russia and Spain it is worth to mention the establishment of a joint cybersecurity group to prevent diplomatic relations from being damaged by the results of misinformation (The Moscow Times, 2018). On the one hand, this agreement intensifies cooperation in information technologies security on the territory of two countries, on the other hand, there is a danger that the Russian-Spanish project may enter into a confrontation with the EU policy of sanctions. Regarding the confrontation between Moscow and Brussels, the Spanish leadership takes a generally Bokeriya, S. Polít. Soc. (Madr.) 56(3) 2019: 737-756 748 neutral attitude, refraining from sharp statements and relying on possible positive developments in the general format of Russia-EU relations. The Foreign Policy Concept of Russia defines anti-terrorist measures as major national concern and key priority in the international security vector. Condemning all terrorist acts in all their formats, Russia actively participates in promoting the idea of joining efforts of all countries and of the world community to prevent this threat, including a liquidation of sources of the terrorist organizations financing (Foreign Policy Concept of the Russian Federation, 2016). Table 1. Ranking of Russia and Spain according to the Global Terrorism Index for 2012-2018 Country Year 2012 2014 2015 2016 2017 2018 Russian Federation 9 11 23 30 33 34 Spain 45 69 65 89 85 50 Total number of countries 158 162 162 163 163 163 Source: Global Terrorism Index reports (2012-2018), economicsandpeace.org Table 1. Ranking of Russia and Spain according to the Global Terrorism Index for 201 Ranking of Russia and Spain according to the Global Terrorism Index for 2012-2018 Source: Global Terrorism Index reports (2012-2018), economicsandpeace.org The Criminal Code of the Russian Federation specifies terrorism as “the carrying out of an explosion, arson or other actions intimidating the population, and creating the threat of human death, of infliction of significant property damage or the onset of other grave consequences, for the purpose of influencing the taking of a decision by authorities or international organizations, and also the threat of commission of the said actions for the same purposes” (Criminal Code of Russia, 1996). As for the Criminal Code of Spain (1995), it does not have an official interpretation of terrorism, but this term is often used within the context of criminal offence or threat. 4. Russia-Spain Cooperation to Counter-Terrorism: Challenges and Legal Approaches The National Security Strategy of Spain 2013 considers terrorism among one of the 12 threats to national security together with cyberattacks, armed conflicts and organized crime. It includes the following definition of terrorism: “A direct threat to the life and security of citizens; it aims to undermine our democratic institutions and jeopardises our vital and strategic interests, infrastructures, supplies and critical services. Preventing, impeding and defeating terrorism, whatever its origin, is a priority aim of the Government” (The National Security Strategy of Spain, 2013). As can be seen, Russia and Spain understand terrorism not just as a complex threat to security, but as a criminal offence as well. In 2006 in Madrid Russia and Spain formulated a joint vision of terrorism which was stated by V. Putin, the President of Russia, and J.L.R. Zapatero, former Prime Minister of Spain: “Terrorism is one of the most serious threats to international peace, security and stability, social and economic development of all countries; it aims to destroy coexistence, mutual understanding, and enrichment of civilizations, cultures, and nations. Any attempts to identify terrorism with any particular nation, culture, or religion, shall not be tolerated. Terrorism is a violation Bokeriya, S. Polít. Soc. (Madr.) 56(3) 2019: 737-756 749 of the fundamental human rights, most significantly, of the right to life and the right to live without fear” (Ministry of Foreign Affairs of the Russian Federation, 2006). The mutual understanding of terrorism as a threat and a destabilization in the world was adopted on a bilateral level, together with the special accent to the human rights and fundamental freedoms protection. g p The Russian national anti-terrorism legal system is more extensive, as compared to Spain. Russia ratified over 40 legal documents dedicated to counter-terrorism and extremism aspects (the President’s Decrees, Federal Laws, Government Regulations, and Codes). Spain adopted 11 documents (Committee of Experts on Terrorism, 2013). The Russian legislation in this area is characterized by a larger scope of legal documents referred to counter-terrorism and extremism measures, while in comparison, the Spanish laws are aimed at fighting local terrorism of a nationalist nature (Gómez Abeja, 2018). The Federal Law “Concerning the Combating of Terrorism” (2006) and Counter-Extremism Strategy of the Russian Federation up to 2025 (2014) are addressed to the analysis of “the extremist activity of nationalist, religious, ethnic, or other organizations and structures”, which destabilize domestic political and social environment of the state. p Art. 4. Russia-Spain Cooperation to Counter-Terrorism: Challenges and Legal Approaches 3 of the Federal Law determines terrorism as “the ideology of violence and practice of influence on decision-making by public authorities, local self- government bodies or international organizations related to frightening the population and/or other forms of unlawful violent actions” (Federal Law “Concerning the Combating of Terrorism”, 2006). Spain does not have a separate law regarding the combating of terrorism, but anti-terrorism issues, and penal measures for terrorist crime are listed in the Criminal Code of Spain, including sanctions as well as the terms and conditions of declaring any act as a terrorist attack (Criminal Code of Spain, 1995). Counter-terrorist pact signed in 2000 is the agreement between the leading Spanish parties to avoid using counter-terrorism methods for political privileges. Besides, Spain has the National Security Law presenting the national security in various aspects, and adopting it for government entities, civil society, etc. , y, Basis of the Russian-Spanish relations comprises over 50 interstate, intergovernmental, and interdepartmental agreements. A number of them can be accessed on the website of the Russian embassy in Spain. Since 1990s a number of the legal framework of cooperation with regard to mutual assistance in counter- terrorism, organized crime, and drug traffic efforts (Embassy of the Russian Federation in Madrid, 2017) have emerged. From 1994 to the present day 8 agreements with regard to counteraction of terrorism were implemented, and 2 of them (1996 Treaty on rendering legal assistance in criminal matters and also the 1999 Agreement between the Government of the Russian Federation and the Government of the Kingdom of Spain on cooperation with regard to informatization matters), while being of great importance with regard to the counter-terrorist agenda, are approved but are not working, as is stated on the website of the MFA of Russia (The Ministry of Foreign Affairs of the Russian Federation, 2017). Despite the prioritization of the threat on the part of these two countries in relation to domestic and foreign policy, considering the scope of the threat, few counter-terrorist agreements and contracts have been implemented. The foregoing does not correspond to the current interaction potential between the states. 750 Bokeriya, S. Polít. Soc. (Madr.) 56(3) 2019: 737-756 Agreement between the Government of the Russian Federation and the Government of the Kingdom of Spain for crime control (1999) marked a new collaboration stage between the countries. 4. Russia-Spain Cooperation to Counter-Terrorism: Challenges and Legal Approaches The purpose of this Agreement was development and acceleration of the efficiency of two-sided cooperation with regard to security. A relevant Road map was adopted in July 1999. It included mechanisms of mutual exchange of information, the cooperation with regard to prevention, investigation, and combating with organized criminal activity, and on- site training, including sharing experiences related to successful problem solving by the law enforcement agencies. y g The establishment of the Russian-Spanish interdepartmental anti-terrorism working group in 2004 serving as the platform for an open dialogue, drawing up of joint arrangements, and elaborating of bilateral initiatives, became a striking example of willingness to eradicate terrorism together. Cooperation with regard to counter-terrorism efforts intensified through latest meetings and conferences of the official circles: i.e. the meeting of the Russian-Spanish anti-terrorism working group in May 2015 in Madrid; in 2016 this meeting took place in Moscow under co-chairmanship of O.V. Syromolotov, Deputy Minister of Foreign Affairs of the Russian Federation, and Mr. E. Mora Benavente, Director-General for Foreign Policy and Security of Spain. y y p Russia and Spain support the principles of the UN anti-terrorism conventions, strategies and protocols, promote counter-terrorism efforts within the European Union. In particular, both countries welcomed ratification of the International Convention for the Suppression of Acts of Nuclear Terrorism (2005), the Council of Europe Convention on the Prevention of Terrorism (2005), and the Security Council resolutions 1566, which organized a working group for practical measures consideration to be applied to the involved in terrorist activity apart from Al-Qaeda and the Taliban, and the Security Council resolution 1624 criminalizing abetment to terrorist attacks. The cooperation of the two-sided Russian-Spanish has been drawn up by similarity of points of view with regard to the key anti-terrorist issues; both countries have certain differences in terms of the approaches to the formation of a relevant national legal system, which affected the bilateral legal base which is in need of improvement. Best domestic counter-terrorism practices may be used in bilateral cooperation. However, much is to be done by Russia and by Spain in order to achieve the level of counter-terrorism cooperation for further strategic partnership. It also seems advisable to activate exchanging of best practices as well as information with this regard. 5. Conclusion Within the context of the growing threat of terrorism influencing over the international security, three major tendencies may be emphasized: the UN counter- terrorism strategy fails to achieve optimum effect in fighting international terrorism; the international legal counter-terrorism framework is being functioning in the lack of a unified approach to the terminology of terrorism; the level of international cooperation between the countries doesn’t correspond the transnational character of terrorism; conflicts with regional and international dimensions in Libya, Afghanistan, Iraq, Syria have been going on for a long time. This is used by terrorist groups— not only those involved in internal conflicts, but also more and more foreign terrorist groups. The changeable context of the international security resulted in the idea of human protection from interrelated challenges of the globalized world. In this line, international control over terrorism is possible via using an “all-of- government”, “all-of-society” and “all-of-UN” approach. Government participation in counter-terrorism performs a key function in normalizing the relations within the system of regional, multinational, and international interaction in the formats of adopting agreements, developing road maps, concepts and strategies and realizing anti-terrorist projects. The events in Ukraine and the EU sanctions started in 2014 have a negative influence on bilateral relations between Russia and Spain, both from an economic point of view and in terms of cooperation in the sphere of countering terrorism. Despite the fact that many European countries, such as Italy, Greece, Austria, Hungary, Spain and others, are experiencing significant disruptions in their economies because of the response of Russian counter-sanctions, they will not be able to soften or at least partially mitigate them. In the light of freezing of political dialogue between Russia and the West, there is a real risk that the common distrust can have a negative result in combating global terrorism. Disunity, lack of coordination of actions becomes another threat to each country, as well as the whole world. These factors are beneficial for terrorists and their destructive plans. The policy of confrontation in relations with Russia, whose participation is necessary to solve many international problems, should be replaced by the logic of cooperation. p y g p Counter-strategy needs the same integrated system of reaction. 4. Russia-Spain Cooperation to Counter-Terrorism: Challenges and Legal Approaches g A comparative analysis of approaches, terrorist threats and radicalization in Russia and Spain leads to two key conclusions: 1) Despite the fact that Russia was more susceptible to terrorist attacks after the end of the Cold War, the EU pays more attention to the topic of de-radicalization, preventive and non-military measures to counter extremism and terrorism. 1) Despite the fact that Russia was more susceptible to terrorist attacks after the end of the Cold War, the EU pays more attention to the topic of de-radicalization, preventive and non-military measures to counter extremism and terrorism. 2) Despite the existing obstacles in the bilateral cooperation between Russia and Spain (sanctions, an atmosphere of mistrust, the problem of human rights violations and data collection), commonality in understanding the seriousness of the terrorist threat, as well as a comparable level and similar type of threat for the Bokeriya, S. Polít. Soc. (Madr.) 56(3) 2019: 737-756 751 two countries can become the basis and catalyst of the Russian-Spanish interaction on anti-terror. two countries can become the basis and catalyst of the Russian-Spanish interaction on anti-terror. 5. Conclusion The absence of a unified approach to terrorism terminology regarding international terrorism, terrorist attack and terrorism on an international level, problems with adopting national legal frameworks to the international legislation catalyzes the development of new, and more effective counter-terrorism formats and intensifies networking on local, regional, and international levels that might provide a chance to stay a step ahead of terrorist plans rather than to take post factum measures. p p Taking into account, that the core responsibility for countering terrorism rests with sovereign states, development of Russia-Spain strategic collaboration in Bokeriya, S. Polít. Soc. (Madr.) 56(3) 2019: 737-756 752 combating terrorism intensifies multipartite interaction in this area. However, despite of the accumulated experience in various spheres, the bilateral counter- terrorism legal framework does not correlate with the current cooperation potential between Russia and Spain, and requires further updating. p q p g As for future forecasts, first, the establishment of a bilateral dialogue between Russia and Spain, including a successful cooperation in the fight against terrorism, which will depend on solving the problem of sanctions and counter-sanctions. To make predictions on this issue is quite difficult, as some researchers believe that the EU at this stage faces a choice: to support Russia and thus go against US policy or continue to follow the policy of Washington (Emelina, 2016). Secondly, the effectiveness of Russian-Spanish cooperation in the field of counter-terrorism will most likely be determined by the interest of the two countries in exchanging best practices in this area. For example, Russia could learn from the experience of working with victims of terrorism, while Spain could benefit from Russia’s experience of living closely and interacting with a large indigenous Muslim population. 6. Bibliography Agreement between the Government of Russia and the Government of the Kingdom of Spain about Cooperation in Fighting with Crime (1999). Available at: http://docs.cntd.ru/document/901711861 [Last consulted: March 18, 2019]. Anikeeva, N.E. (2019): Russia-Spain: Dialogue with Acсents. ents. MGIMO Website. Available at: https://mgimo.ru/about/news/experts/rossiya-ispaniya-dialog-s-aktsentami/ [Last consulted: March 18, 2019] ] Aoláin, F.N. 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W2027649407.txt	https://downloads.hindawi.com/journals/sci/2012/709763.pdf	en		Amnion Epithelial Cells as a Candidate Therapy for Acute and Chronic Lung Injury	Stem cells international	2,012	cc-by	6,233	Hindawi Publishing Corporation Stem Cells International Volume 2012, Article ID 709763, 7 pages doi:10.1155/2012/709763 Review Article Amnion Epithelial Cells as a Candidate Therapy for Acute and Chronic Lung Injury Ryan J. Hodges, Rebecca Lim, Graham Jenkin, and Euan M. Wallace The Ritchie Centre, Monash Institute of Medical Research and Department of Obstetrics and Gynaecology, Southern Clinical School and Monash Medical Centre, Monash University, 246 Clayton Road, Clayton, VIC 3168, Australia Correspondence should be addressed to Euan M. Wallace, euan.wallace@monash.edu Received 19 December 2011; Revised 8 February 2012; Accepted 15 February 2012 Academic Editor: Markus Hengstschläger Copyright © 2012 Ryan J. Hodges 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. Acute and chronic lung injury represents a major and growing global burden of disease. For many of these lung diseases, the damage is irreparable, exhausting the host’s ability to regenerate new lung, and current therapies are simply supportive rather than restorative. Cell-based therapies oﬀer the promise of tissue regeneration for many organs. In this paper, we examine the potential application of amnion epithelial cells, derived from the term placenta, to lung regeneration. We discuss their unique properties of plasticity and immunomodulation, reviewing the experimental evidence that amnion epithelial cells can prevent and repair lung injury, oﬀering the potential to be applied to both neonatal, childhood, and adult lung disease. It is amazing to suggest that the placenta may oﬀer renewed life after birth as well as securing new life before. 1. Introduction Chronic lung diseases, in both children and adults, are leading causes of morbidity and mortality worldwide, estimated to account for about 10% of global mortality [1]. It has been estimated by the World Health Organization that, by 2030, chronic lung disease, mainly caused by tobacco smoking, occupational irritant exposure and pollution, will become the third most common cause of death worldwide [1]. However, mortality is just the tip of the iceberg. A recent economic analysis of the burden of chronic lung disease in Australia revealed that almost 1 in 5 adults aged 40 or older have chronic obstructive pulmonary disease (COPD) to some degree, with half of these individuals having advanced disease [2]. The estimated financial cost of COPD in Australia in 2008 was nearly $9 billion. Further, chronic lung disease does not only aﬀect adults. About 1 in 80 children aged under 10 suﬀer morbidity from COPD. In particular, over recent decades advances in perinatal care have greatly improved the survival chances of very preterm babies, principally through the reduction of acute respiratory distress syndrome (RDS) by antenatal corticosteroids and postnatal surfactant therapies [3, 4]. However, almost a third of these survivors develop chronic neonatal lung disease, so called bronchopulmonary dysplasia (BPD), a disease with the consequent long-term burdens of childhood respiratory dysfunction and neurodevelopmental delay [5]. Unfortunately, both neonatal BPD and adult COPD have an important feature in common. Neither have an eﬀective treatment. Accordingly, together, these childhood and adult chronic lung conditions represent a significant and growing burden of disease for which there is no targeted intervention that might restore lung function and thereby reduce morbidity and mortality. However, while the causes of childhood and adult COPD diﬀer, the fundamental lung injury is similar— chronic inflammation, fibrosis, and scarring [6–8]—and the clinical end result—loss of functional lung tissue— is identical. As such these chronic lung diseases may be amenable to regeneration, which may be aﬀorded by cellbased therapies. In this paper we review the recent advances in the application of placenta-derived cells as a potential therapy for human lung disease. Specifically, we will review the unique properties of amnion, the eﬀect of amnion cells on diﬀerent models of lung injury and explore the likely mechanisms of action of amnion cells in lung repair with a view to human clinical trials. 2 2. Unique Therapeutic Properties of Amnion Epithelial Cells At the first international workshop on placenta-derived stem cells, convened in Brescia, Italy in 2007, two key properties of placental cells that make them attractive for regenerative medicine were highlighted: plasticity and immunomodulation [9]. Human amnion epithelial cells (hAECs) are a subset of placental-derived stem cells that display both of these key features and, perhaps, possess advantages over the other populations of stem cell-like cells in the placental tissues. First, the amnion itself is derived from the embryonic epiblast prior to gastrulation. This is important because cells derived from the epiblast prior to gastrulation are thought to retain multipotent memory or plasticity, reflecting the capability of the epiblast itself to diﬀerentiate into the ectoderm, endoderm, and mesoderm of the definitive embryo. Thus, at least theoretically, amnion cells should be capable of diﬀerentiation down each primary lineage. Over the past 6 years or so we, and others, have carefully characterized human amnion epithelial cells (hAECs), defining cell types and asking whether they share any transcriptional factors with embryonic stem cells that might confer pluripotentiality. First, cells isolated from amniotic membranes by simple digest are essentially exclusively epithelial cells [10]. These human amnion epithelial cells (hAECs) do not express mesenchymal or haematopoietic cell markers and diﬀer from cells derived from amniotic fluid in early to midpregnancy [10–12]. This distinction between amniotic membrane-derived amnion epithelial cells, and amniotic fluid stem cells is that the former are a pure population of epithelial cells while the latter are a mixed cell population of mesenchymal, stromal, and epithelial cells. This diﬀerence is important to keep in mind when assessing possible therapeutic and regenerative medicine applications for each of these cell populations, as will be discussed later. However, while hAECs are all epithelial cells they are still a heterogeneous population of epithelial cells with diverse cell marker expression. Importantly, these cell lineage markers include early “stem cell” markers such as the POU domain, class 5, transcription factor, Nanog homeobox; SRY-2 box, the stage-specific embryonic antigen-4 (SSEA4) [10, 13]. For example, in one report 44% of hAECs expressed SSEA4, 5–15% of cells expressed Oct-3/4, and 5–15% expressed Nanong and/or Sox-2 [10]. Consistent with the expression of such early lineage markers, the diﬀerentiation repertoire of hAECs has been confirmed in vitro using various techniques (phenotypic, mRNA expression, immunocytochemical, and/or ultrastructural characteristics), demonstrating that hAECs derived from term placental membranes can be successfully diﬀerentiated into cardiomyocytic, myocytic, osteocytic, adipocytic (mesodermal), pancreatic, hepatic, lung (endodermal), neural, and astrocytic (neuroectodermal) cells [13, 14]. With regard to the lung, hAECs express thyroid transcription factor or Nkx 2.1 mRNA, one of the earliest lineage markers of the developing lung that is essential for branching lung morphogenesis and type II alveolar cell formation [14]. Stem Cells International However, while hAECs express many markers of early stem cells, they are not omnipotent like embryonic stem cells. Indeed, there are a number of key diﬀerences between hAECs and embryonic stem cells that suggest that hAECS may be more suitable for clinical application. For example, unlike embryonic stem cells (ESCs) and human induced pluripotent stem (IPS) cell lines [15, 16], hAECs do not form teratomas when injected into the testes of mice with severe combined immunodeficiency (SCID) [13, 17] and they maintain a normal karyotype and cell cycle distribution with telomere stability over prolonged in vitro passaging [10]. These observations suggest that the more limited pluripotency displayed by hAECs, compared with ESCs or IPS cells, will pose less risks for in vivo tumour formation after-transplantation than those other stem cells. Furthermore, hAECs express no, or very little, class IA and class II human leukocyte antigens (HLAs) [13, 18]. In fact, likely reflecting their functions during pregnancy, hAECs express the immunosuppressive human leukocyte antigen G (HLAG) that confers a degree of immune privilege by suppressing NK cells, inducing apoptosis of activated CD8+ T-cells and inhibiting CD4+ T cell proliferation [18, 19]. Such findings are consistent with promoting maternal tolerance of a fetal allograft (including its membranes) for the nine months of human pregnancy. Such a property might also suggest a low risk of tissue rejection when given therapeutically. So far this indeed appears true. Following xenotransplantation hAECs can survive for prolonged periods in immune competent monkeys, rabbits, guinea pigs, rats, and swine without immunorejection, albeit without confirmed in vivo diﬀerentiation [19–22]. In one study hAECs transplanted into neonatal swine and rats resulted in human microchimerism in various organs and tissues without immune clearance [19]. Furthermore, hAECs injected into healthy human volunteers did not elicit any clinical signs of acute rejection and recipients did not produce antibodies against HLA antigens [23]. However, while undiﬀerentiated hAECs do not express HLAs, apart from HLA-G, it would appear that as they are made to diﬀerentiate, at least in vitro, this immune privileged state may be lost. For example, recently we showed that, following diﬀerentiation into hepatic and pancreatic lineages, significant numbers of hAECs began to express Class IA, but not Class II HLA [13]. The clinical significance of this finding for future cell transplantation remains unclear but suggests that cells diﬀerentiated in vitro prior to transplantation may be less suitable for allogeneic use than primary undiﬀerentiated cells. We will revisit this theme later. 3. Endogenous Lung Stem Cells Before any discussion of stem-cell-mediated lung repair, it is useful to distinguish the roles and activities of exogenous stem cells, such as hAECs, from those of resident endogenous lung stem cells. While the very slow natural turnover of lung and bronchial epithelia and the multiple distinct anatomical zones of the lung have made the identification of lung stem cells diﬃcult [24–26], a number of diﬀerent resident lung stem or progenitor cells have been identified Stem Cells International [27, 28]. It is thought that each of these progenitor cell niches provides specific repair mechanisms for the diﬀerent parts of the respiratory tract and that diﬀerent injuries may trigger diﬀerential responses from the various progenitor cell populations [25, 27, 28]. Specifically, distinct progenitor cells have been identified in the proximal trachea, the bronchi, the bronchioli, the bronchiolar-alveolar junction, and the alveoli [24, 25, 28]. Of these various lung progenitor/stem cells, specialized nonciliated airway epithelial cells called Clara cells, or variant Clara cells, respond to airway injury by replenishing the ciliated epithelium, particularly in the bronchioli and at bronchiolar-alveolar junction, while alveolar type II (ATII) cells are thought to be the principle repair mechanism in the alveoli [25, 26, 28]. With regard to exogenous stem cells and lung repair, the endogenous “resident” lung progenitor/stem cells are likely to be important. As will be discussed later, while it was initially thought that exogenous stem cells aﬀected lung repair by integrating into the damaged epithelium and diﬀerentiating into lung cells, it is more likely that the principal mechanism whereby they eﬀect repair is via immunomodulation [29] and by supporting endogenous lung stem cell activity. Indeed, while the lung contains its own population of resident endogenous stem cells, it is thought that their regenerative eﬀorts become exhausted during severe injury, leading to both acute and chronic respiratory embarrassment. It is this feature that makes the lung a particularly receptive organ for exogenous cell therapy. 4. Amnion and Models of Lung Injury The first report of using amnion cells for repairing lung injury was by Carraro and his colleagues [30] who used amniotic fluid stem cells (hAFSCs) obtained from amniocentesis in midpregnancy. They assessed this mixed population of cells that included hAECs, other epithelial cells, and mesenchymal cells with regard to their reparative abilities in two diﬀerent murine models of lung injury. First, they demonstrated that hAFSCs could engraft into mouse embryonic lung explants in vitro and diﬀerentiate into a lung-type cell, as evidenced by the expression of thyroid transcription factor 1 (TTF1). Then, to assess the ability of hAFSCs to repair alveolar lung injury they administered the cells to mice following short-term hyperoxia. The hAFSCs migrated to the distal lung and expressed both TTF1 and the type II alveolar cell product surfactant protein C. Next, hAFSCs were administered to mice which had undergone naphthalene lung injury. Naphthalene targets Clara cells in the airways. As with the hyperoxia alveolar injury model, the hAFSCs traﬃcked to the sites of injury—this time in a bronchoalveolar junction and bronchial distribution rather than the alveoli—and expressed the Clara cell 10 kDa protein. This first report highlighted two key properties of cells derived from amniotic fluid: their ability to track to specific sites of injury and their plasticity to respond specifically to the nature of the lung insult itself, diﬀerentiating into the cell type that had been injured. Importantly, while the origin of these cells was from a mixed population isolated from 3 amniotic fluid, the cells had been sorted by c-kit positivity— a stem cell marker—and derived from clonal cultures to further select for stem cell-like behaviour. Therefore, it was not surprising that the cells displayed pluripotency. However, in neither of the two injury models was there evidence of amelioration of injury. This suggests that integration and diﬀerentiation in vivo are not suﬃcient for exogenous stem cells to eﬀect repair. Furthermore, translating this cell therapy into clinical practice may have some limitations. The cells were derived from amniotic fluid by amniocentesis, selected by c-kit expression and then expanded and purified through clonal isolation. Such a source is not likely to be a ready source of suﬃcient cells for widespread application because amniocentesis is an invasive procedure that carries a risk of miscarriage. It is unlikely that women will be prepared to expose their pregnancy to such risks for the benefits of others. It is also unlikely that suﬃcient numbers of cells will be able to be derived for widespread application, although expansion would be feasible. Utilising placentae from term births is one strategy to circumvent these problems entirely. Cargnoni and her coworkers [31] transplanted a mixed population of fetal membrane-derived cells from the amnion and chorion from both allogeneic and xenogeneic (50% human mesenchymal cells, 50% hAECs) sources, to a murine bleomycin model of adult idiopathic pulmonary fibrosis (IPF). Importantly, unlike the amniotic fluid derived cells used by Carraro and colleagues, the cells used by Cargnoni were primary cells that had not been selected for either c-kit expression or clonal activity. Bleomycin produces many of the histological hallmarks of IPF such as intra-alveolar buds, mural incorporation of collagen, and obliteration of the alveolar space [32]. Both allogeneic and xenogeneic populations of mixed primary cells, administered either systemically or intraperitoneally, mitigated lung fibrosis to a similar extent and markedly reduced neutrophil infiltration, a key prognostic determinant of IPF. Persistence of both cell populations was detected in the lung 14 days after administration confirming engraftment, albeit without confirmed diﬀerentiation into a lung phenotype. Indeed, it is unclear if engraftment is even essential for their therapeutic eﬀect. The same group had earlier demonstrated microchimerism in the lung at 90 days only by PCR after intraperitoneal injection of a similar xenogeneic population of cells. That finding suggests that the significance of the engraftment seen in the second study was likely to be modest [19, 31]. Extending these early reports, Moodley and colleagues [14] also used the bleomycin model of lung injury in SCID mice with the aim of examining diﬀerentiation of the cells into lung phenotypes in more detail. In this study a pure population of primary, unselected hAECs derived from term placental membranes were used. First, primary undiﬀerentiated hAECs were cultured in small airway growth media (SAGM), known to induce diﬀerentiation of umbilical mesenchymal cells and embryonic stem cells into type II pneumocytes [33, 34], to explore whether hAECs could be directed down an alveolar epithelial phenotype lineage. After prolonged culture in SAGM, hAECs appeared to 4 partially diﬀerentiate into lung cells, producing surfactant proteins A, B, C, D and displaying ultrastructural evidence of lamellar bodies, an organelle of type II pneumocytes. Further, these cells responded to a glucocorticoid trigger and were capable of secreting surfactant D [14]. None of these features were present in freshly isolated hAECs. Primary, undiﬀerentiated hAECs (lacking surfactant proteins) were also injected intravenously into bleomycin treated SCID mice and were shown to engraft and produce all surfactant proteins [14]. This suggested an ability of primary hAECs to diﬀerentiate into lung cells in vivo—similar to the observations made by Carraro of selected and purified amniotic derived cells [30]. However, unlike the amniotic fluid derived cells, hAEC administration significantly reduced bleomycin-induced lung fibrosis and inflammation [14]. Specifically, levels of pro-inflammatory cytokines were reduced (monocyte chemo-attractant protein-1, tumor necrosis factor-alpha, IL-1 and IL-6) and anti-inflammatory cytokines were increased (IL-10 and macrophage migration inhibitory factor). Expression of the profibrotic cytokine transforming growth factor-beta was also reduced by hAECs administration. In keeping with these cytokine changes, lung collagen content was reduced, reported to be a consequence of increased action of matrix metalloproteinase2 and down-regulation of the tissue inhibitors of matrix metalloproteinase-1 and 2 consistent with lung repair. hAECs also seemed to reduce established fibrosis in one small group of mice, with a reduced collagen content confirmed with the delayed administration of hAECs two weeks after the bleomycin insult. This comprehensive study demonstrated that a pure population of primary hAECs derived from term placenta after completion of a pregnancy had the ability to prevent and repair acute lung injury induced by bleomycin. The authors suggested that the cells exerted these aﬀects via modulation of the host response to injury and by in vivo diﬀerentiation [14]. However, while subsequent studies have confirmed the injury prevention abilities of hAECs, they have cast some doubt on the mechanisms by which hAECs eﬀect this. Using the bleomycin model of lung injury, Murphy et al. [29] advanced the field by the administration of a pure population of undiﬀerentiated hAECs to immune competent, as opposed to immune compromised, mice. In this study, intraperitoneal administration of hAECs 24 hrs after bleomycin administration decreased lung fibrosis, evidenced by reduced collagen deposition and alpha-smooth muscle actin, and decreased lung inflammation and the expression of proinflammatory cytokines [29]. Moving one step further, this was to the first study to demonstrate that the mitigation of structural lung injury by hAECs was associated with a partial restoration of physiological lung function, as assessed by whole body plethysmography. However, in contrast to the previous studies—all performed in immune compromised mice—Murphy and his colleagues were unable to demonstrate any engraftment of hAECs in the lung [29]. Based on this observation, some doubt was cast on the likely mechanism(s) by which hAECs may work. Stem Cells International 5. Mechanisms of Action of hAECs: Engraftment versus Immunomodulation It is fair to say that, at present, there remains some uncertainty about the primary mechanism(s) by which hAECs aﬀect lung injury prevention/repair. Specifically, whether in vivo engraftment and diﬀerentiation are necessary or whether modulation of the host response to injury that then reduces inflammation and fibrosis, either directly or indirectly, is key. At this stage it would appear that the latter is the more likely. Our recent report [29] clearly demonstrated that hAECs can exert a reparative eﬀect without the need for engraftment or diﬀerentiation and the work of Carraro and his colleagues showed that engraftment and diﬀerentiation per se was not suﬃcient for injury prevention/repair [30]. The apparent inconsistencies between studies regarding whether in vivo integration and diﬀerentiation of hAECS following injury actually occurs may be explained by the methods used to identify hAECs in vivo. Murphy et al. [29] chose fluorescence-activated cell sorting (FACS) with gating applied to exclude dead cells to detect only live resident human cells. Previous studies relied on PCR or in situ hybridization for human DNA or immunohistochemistry. These latter methods are all unable to discern living from dead cells [14, 30, 31] and so it is possible that those studies were simply reporting dead cells. Of course, this does not explain the in vivo identification of surfactant proteinexpressing hAECs [14]. Nonetheless, we have suggested that the primary mechanism of injury repair in their study was likely to be paracrine signaling to the surrounding tissues to reduce proinflammatory and profibrotic mediators [29]. This is consistent with previous reports of a beneficial eﬀect of amnion where cellular diﬀerentiation has not been confirmed, such as in brain ischaemia, Parkinson’s disease, spinal cord injury, myocardial ischaemia, critical limb ischemia, burns and skin wounds [20, 35–39]. The exact identity of antiinflammatory and anti-fibrotic factors that might be released by amnion cells remains to be elucidated. However, the ophthalmology literature has described for some time the beneficial eﬀect of amnion for corneal ulcers mediated through a reduction in HLA Class II antigen presenting cells at the site of injury, reduction in apoptosis and inflammation [40]. This literature suggests hAECs are able to inhibit the chemotactic migration of neutrophils and macrophages to the site of injury, possibly via MIF and suppression of IL-1α, IL-1β, and proteinase [41], similar to that shown by Moodley et al. [14]. Indeed, hAEC-induced suppression of macrophage and neutrophil migration into the injured lung has been a consistent finding [29, 31]. This is supported by the observation that hAECs decrease macrophage migration in vitro [42]. That hAECs modulate macrophage and/or neutrophil migration is likely to be important in the context of lung injury because both macrophages [43] and neutrophils [44] play important roles in mediating such injury. In this regard, very recently we showed that hAECs were unable to mitigate bleomycininduced lung injury in SP-C knock-out mice [42]. This strain of mouse is known to have deficient macrophage function Stem Cells International with macrophages unable to switch from an M1 (profibrotic) phenotype to an M2 (reparative) phenotype. We suggested that this observation was consistent with hAECs exerting their reparative eﬀects via macrophages rather than directly [42]. Of course, hAECs may operate through other mechanisms too. For example, they express the anti-inflammatory IL-1 receptor antagonist, IL-10, collagen XVIII, thrombospondin-1 and all four tissue inhibitors of metalloproteinase (TIMPs) [45]. Apoptosis of leucocytes has been reported and hAECs express apoptosis-inducing genes Fas L, TNF, and TRAIL [41, 46]. Furthermore, there is evidence to support an anti-angiogenenic eﬀect through release of endostatin, TSP-1 and TIMPs and the antibacterial protein lactoferrin [47]. The relative contributions of these pathways to amnion cell-mediated tissue repair will clearly require a considerable amount of working through. In relation to fibrosis, human corneal and limbal fibroblasts grown on the matrix surface of amniotic membranes displayed marked down regulation of TGFβ-signalling system with decreased expression of TGFβ-1, β-2 and β-3 isoforms and reduced expression of TGF-Receptor II preventing fibroblast activation into myofibroblasts [48]. Furthermore, amniotic membrane is also capable of reversing already diﬀerentiated myofibroblasts back into a fibroblast phenotype, which may be particularly useful for ameliorating more established disease [49]. Since TGF-β signalling plays a central role in pulmonary fibrosis [50] the ability of hAECs to decrease TGF-β signalling and prevent fibroblast activation is likely to be an important eﬀector mechanism in their reparative properties. 6. Towards Clinical Trials Before hAECs can be eﬀectively translated into a future cellular therapy for lung injury a number of questions need resolving. It would be useful if the identity of the antiinflammatory and antifibrotic signaling pathways at play are characterized. It may be possible that these pathways can be manipulated pharmacologically rather than necessitate cell delivery. In this way, the reparative abilities of hAECs would have been used to develop new drug-based therapies by revealing the key pathways that need targeting. However, such an endeavour will be considerable and more immediate therapy may be aﬀorded by simply administering cells. If this is the case then it will also be necessary to determine the optimum cell type for transplantation, whether that be undiﬀerentiated hAECs, or hAEC-derived partially diﬀerentiated lung progenitor cells, or even a mixed cell population. As detailed earlier, the hAECs used in the studies to date have been unselected, primary cells—a heterogeneous population of epithelial cells. It is possible that only a subset of these cells are reparative and that by identifying and purifying those cells more eﬀective regenerative therapies can be developed. Of course, it is also possible that it is the heterogeneous nature of the population that is a key attribute of the therapy, providing diverse cells to undertake diverse roles at diﬀerent stages of tissue injuryrepair-resolution. If this is so then purified subpopulations 5 of cells may prove to be less, rather than more, eﬀective. It is also unknown whether both undiﬀerentiated and partially diﬀerentiated cells equally eﬀect immunomodulation. This would be important to define before embarking upon clinical trials. From a pragmatic perspective, regulatory authorities are more likely to approve primary, unmanipulated cells than cells that have undergone extensive purification, selection, and diﬀerentiation. However, if diﬀerentiated cells are more eﬀective then these should form the basis of future therapies. With regard to regulatory approval and cell handling, it will also be necessary for future studies to adhere to good manufacturing practice (GMP) processes suitable for clinical use, as has already been described [10, 51], to readily ensure standardization and clinical applicability moving forward. Should hAECs prove useful clinically, such standardization will be a foundation of cell banks. We believe that while autologous use of hAECs may oﬀer a safe first step application, for example in preterm neonates with bronchopulomonary dysplasia [26], ultimately widespread use of hAECs will require the development of biobanks of high quality cells for allogeneic application, most likely in an alpha clinic setting [52]. The clinics and cell banks will be necessary simply because the majority of patients do not have their own amnion cells in storage. Finally, and possibly most importantly, all of the studies performed to date have administered hAECs very early during the injurious process in the lung. There has been no comprehensive assessment of whether hAECs are able to repair established and long-standing lung injury. This is clearly critical because the most common clinical application of cell therapy for lung disease will be to those individuals with chronic, established and extensive lung injury. This is where the current clinical burden lies and where current therapies desperately fail. 7. Conclusion There is no question that acute and chronic lung disease require novel therapies. Preclinical studies have shown that amnion cells are able to reduce fibrosis and inflammation, and thereby improve lung function. However, several questions remain unanswered, including whether how these cells work, whether there are subpopulations of cells that are most eﬀective, and whether amnion cells are able to repair established disease. We are indeed hopeful that the immunomodulatory concert that is present at the maternalfetal interface during pregnancy may soon extend long after birth to oﬀer new therapies for suﬀerers of chronic lung disease. 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https://openalex.org/W2061148349	http://eprints.gla.ac.uk/25747/1/25747.pdf	English	null	Bayesian salamanders: analysing the demography of an underground population of the European plethodontid Speleomantes strinatii with state-space modelling	BMC ecology	2,010	cc-by	7,559	RESEARCH ARTICLE RESEARCH ARTICLE Open Access © 2010 Lindström 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. * Correspondence: j.lindstrom@bio.gla.ac.uk 1Boyd Orr Centre for Population and Ecosystem Health Division of Ecology and Evolutionary Biology Faculty of Biomedical and Life Sciences University of Glasgow Glasgow G12 8QQ, UK Bayesian salamanders: analysing the demography of an underground population of the European plethodontid Speleomantes strinatii with state- space modelling Jan Lindström1, Richard Reeve1, Sebastiano Salvidio2 Jan Lindström1, Richard Reeve1, Sebastiano Salvidio2 Abstract Background: It has been suggested that Plethodontid salamanders are excellent candidates for indicating ecosystem health. However, detailed, long-term data sets of their populations are rare, limiting our understanding of the demographic processes underlying their population fluctuations. Here we present a demographic analysis based on a 1996 - 2008 data set on an underground population of Speleomantes strinatii (Aellen) in NW Italy. We utilised a Bayesian state-space approach allowing us to parameterise a stage-structured Lefkovitch model. We used all the available population data from annual temporary removal experiments to provide us with the baseline data on the numbers of juveniles, subadults and adult males and females present at any given time. Results: Sampling the posterior chains of the converged state-space model gives us the likelihood distributions of the state-specific demographic rates and the associated uncertainty of these estimates. Analysing the resulting parameterised Lefkovitch matrices shows that the population growth is very close to 1, and that at population equilibrium we expect half of the individuals present to be adults of reproductive age which is what we also observe in the data. Elasticity analysis shows that adult survival is the key determinant for population growth. Conclusion: This analysis demonstrates how an understanding of population demography can be gained from structured population data even in a case where following marked individuals over their whole lifespan is not practical. Lindstrom, J. and Reeve, R. and Salvidio, S. (2010) Bayesian salamanders: analysing the demography of an underground population of the European plethodontid Speleomantes strinatii with state-space modelling. BMC Ecology, 10 (4). Lindstrom, J. and Reeve, R. and Salvidio, S. (2010) Bayesian salamanders: analysing the demography of an underground population of the European plethodontid Speleomantes strinatii with state-space modelling. BMC Ecology, 10 (4). http://eprints.gla.ac.uk/25747/ Deposited on: 16 April 2010 http://eprints.gla.ac.uk/25747/ Deposited on: 16 April 2010 Enlighten – Research publications by members of the University of Glasgow http://eprints.gla.ac.uk Lindström et al. BMC Ecology 2010, 10:4 http://www.biomedcentral.com/1472-6785/10/4 Background Two examples of different possible parameteri- sations of the Lefkovitch matrix are: It has been suggested that Plethodontid salamanders, which comprise about 70% of the living urodelan spe- cies, are a suitable group to use as an indicator of eco- system health [18,19]. However, long-term studies on them are scarce [20] and demographic information for them is mainly based on life table and time series ana- lyses of few terrestrial species from the USA and Europe [for a recent review, see [21]]. In general, these studies suggest that limiting factors, such as moisture, food and retreat or nesting sites may contribute to density-depen- dent population regulation [21]. Time series analysis on a population of the NW Italian Speleomantes strinatii showed that the population trajectory is stable and that the population was probably fluctuating near the envir- onmental carrying capacity [22]. M M i j             0 10 0 0 47 0 50 0 43 0 0 0 09 0 93 0 31 0 0 30 0 26 0 . . . . . . . . . .556 0 0 0 27 0 90 . . ,           (1) (1) corresponding to population growth rates, l, of 0.97 and 0.98 respectively. The medians and 95% confidence intervals for the expected stable age-structure are 24% (20%,28%), 27% (22%,31%), and 49% (44%,54%) for juve- niles, sub-adults and adults, respectively, reproductive values are 1.18 (0.93, 2.44) and 1.51 (0.92, 5.56) for sub- adults and adults relative to juveniles (whose reproduc- tive value is scaled to 1), and the matrix elasticities (medians and 95% confidence intervals) are As most of the long-term population data on salaman- ders are from a small number of species, mainly North American Plethodon and Desmognathus species [e.g. [23,24]], and from a single species of the European genus Speleomantes [25,26], our understanding of the population regulation and demography of terrestrial sal- amanders is rather limited. Here we present an analysis based on a long-term data set on an underground popu- lation of Speleomantes strinatii using state-space model- ling. This approach allows us to take into account both the inherent stochasticity in the demographic processes, and the uncertainty in the parameter estimates while using all of the available data to inform the model fit [27]. Background of a larger ecosystem [4,5]. Biological properties that make a species or a taxonomic group attractive for use as indicators of ecosystem health are, for instance, clear taxonomy, existing information on basic biology, some measurable correlations to ecosystem health, limited mobility, practicality of sampling and relatively low variability of population fluctuations [5]. The human population relies on ecosystem services for its maintenance and well-being, and these can only be produced by functioning ecosystems [e.g. [1,2]]. It has been argued that conserving nature and thereby retain- ing ecosystem functions is often vastly more profitable in economic terms than converting it, for instance, to forestry or agriculture [3]. Effective ecosystem conserva- tion requires noticing changes both initially before active management has taken place and later to assess whether an employed conservation strategy is adequate. It is not realistic to have monitoring programmes for everything so indicator species are often used to gauge the health Once an indicator species has been identified, it is then necessary to know whether its population is stable, increasing or decreasing [6-8]. Such assessments are often based on statistical analyses of long-term popula- tion trends [9-11]. However, a much more thorough understanding of the situation can be gained by a more detailed demographic analysis whenever the extant data allow, especially when relatively small populations are considered [12-14]. This is because they are more sensi- titive to demographic and environmental stochasticity Lindström et al. BMC Ecology 2010, 10:4 http://www.biomedcentral.com/1472-6785/10/4 Page 2 of 9 Page 2 of 9 results indicate that the abundance estimates were reli- able and should be considered with relatively high confi- dence as CP ≥0.40 provide “...really good results in the typical removal study, having N of a few hundred and t = 3 to 6” [32:108, where t is the number of removal occasions]. than larger populations and it is therefore important to understand how changes in population structure trans- late to changes in population growth rate [8]. Individual differences, such as age, sex, and condition, can greatly affect their contribution to population growth and con- sequently the same population size can lead to different population trajectories [15,16]. Understanding the evolu- tionary selection pressures operating in a species also requires understanding the demography [17]. The demographic parameter estimates are illustrated in Fig. 1. Background We explicitly model the underlying stochastic pro- cess, using Markov chain Monte Carlo (MCMC) simula- tion in a Bayesian framework [28-30]. 0 036 0 0 136 0 136 0 108 0 . . . . 0.002,0.104 0.052,0.198 0.052,0.19     8 0.029,0.217 0.052,0.198 0.278,0.760             0 0 136 0 420 . .                 [8]. These results indicate a stable population as the l value was very close to 1 (median and 95% confidence interval: 0.95, 0.91, 0.99). At population equilibrium, we therefore expect half of the individuals among these three stage-classes to be reproductive adults. This agrees with the observed population structure, where the over- all mean proportion of the modelled stage-classes (i.e. juveniles, subadults and adults) was 20%, 27% and 53%, respectively. The adult stage appears very important to population growth, having both the highest reproductive value (1.51) and the highest elasticity of all the matrix entries (0.42 for adult survival). Results Abundance estimates were obtained for each age class and sex group in all years (N = 13). Mean capture prob- abilities (CP) were relatively high ranging from 0.60 to 0.72, and there were no significant differences among groups (repeated measures ANOVA, F = 1.37, DF = 4, P = 0.335) or years (repeated measures ANOVA, F = 0.97, DF = 12, P = 0.492), as noted by Salvidio [31]. There was no relationship between mean snout vent length (SVL) and CP (Spearman’s r = 0.10, n = 5, P = 0.87), but it may be noteworthy that the smallest age group (i. e. first year juveniles) had the lowest CP. Overall these Fig. 1 shows the prior and posterior distributions of all the estimated population parameters, and Fig. 2A shows the observed female population sizes, and the 95% confidence intervals on the population sizes when the population renewal process was simulated using Lindström et al. BMC Ecology 2010, 10:4 http://www.biomedcentral.com/1472-6785/10/4 Lindström et al. BMC Ecology 2010, 10:4 Page 3 of 9 0 0.2 0.4 0.6 0.8 1 1.2 0 1 2 3 4 5 0 0.2 0.4 0.6 0.8 1 1.2 0 1 2 3 4 5 0 0.2 0.4 0.6 0.8 1 1.2 0 1 2 3 4 5 0 0.2 0.4 0.6 0.8 1 1.2 0 1 2 3 4 5 0 0.2 0.4 0.6 0.8 1 1.2 0 1 2 3 4 5 0 0.2 0.4 0.6 0.8 1 1.2 0 1 2 3 4 5 0 0.2 0.4 0.6 0.8 1 1.2 0 1 2 3 4 5 Density Parameter estimate (A) j1 (B) j2 (C) p1 (D) s (E) p2 (F) a (G) f 0 0.2 0.4 0.6 0.8 1 1.2 0 1 2 3 4 5 (H) pobs Figure 1 Probability densities of the last 1,000 iterations of the WinBUGS estimation for the estimated demographic parameters. (see Fig. 3 and Equation 2): j1 (A), j2 (B), p1 (C), s (D), p2 (E), a (F) and f (G). The medians, 2.5 and 97.5 percentiles are: j1: 0.45, 0.29, 0.61; j2: 0.20, 0.02, 0.44; p1: 0.60, 0.27, 0.89; s: 0.42, 0.17, 0.69; p2: 0.42, 0.14, 0.69; a: 0.72, 0.57, 0.90; f: 0.82, 0.68, 0.98, and pobs: 0.40, 0.29, 0.55. The priors are indicated by dotted lines. Results 0 0.2 0.4 0.6 0.8 1 1.2 0 1 2 3 4 5 0 0.2 0.4 0.6 0.8 1 1.2 0 1 2 3 4 5 0 0.2 0.4 0.6 0.8 1 1.2 0 1 2 3 4 5 0 0.2 0.4 0.6 0.8 1 1.2 0 1 2 3 4 5 Density Paramet (A) j1 (C) p1 (E) p2 (G) f Figure 1 Probability densities of the last 1,000 iterations of the WinB Fig. 3 and Equation 2): j1 (A), j2 (B), p1 (C), s (D), p2 (E), a (F) and f (G). The m 0.44; p1: 0.60, 0.27, 0.89; s: 0.42, 0.17, 0.69; p2: 0.42, 0.14, 0.69; a: 0.72, 0.57, 0.9 by dotted lines. Density Parameter estimate Figure 1 Probability densities of the last 1,000 iterations of the WinBUGS estimation for the estimated demographic parameters. (see Fig. 3 and Equation 2): j1 (A), j2 (B), p1 (C), s (D), p2 (E), a (F) and f (G). The medians, 2.5 and 97.5 percentiles are: j1: 0.45, 0.29, 0.61; j2: 0.20, 0.02, 0.44; p1: 0.60, 0.27, 0.89; s: 0.42, 0.17, 0.69; p2: 0.42, 0.14, 0.69; a: 0.72, 0.57, 0.90; f: 0.82, 0.68, 0.98, and pobs: 0.40, 0.29, 0.55. The priors are indicated by dotted lines. Figure 1 Probability densities of the last 1,000 iterations of the WinBUGS estimation for the estimated demographic parameters. (see Fig. 3 and Equation 2): j1 (A), j2 (B), p1 (C), s (D), p2 (E), a (F) and f (G). The medians, 2.5 and 97.5 percentiles are: j1: 0.45, 0.29, 0.61; j2: 0.20, 0.02, 0.44; p1: 0.60, 0.27, 0.89; s: 0.42, 0.17, 0.69; p2: 0.42, 0.14, 0.69; a: 0.72, 0.57, 0.90; f: 0.82, 0.68, 0.98, and pobs: 0.40, 0.29, 0.55. The priors are indicated by dotted lines. Page 4 of 9 Lindström et al. BMC Ecology 2010, 10:4 http://www.biomedcentral.com/1472-6785/10/4 the different age and sex groups were obtained calcu- lating specific capture probabilities, that, in the study population, were relatively high. Thus these basic demographic data can be considered reliable [32,33]. parameter estimates drawn from those posteriors (Equations 3-5). The resulting distribution of popula- tion growth rates is presented in Fig. 2B. Age-specific survival and fertility calculated as the expected values for a newborn individual are shown in Fig. 2C and 2D, and the age-within-stage distribution is shown in Fig. 2E. Results We found that the population growth rate estimate based on the parameterised Lefkovitch matrix model is very close to 1 giving us confidence that the further cal- culations based on this matrix assuming stable popula- tion are reliable [8]. This was further corroborated by the good match between the predicted and observed population stage-structure. The model also predicted the observed population reasonably well (Fig. 2B). We can therefore be reasonably confident that the other measures derived from the parameterised Lefkovitch Conclusion The modelling approach presented here is potentially very valuable in situations where following marked individuals over their whole lifetime is not feasible. For instance, the method introduced by Ricklefs [[40]; for use in Plethodon- tids, see [41])] calculate survival on the assumption that population growth rate is 1, an assumption we do not have to make. It was also recently shown [42] that state- space modelling approach produces results in close agree- ment with those based on mark-recapture techniques, highlighting further the potential of this approach. In terms of population growth, adult survival emerges as the most important determinant of population growth rate as indicated by the elasticity analysis. This finding is similar to an analysis on Salamandra sala- mandra (L) by Schmidt et al. [34]. This demographic feature may be common in salamander populations that possess high adult survival and lengthy reproduction, such as Salamandra and many large and medium-sized plethodontid species. In the case of terrestrial salaman- ders, such as Speleomantes, there is a considerable over- lap in habitat and resource use by juveniles and adults, in contrast with the biphasic species that have aquatic larvae. This may enhance intraspecific density-depen- dent regulation in the terrestrial environment as sug- gested by Bruce [21]. However, the notion of the importance of adult survival to population growth based on this analysis has to be treated somewhat cautiously as the technique of basic elasticity analysis has been cri- ticised for ignoring density dependence, stochasticity and correlations between the transition matrix entries [35,36]. Calculating the integrated elasticities [37,38] would be better than the more simplistic figures pre- sented here, but we do not have annual estimates for all the demographic parameters in order to calculate corre- lations between the matrix entries and their variances as our approach needs all the data for estimating the para- meters. In our case, the correlation estimates between matrix entries are based on different possible parameter- isations of the demography (Table 1), not due to observed annual variation in those parameters. So, for instance the strong negative correlation between adult survival, a, and the transition probability from subadults to adults, p2 is probably more of an identifiability pro- blem of the model [e.g. Discussion We would like to emphasise, however, that despite these shortcomings, we have used all the avail- able data efficiently and parameterised a full demo- graphic model for this species for the first time, simultaneously addressing the parameter uncertainty explicitly, and have therefore improved the current understanding of Speleomantes demography significantly. model (age-specific survival and fertility and the age- within-stage distribution) are also reliable. As this sub-population is part of a relatively dense population inhabiting a larger area, there is no reason to rule out immigration and emigration between years but without data on this we simply have to combine these processes with the survival and fecundity parameters. Better data on immigration and emigration would help to further narrow posterior distributions of all the para- meter estimates. We also assumed 50:50 sex-ratio for births, and that the female fecundity is not limited by availability of mates. This is likely to be the case, as the adult population sex ratio (males/(males + females)) stu- died over 12 years was slightly male biased, being on average 0.57 (bootstrap 95% CI 0.53-0.65). This is prob- ably due to the females entering the reproductive popu- lation one year later than males [25]. Discussion Here we present the basic population demography of a population of the European plethodontid Speleomantes strinatii using a Bayesian state-space model built from long-term population data. Although not obtained through individual marking, abundance estimates of Figure 2 Summary of the fitted demographic model. (A) Observed female population size (open dots and solid line) and 95% confidence limit from the simulated population process (dotted line). (B) Frequency histogram of 1,000 rounds of the simulated population growth rate (calculated for each time step, i.e. 12 values for each simulation round). (C) Age-spefic survivorship and (D) fertility (medians denoted with solid line and filled dots, 95% confidence limit shown by dotted line) calculated from the transition matrix parameterised by sampling 1,000 combinations of parameter values from the last 5,000 posterior chains. (E) Age-within-stage distributions (the three stages are marked with small, medium and large dots for J2, S and A, respectively). These are scaled to proportions of individuals expected to be of a given age in each stage. Figure 2 Summary of the fitted demographic model. (A) Observed female population size (open dots and solid line) and 95% confidence limit from the simulated population process (dotted line). (B) Frequency histogram of 1,000 rounds of the simulated population growth rate (calculated for each time step, i.e. 12 values for each simulation round). (C) Age-spefic survivorship and (D) fertility (medians denoted with solid line and filled dots, 95% confidence limit shown by dotted line) calculated from the transition matrix parameterised by sampling 1,000 combinations of parameter values from the last 5,000 posterior chains. (E) Age-within-stage distributions (the three stages are marked with small, medium and large dots for J2, S and A, respectively). These are scaled to proportions of individuals expected to be of a given age in each stage. Lindström et al. BMC Ecology 2010, 10:4 http://www.biomedcentral.com/1472-6785/10/4 Page 5 of 9 Page 5 of 9 Page 5 of 9 Page 5 of 9 Addressing this directly would require mark-recapture data where the individuals are followed over their whole lifespan. Unfortunately, this is not easily achieved with this species; newborns are less than 25 mm long includ- ing tail creating a major obstacle for marking with, for instance, elastomers (VIE) or pit tags which are suitable for adults. Conclusion [39]] than reflecting the underly- ing biology of the species; it is possible to have a very similar fit to the data with a model with a relatively high a and low p2 as it is the other way round. Parameter estimation We modelled the female population using a standard Lefkovitch model for population growth with birth-pulse dynamics and pre-breeding census [8]. The salamander life cycle (Fig. 3) corresponds to transition matrix M: j j f p s p a p p f p p p j j j s s 2 1 1 2 1 1 2 2 0 0 0 1 0 1 0 0 2 1 2                      pa           (2) During July, from 1996 to 2008, the population abun- dance was estimated by a standardised temporary removal experiment [32], with three removal samples obtained every other day (i.e. the total sampling period was 96 hours). During sampling the population was con- sidered demographically closed, because dispersion from and towards the underground cavity was prevented by high temperatures and dry weather conditions, typical of summer climate in the region. (2) where the parameters represent transitions between the different stages J2 (juvenile), S (subadults) and A (reproductive adults), from one year to the next (Fig. 3). Note that as we had data available for the annual num- ber of juveniles born in the year of the census, we uti- lised this information to introduce a transitional stage J1 and wrote the fecundity contribution of the adults as j1f, where f is the expected number of female offspring pro- duced by a female per year and j1 is the probability that this newly produced offspring survives until the age of one year, and therefore completes the transition from newly born to juvenile, J2. The parameters p1, p2, j1, j2, s and a were estimated in such a way that each year adults first reproduce (at rate f), then all individuals sur- vive or die (with stage-dependent survival probabilities Salamanders caught on the cavity walls were measured from the snout to the posterior end of the cloaca to the nearest mm (snout-vent length, SVL) and kept in venti- lated plastic boxes inside the cavity. Sex was determined only in adults, as mature males possess a conspicuous mental gland that is absent in females and immatures of both sexes. Study species Speleomantes strinatii is a medium-sized (total length < 115 mm) plethodontid salamander endemic to S France and NW Italy. It is found from the sea level up to about 2000 m a.s.l. on humid rocky outcrops, in the forest talus and in caves [43]. The aquatic larval stage is lack- ing and, during winter, females lay about 10 large terres- trial eggs that are attended for several months until hatching [43]. There are no field data on the proportion of successfully hatching embryos, and the only available data are from captive bred individuals. According to Durand [44], females lay 6-14 eggs but only half of them develop completely as females eliminate unferti- lized or infected eggs from the clutch. In the case of a video surveilled female observed during the entire brooding period, only two out of nine attended eggs produced viable offsprings (Oneto et al. unpublished data). In this species, recruitment is seasonal and three immature body size groups (i.e. newborns, yearlings and subadults) may be recognised as separate age classes [45]. Males become sexually active at a snout-vent length (SVL) of about 50 mm when a mental gland, lacking in females and juveniles, becomes evident. A previous study based on dissections [25], demonstrated that females begin yolking at a SVL of 58 mm, and are probably reproductive at an older age than males. Lindström et al. BMC Ecology 2010, 10:4 http://www.biomedcentral.com/1472-6785/10/4 Page 6 of 9 Table 1 Correlation coefficients between the parameter estimates in the posterior chains. Parameter j1 j2 p1 s p2 a f pobs j1 1.00 j2 -0.76 1.00 p1 0.30 -0.43 1.00 s -0.35 0.32 -0.87 1.00 p2 0.11 -0.08 0.43 -0.55 1.00 a -0.12 0.10 -0.44 0.48 -0.91 1.00 f -0.20 -0.01 -0.01 0.02 -0.02 -0.05 1.00 pobs -0.05 0.08 -0.15 0.16 -0.17 0.13 -0.13 1.00 Table 1 Correlation coefficients between the parameter estimates in the posterior chains. salamanders were released. To delimit body size compo- nents of each year sample correctly, the population structure was separated into first and second year juve- niles, subadults and adults by means of FiSAT software [47]. This procedure gives more reliable results than using fixed SVL values, because the population structure shows year to year variations. Thus the number of dif- ferent age class individuals was estimated separately with the generalized removal model Mbh of CAPTURE software that allows for heterogeneity in capture prob- abilities [32]. Data collection and field methods The study site is an artificial cavity excavated during World War II near Busalla (province of Genova, NW Italy) and naturally colonised by salamanders [26]. Cre- taceous marlstones constitute the geological substrate of the area and a true natural karstic system is lacking, thus artificial underground retreats constitute an exten- sion of the natural superficial underground compart- ment available to salamanders. During dry summer periods salamanders concentrate inside these buffered habitats in which low temperatures and high moisture levels are present, while they are less active on the soil surface. Study species For further details on the methods and the study population, see [22,31,48]. From a conservation point of view, the species appears widespread and it is not considered globally endangered [43]. However, populations living in Mediterranean kars- tic areas that tend to concentrate inside caves or other artifical underground habitats, at least during unfavour- able external environmental conditions, are highly exposed to disturbance, predation and or collection, and can thus be considered locally threatened. Data collection and field methods Parameter estimation J t J t nbin J t p J t J t n i i o i o obs i i i o 1 1 1 2 2 , * , , , , * , ,       bin J t p S t S t nbin S t p A t i o obs i i i o i o obs i i 2, , * , * , ,              A t nbin A t p i o i o obs i , , (3) We used a Bayesian framework for estimating the parameters of this demographic model, fitting the mod- els using Monte Carlo Markov Chain (MCMC) techni- ques with the aid of the WinBUGS (version 1.4.3) statistical programming environment [28]. As very little prior knowledge on these demographic parameters is available, we used uninformative priors, using continu- ous uniform distributions as a starting point for each parameter. These were limited to 0 and 1 for all the other parameters except the fecundity parameter f for which the upper limit was set to 2 (as exploratory ana- lyses indicated much smaller values for it). Note that this does not reflect the number of eggs laid, typically 6- 14 in this species [44], but rather the number of hatched young per female. We also modelled the observation process explicitly, with a constant observation probabil- ity across all age classes and time periods [31], and hence the observed number in any class as binomially distributed from the true population at that time, with the probability of observation being a parameter of the model which is estimated from the data. (3) Equation 3 represents the observation process in year t, where J1 o, J2 o, So and Ao are the observed numbers in the different stages, J1 , J2 , S* and A* are estimates of the true (unobserved) population sizes, pobs is the obser- vation rate or capture probability and nbin is the nega- tive binomial distribution formulation representing the number of unobserved individuals before J1 o, J2 o, So or Ao individuals are observed with this capture rate; since we separate out the observation model from the under- lying process model, this constitutes a state-space model [29]. Parameter estimation In each year sample the population was well structured, as all age groups were present, indicating that there was no transient emigration, a phenomenon that may bias the estimates of population demographic parameters [46]. At the end of the experiment, all Page 7 of 9 Page 7 of 9 Lindström et al. BMC Ecology 2010, 10:4 http://www.biomedcentral.com/1472-6785/10/4 J2 A S p1 p2 j1 f a j2 s Figure 3 Life cycle graph for Speleomantes strinatii. The stage- classes are J2 (juveniles born in the previous year), S (subadults) and A (adults), see text for further details. J2 A S p1 p2 j1 f a j2 s subsequent simulations were run using MatLab 7.5.0 R2007b. In addition to the estimation of the demographic para- meters, we also simulated the population growth using the posterior distributions of the parameters (Table 1). Using the parameterised Lefkovitch matrix (Eq. 2) as a starting point, we then calculated the transition elastici- ties, stage-specific reproductive value (indicating the expected long-term contribution by each stage, given by the left eigenvector of the transition matrix), stable population structure, age-specific survivorship and ferti- lity, and the age-within-stage distributions using the methods described in [8]. The population renewal pro- cess was simulated by taking all the variability associated with the estimated population parameters into account. This procedure was repeated with the 1,000 posterior samples, so that the population at round i would renew itself according to the following equations. s a s Figure 3 Life cycle graph for Speleomantes strinatii. The stage- classes are J2 (juveniles born in the previous year), S (subadults) and A (adults), see text for further details. pj1, pj2, ps and pa), and finally individuals in stages J1, J2 and S will either continue to the next stage or stay where they are (with transition probabilities 1, g1 and g2 respectively - i.e. all new juveniles in J1 transition imme- diately to J2 before the end of the year). See Equation 4 for a mathematical description of this process. pj1, pj2, ps and pa), and finally individuals in stages J1, J2 and S will either continue to the next stage or stay where they are (with transition probabilities 1, g1 and g2 respectively - i.e. all new juveniles in J1 transition imme- diately to J2 before the end of the year). See Equation 4 for a mathematical description of this process. Parameter estimation Then the following three sets of equations govern the birth, death, and stage-transition of the individuals in year t: J t pois A t f J t bin J t p J t i i i i j i 1 1 1 1 2 , , ’ , , ’ , ,            bin J t p S t bin S t p A t bin A t p i j i i s i i a 2 2 , ’ ’ , , ,                         J t bin J t S t bin S t i i i i 2 2 1 2 , , ’ ’ , ,   (4) J t pois A t f i i 1, ,       The WinBUGS model was run for 20,000 iterations with 5 chains allowing a “burn-in period” for 5,000 iterations. Convergence of the parameter estimates was confirmed with the Gelman-Rubin convergence statistic provided by the WinBUGS software, and in all cases the Monte Carlo error was less than 5% of the sample stan- dard deviation (max. 2.2%). The observed autocorrela- tion in samples disappeared by setting the “thinning” to 50. In the analyses, we used the last 5,000 observations of the converged posterior chains. Since there was strong correlation between some of the model para- meters (Table 1), we took 1,000 samples from these pos- teriors to parameterise the matrix model. All the (4) Lindström et al. BMC Ecology 2010, 10:4 http://www.biomedcentral.com/1472-6785/10/4 Page 8 of 9 Lindström et al. BMC Ecology 2010, 10:4 http://www.biomedcentral.com/1472-6785/10/4 Working from top to bottom, the first equation gov- erns the birth of new offspring into the transitional stage J1; this is used here in the MCMC model and informs estimates of the parameter f, but in the simula- tion it is used in year t-1, together with an observation process to assess the accuracy of observed juveniles in the following year (see below). In the middle four equa- tions, J1’, J2’, S’ and A’ denote the number of individuals in stages J1, J2, S and A that survive that stage. Parameter estimation In the final two equations, J2 + and S+ are the number of those survivors which move to the next stage (all survivors of stage J1 transition immediately to J2); bin and pois are the Binomial and Poisson distributions respectively. Finally: 4. Simberloff D: Flagships, umbrellas, and keystones: is single-species management passé in the landscape era?. Biol Conserv 1998, 83:247-257 5. 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BMC Ecology 2010, 10:4 http://www.biomedcentral.com/1472-6785/10/4 Salvidio S: Estimating abundance and biomass of a Speleomantes strinatii (Caudata: Plethodontid) population by temporary removal sampling. Amphibia-Reptilia 1998, 19:113-124. 48. Salvidio S: Estimating abundance and biomass of a Speleomantes strinatii (Caudata: Plethodontid) population by temporary removal sampling. Amphibia-Reptilia 1998, 19:113-124. do : 0. 86/ 6 85 0 Cite this article as: Lindström et al.: Bayesian salamanders: analysing the demography of an underground population of the European plethodontid Speleomantes strinatii with state-space modelling. BMC Ecology 2010 10:4. 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https://openalex.org/W2080911106	https://eprint.ncl.ac.uk/fulltext.aspx?url=209078/8F1C9D15-5A2C-45D8-9C94-797526B31A0D.pdf&pub_id=209078	English	null	Regionally based alarm index to mitigate ionospheric scintillation effects for GNSS receivers	Space weather	2,015	cc-by	12,799	Copyright: ©2015. 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. Tiwari R, Strangeways HJ. Regionally based alarm index to mitigate ionospheric scintillation effects for GNSS receivers. Space Weather 2015, 13(1), 72-85. Tiwari R, Strangeways HJ. Regionally based alarm index to mitigate ionospheric scintillation effects for GNSS receivers. Space Weather 2015, 13(1), 72-85. Link to published article: http://dx.doi.org/10.1002/2014SW001115 Date deposited: Newcastle University ePrints - eprint.ncl.ac.uk 1. Introduction Tiwari, R., and H. J. Strangeways (2015), Regionally based alarm index to mitigate ionospheric scintillation eﬀects for GNSS receivers, Space Weather, 13, 72–85, doi:10.1002/2014SW001115. According to Yeh and Liu [1982], ionospheric scintillation can be modeled based on a phase changing irregular screen which diﬀracts the wave while propagating through it. The distortion of the wavefront results in constructive or destructive interference of diﬀerent “wavelets” at a ground-based receiver resulting in amplitude fading and phase changes. The rapid ﬂuctuation in the phase of the received signal can stress the PLL (phase-locked loop) while it is tracking the signal. Neglecting the eﬀect of the geomagnetic ﬁeld and collisions, the phase advance of the GNSS (Global Navigation Satellite System) signal along the line of the complete (assumed straight line) path through the ionosphere can be determined as given by equation (1). Received 3 AUG 2014 Accepted 30 DEC 2014 Accepted article online 7 JAN 2015 Published online 28 JAN 2015 𝜙= q2 2c𝜖omef(2𝜋)2 ∫path nedl (1) (1) The copyright line for this article was changed on 9 FEB 2015 after original online publication. where q is charge of electron, me mass of electron, 𝜖o permittivity of free space, c the speed of light in vacuum, and f the carrier frequency in Hz; ∫path nedl is the TEC where ne is the electron density at any point distance l along the length of the satellite to receiver path. Inserting the numerical values of the physical constants in equation (1) yields the phase of the GNSS signal as given in equation (2), where it can be seen that the phase depends upon the TEC for the path 𝜙= 40.3 cf TEC (2) 𝜙= 40.3 cf TEC (2) and can be measured experimentally using equations and observables as given below, nd can be measured experimentally using equations and observables as given below, TEC = f 2 1 f 2 2 40.3 (f 2 2 −f 2 1 ) (P1 −P2 ) (3a) TEC = −f 2 1 f 2 2 40.3 ( f 2 2 −f 2 1 ) (Φ1 −Φ2 ) (3b) (3a) (3b) here f1 and f2 are L1 and L2 GPS frequencies, pseudoranges (Ps), and phase observations (Φs) for the corresponding frequencies. RESEARCH ARTICLE 10.1002/2014SW001115 R. Tiwari1 and H. J. Strangeways1 Key Points: Key Points: • Derivation of ionospheric scintillation warning grid map for GNSS receivers • Ionospheric scintillation mitigation for precise GNSS positioning • Derivation of regional analogous phase scintillation index • Derivation of ionospheric scintillation warning grid map for GNSS receivers • Ionospheric scintillation mitigation for precise GNSS positioning • Derivation of regional analogous phase scintillation index 1School of Electrical and Electronic Engineering, Newcastle University, Newcastle upon Tyne, UK Abstract An approach to mitigate the eﬀect of ionospheric scintillation on GNSS (Global Navigation Satellite System) users in the European region using TEC (total electron content) at 1 Hz rate is presented. The TEC in the study is derived using raw GPS (Global Positioning System) observations obtained from the EUREF networks. The study also presents derivation of a geographic mesh-map warning of the expected standard deviation of phase jitter in receiver carrier tracking loops, information which would help to mitigate scintillation eﬀects in GPS software receivers. The copyright line for this article was changed on 9 FEB 2015 after original online publication. Space Weather The C/No can be computed using the WBP represented as shown in equation (7) [Kintner et al., 2007] C∕No = 10 log [( WBP 𝜂 −1 ) 50 ] (7) (7) where 𝜂is the noise term and is assumed to be the addition of the noise of the signal and the replica signal. The constant 50 in equation (7) is the sampling rate, here assumed to be 50 Hz. The above equation will be used in computing the threshold for the receiver locking. The C∕No is the deciding parameter for signal acquisition, and it depends on the WBP as shown in equation (7). As the amplitude and phase changes resulting from scintillation are random, they cannot be corrected, and thus, the best policy is to consider a mitigation method. During the scintillation, the intensity of a received GPS signal can be reduced so that it sometimes falls below the sensitivity (threshold) of the receiver. The extra disturbance of the phase of the signal, also associated with the scintillation, can cause the carrier tracking loop of a GPS receiver to lose lock so that the pseudorange from that satellite cannot be utilized in the receiver position determination. This then can have very serious consequences, e.g., in aviation and marine navigation, and can introduce positional errors in geodetic surveying. The next section discusses the carrier tracking loop of a generic GPS receiver and the eﬀects of ionospheric scintillation on the carrier tracking loop. where 𝜂is the noise term and is assumed to be the addition of the noise of the signal and the replica signal. The constant 50 in equation (7) is the sampling rate, here assumed to be 50 Hz. The above equation will be used in computing the threshold for the receiver locking. The C∕No is the deciding parameter for signal acquisition, and it depends on the WBP as shown in equation (7). As the amplitude and phase changes resulting from scintillation are random, they cannot be corrected, and thus, the best policy is to consider a mitigation method. During the scintillation, the intensity of a received GPS signal can be reduced so that it sometimes falls below the sensitivity (threshold) of the receiver. Space Weather The extra disturbance of the phase of the signal, also associated with the scintillation, can cause the carrier tracking loop of a GPS receiver to lose lock so that the pseudorange from that satellite cannot be utilized in the receiver position determination. This then can have very serious consequences, e.g., in aviation and marine navigation, and can introduce positional errors in geodetic surveying. The next section discusses the carrier tracking loop of a generic GPS receiver and the eﬀects of ionospheric scintillation on the carrier tracking loop. Space Weather Space Weather 10.1002/2014SW001115 clock drift, and, at least partially, the multipath. This is achieved when, for example, the ADR is detrended using a sixth-order Butterworth high-pass ﬁlter with a 0.1 Hz cutoﬀfrequency [Van Dierendonck et al., 1993]. clock drift, and, at least partially, the multipath. This is achieved when, for example, the ADR is detrended using a sixth-order Butterworth high-pass ﬁlter with a 0.1 Hz cutoﬀfrequency [Van Dierendonck et al., 1993]. Depending on the sensitivity of the receiver, a threshold value of power is required for any GPS receiver to lock a satellite; the quality of the received signal can be represented in terms of C/No (Carrier to Noise ratio). The typical value of C/No for a GPS receiver acquisition is 33 dB-Hz, while between 26 and 30 dB-Hz of C/No is required to maintain lock. The receiver correlates the carrier and code replicas with the incoming signal to compute I and Q pairs of the signal over 1 ms. The I and Q pairs are used to compute the WBP (wideband power) and NBP (narrowband power) over 20 ms as deﬁned in equations (4a) and (4b), respectively [Van Dierendonck et al., 1996]. WBP = 20 ∑ i=1 (Ii )2 + (Qi )2 (4a) NBP = ( 20 ∑ i=1 Ii )2 + ( 20 ∑ i=1 Qi )2 (4b) (4a) (4b) The amplitude ﬂuctuation can be measured in terms of the amplitude scintillation index which depends upon the received signal strength/intensity (I = A2∕2). The signal strength as in equations (4a) and (4b) can be determined using detrended data of WBP and NBP which can be accomplished by using a sixth order Butterworth ﬁlter with 0.1 Hz cutoﬀfrequency [Van Dierendonck et al., 1993]. The signal strength can then be found using equation (5), and then the amplitude scintillation (S4) can be obtained from equation (6) [Van Dierendonck et al., 1993]. I = NBP −WBP (NBP −WBP)LPF (5) (5) S4 = √ ⟨I2⟩−⟨I⟩2 ⟨I⟩2 (6) (6) The <.> denotes the mean of the corresponding value for a 1 min time interval. To study the eﬀect of the ionosphere on a GPS receiver, the S4 index can be used. Correspondingly, the phase scintillation is quantiﬁed by using the phase scintillation index which is usually obtained from the detrended carrier phase [Van Dierendonck et al., 1993; Forte and Radicella, 2002]. 1. Introduction During ionospheric storms, time-varying ionospheric irregularities which drift through the ionosphere with velocities up to about 1000 m/s alter the TEC values, so altering the received phase as given by equation (2). In the last two decades, some studies [Pi et al., 1997; Basu et al., 1988, 1999; Beach and Kintner, 1999; Mendillo et al., 2000; Krankowski et al., 2005; Alfonsi et al., 2011; Tiwari et al., 2013a; Pi et al., 2013] have shown strong correlation of the time derivative of TEC with scintillation indices. The scintillation indices also quantify the level of stress in the tracking loop. In the GISTM (GPS Ionospheric Scintillation and TEC Monitoring) receivers, the 50 Hz ADR (accumulated Doppler range) samples, output from PLL, are used for scintillation index estimation; the higher the phase scintillation, the higher the PLL stress. In order to only measure the ionospheric scintillation eﬀect, the 50 Hz ADR samples require detrending which eliminates the lower fading frequencies due to satellite motion, receiver and satellite here f1 and f2 are L1 and L2 GPS frequencies, pseudoranges (Ps), and phase observations (Φs) for the corresponding frequencies. During ionospheric storms, time-varying ionospheric irregularities which drift through the ionosphere with velocities up to about 1000 m/s alter the TEC values, so altering the received phase as given by equation (2). In the last two decades, some studies [Pi et al., 1997; Basu et al., 1988, 1999; Beach and Kintner, 1999; Mendillo et al., 2000; Krankowski et al., 2005; Alfonsi et al., 2011; Tiwari et al., 2013a; Pi et al., 2013] have shown strong correlation of the time derivative of TEC with scintillation indices. The scintillation indices also quantify the level of stress in the tracking loop. In the GISTM (GPS Ionospheric Scintillation and TEC Monitoring) receivers, the 50 Hz ADR (accumulated Doppler range) samples, output from PLL, are used for scintillation index estimation; the higher the phase scintillation, the higher the PLL stress. In order to only measure the ionospheric scintillation eﬀect, the 50 Hz ADR samples require detrending which eliminates the lower fading frequencies due to satellite motion, receiver and satellite 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. ©2015. The Authors. 72 TIWARI AND STRANGEWAYS Space Weather Space Weather 10.1002/2014SW001115 ionospheric scintillation. However, during ionospheric scintillation, altered phase and/or amplitude distorts the GPS signal S(t), modifying it to the modiﬁed signal R(t) as shown in equation (9): S(t) = AC(t)D(t)cos (𝜔IFt + 𝜙) (8) (8) where A is the received amplitude, C(t) is C/A code, 𝜔IF is the down converted (from L1/L2) carrier frequency, D(t) is the navigation data bit at a 50 Hz sample rate, and 𝜔IFt + 𝜙together is the nominal phase. where A is the received amplitude, C(t) is C/A code, 𝜔IF is the down converted (from L1/L2) carrier frequency, D(t) is the navigation data bit at a 50 Hz sample rate, and 𝜔IFt + 𝜙together is the nominal phase. R(t) = A′C(t)D(t)cos (𝜔IFt + 𝛿𝜙) (9) (9) R(t) = A′C(t)D(t)cos (𝜔IFt + 𝛿𝜙) where A′ and 𝛿𝜙characterize the modiﬁed amplitude and extra carrier phase perturbation that can result from ionospheric scintillation. The R(t) signal in equation (9) passes through the code and carrier tracking loop, where the C/A is removed using the code delay loop. During phase scintillation, 𝛿𝜙(the phase perturbation in equation (9) can be very large and sometimes, according to the Thumb rule in [Kaplan and Hegarty, 2006], cycle slips occur when 𝛿𝜙exceeds a given threshold, i.e., the standard deviation of 𝛿𝜙(1𝜎), phase jitter above 15◦(𝜎𝛿𝜙> 15◦). The level can, however, vary with the bandwidth of the PLL which can be appropriately altered if the scintillation level is known or can be at least be approximately predicted by, for example, using a regional alarm index. Although it is not possible for a generic GPS receiver to mitigate scintillation eﬀects on its own, studies have shown that ionospheric scintillation can be mitigated using GPS software receivers [Ganguly et al., 2004; Tiwari et al., 2011] which basically update their loop parameters, e.g., increasing the loop bandwidth based on some prediction model such as Wide Band Modeling (WBMOD) or a regional alarm index [Tiwari et al., 2011]. 2.1. Eﬀect of Scintillation on Carrier Tracking Loop The performance of a GPS receiver depends upon phase error measurements because it is directly related to the tracking threshold; if the measured phase error exceeds the tracking threshold then the receiver loses lock. In Kaplan and Hegarty [2006], the major sources of phase error are stated to be the phase jitter and dynamic stress error and, as a rule of thumb, the threshold 3𝜎jitter given in equations (10a) and (10b) must not exceed one fourth of the pull-in of the PLL discriminator as 3𝜎PLL = 3𝜎j + 𝜃e ≤90◦ (10a) 3𝜎PLL = 3𝜎j + 𝜃e ≤45◦ (10b) (10a) (10b) (10b) where 𝜎j is the 1𝜎phase jitter from all sources and 𝜃e is the dynamic stress in terms of threshold values. The pull-in range for dataless is 360◦and for data modulated is 180◦; therefore, the 1𝜎threshold for dataless is 90◦and 45◦for data modulated. Hence, 𝜎PLL, the threshold for the arctangent discriminator is given by where 𝜎j is the 1𝜎phase jitter from all sources and 𝜃e is the dynamic stress in terms of threshold values. The pull-in range for dataless is 360◦and for data modulated is 180◦; therefore, the 1𝜎threshold for dataless is 90◦and 45◦for data modulated. Hence, 𝜎PLL, the threshold for the arctangent discriminator is given by 𝜎PLL = √ 𝜎2 tPLL + 𝜎2 v + 𝜎2 A + 𝜃e 3 ≤15◦ (11) (11) where 𝜎PLL is the 1𝜎thermal noise, 𝜎v is the 1𝜎vibration-induced oscillation, and 𝜎A is the Allan variance- induced oscillator jitter. In the above equation, the phase jitter due to scintillation was not speciﬁcally considered although the eﬀects of ionospheric scintillation are one of the major sources of error in carrier tracking of any GPS receiver [Conker et al., 2003; Hegarty, 1997; Knight and Finn, 1998]. Further, Tiwari et al. [2011], Forte [2012], and Aquino and Sreeja [2013] show that the calculated PLL error, using the Conker et al. [2003] formula due to phase and amplitude scintillation, is signiﬁcant. The relationship between amplitude and phase scintillation and tracking error variance at the output of the PLL was derived by Hegarty [1997] and Knight and Finn [1998], and is given in equation (12): where 𝜎PLL is the 1𝜎thermal noise, 𝜎v is the 1𝜎vibration-induced oscillation, and 𝜎A is the Allan variance- induced oscillator jitter. 2. Ionospheric Scintillation and Generic GPS Receiver In the GPS receiver, the front-end down-converts the GPS frequency to a lower intermediate frequency (IF) which is then used in acquisition and demodulation of the navigation data stream in the code and carrier tracking loops in order to determine the receiver’s position. Equation (8) represents the GPS signal (single-frequency CA (Coarse Acquisition) code) S(t) at the IF frequency without any additional noise or TIWARI AND STRANGEWAYS 73 ©2015. The Authors. Space Weather The prediction integration time is 𝜂and is equal to 0.02 s for PLL tracking. The phase scintillation is obtained from the power spectra parameters (T, p) of the carrier phase as employed in, e.g., Conker et al. [2003] and Rino [1979]. In the carrier tracking loop, the numerically control oscillator generates the replica of the incoming carrier wave, and the discriminator compares the incoming and the replica carrier waves. The output of the discriminator is fed to the low-pass ﬁlter to reduce the phase error in the loop. Usually, the PLL ﬁlter takes time to minimize based on the phase error or variance. If the variance of the phase error is large, then the time to lock will be larger, and during conditions where the variance of phase error exceeds the threshold value, the PLL may lose lock. During an ionospheric storm, the rapid random ﬂuctuations in electron density increase the phase error in the PLL. The PLL ﬁlter takes a longer time to minimize and ﬁnally causes loss of lock if it crosses the threshold time lock. In equation (13), it can be seen that, S4 and the spectral parameters T, p are required in order to estimate phase jitter, which are not available in normal generic and surveying GPS receivers although C/No can be used to derive thermal phase jitter but not phase jitter due to phase scintillation. In this study, the time derivative TEC index derived in Tiwari et al. [2013a] is used to estimate phase jitter due to phase scintillation (discussed in next section). Furthermore, a regional area mitigation approach is also proposed using the expected standard deviation of PLL error mapped into a latitude-longitude (10◦× 5◦) mesh of grid blocks for the European region, which can help users to update their tracking parameters for robust tracking. Space Weather Space Weather 10.1002/2014SW001115 where Bn is the bandwidth in a generic GPS receiver, with third-order PLL, the Bn, noise bandwidth ranging between 10 and 15 Hz [Van Dierendonck et al., 1996]. S4, the amplitude scintillation index, must have a value less than 0.707 for the formula to be valid. The prediction integration time is 𝜂and is equal to 0.02 s for PLL tracking. The phase scintillation is obtained from the power spectra parameters (T, p) of the carrier phase as employed in, e.g., Conker et al. [2003] and Rino [1979]. In the carrier tracking loop, the numerically control oscillator generates the replica of the incoming carrier wave, and the discriminator compares the incoming and the replica carrier waves. The output of the discriminator is fed to the low-pass ﬁlter to reduce the phase error in the loop. Usually, the PLL ﬁlter takes time to minimize based on the phase error or variance. If the variance of the phase error is large, then the time to lock will be larger, and during conditions where the variance of phase error exceeds the threshold value, the PLL may lose lock. During an ionospheric storm, the rapid random ﬂuctuations in electron density increase the phase error in the PLL. The PLL ﬁlter takes a longer time to minimize and ﬁnally causes loss of lock if it crosses the threshold time lock. In equation (13), it can be seen that, S4 and the spectral parameters T, p are required in order to estimate phase jitter, which are not available in normal generic and surveying GPS receivers although C/No can be used to derive thermal phase jitter but not phase jitter due to phase scintillation. In this study, the time derivative TEC index derived in Tiwari et al. [2013a] is used to estimate phase jitter due to phase scintillation (discussed in next section). Furthermore, a regional area mitigation approach is also proposed using the expected standard deviation of PLL error mapped into a latitude-longitude (10◦× 5◦) mesh of grid blocks for the European region, which can help users to update their tracking parameters for robust tracking. where Bn is the bandwidth in a generic GPS receiver, with third-order PLL, the Bn, noise bandwidth ranging between 10 and 15 Hz [Van Dierendonck et al., 1996]. S4, the amplitude scintillation index, must have a value less than 0.707 for the formula to be valid. 2.1. Eﬀect of Scintillation on Carrier Tracking Loop In the above equation, the phase jitter due to scintillation was not speciﬁcally considered although the eﬀects of ionospheric scintillation are one of the major sources of error in carrier tracking of any GPS receiver [Conker et al., 2003; Hegarty, 1997; Knight and Finn, 1998]. Further, Tiwari et al. [2011], Forte [2012], and Aquino and Sreeja [2013] show that the calculated PLL error, using the Conker et al. [2003] formula due to phase and amplitude scintillation, is signiﬁcant. The relationship between amplitude and phase scintillation and tracking error variance at the output of the PLL was derived by Hegarty [1997] and Knight and Finn [1998], and is given in equation (12): 𝜎2 𝜙= 𝜎2 𝜙S + 𝜎2 𝜙T + 𝜎2 𝜙osc (12) (12) where 𝜎2 𝜙is the tracking error variance; 𝜎2 𝜙S is error variance component related to phase scintillation; 𝜎2 𝜙T is error variance component related to thermal noise and intensity scintillation, and 𝜎𝜙osc is the error variance component from oscillator noise. The tracking error variance in equation (13) is deﬁned in Conker et al. [2003], who, considering phase and weak amplitude scintillation (S4 < 0.707), derived the thermal noise tracking error in the presence of scintillation as given in the second term of equation (13). 𝜎2 𝜙= 𝜋T kf p−1 n sin ( [2k+1−p]𝜋 2k ) + Bn [ 1 + 1 2𝜂(C∕No)L1−C∕A(1−2S2 4(L1)) ] (C∕No)L1−C∕A (1 −2S2 4(L1)) + 𝜎2 𝜙osc (13) (13) 74 ©2015. The Authors. TIWARI AND STRANGEWAYS 74 TIWARI AND STRANGEWAYS 3. Experimental Setup and Methodology In this study, GPS data at 1 Hz archived from the EUREF network stations for deriving TEC values over Europe have been used in deriving an analogous phase scintillation index which is further used in the estimation of the expected PLL error for a generic GPS receiver with given speciﬁcation: third-order PLL and 15 Hz bandwidth. The overall method is executed in four steps: (i) estimation of TEC at a 1 Hz rate from the EUREF network of GPS receivers, (ii) estimation of the analogous phase scintillation index using the TEC for every minute, (iii) determination of the expected PLL error variance using the analogous phase scintillation index, and (iv) determination of the expected values of the PLL error over a latitude-longitude area region divided into 10◦× 5◦(longitude × latitude) area grid blocks each averaged over 5 min. These four steps are discussed below. Essentially, the program designed in this study requires raw observations (phase and pseudorange) and the ephemeris ﬁles as input. First, the TEC is estimated using equation (3a) after which the observation data passes through the quality control processing unit which includes cycle slip detection in the phase observation. The phase measurements are susceptible to cycle slips which can be due to strong ionospheric scintillation. At high latitudes, this generally results from small-scale ionospheric electron density irregularities causing rate of change of phase variations, which exceed the tracking loop bandwidth. Generally, more cycles slips are observed on L2 in comparison to L1. In order to estimate TEC using equation (3b), the cycle slips have to be detected and corrected, if possible. The Widelane detection criterion is used for cycle slips detection [Blewitt, 1990] for the case that the cycle slip occurs in only one of the L1 and L2 signals. It is corrected based on the trend (we used Kalman ﬁlter) of the collected observa- tions except in case of phase jumps in the L1 or L2 phase observation that last for 5 s or more as then the data are discarded. The diﬀerential code biases for all satellites are also provided for further adjustment. Tiwari et al. [2013a] derived an analogous phase scintillation index, 𝜎𝜙a given in equation (14). TIWARI AND STRANGEWAYS Space Weather 𝜎2 𝜙p = r × 𝜎2 VTEC′ HPF (17) (17) 𝜎2 𝜙p = r × 𝜎2 VTEC′ HPF where r is the scaling factor derived from straight line ﬁtting of 𝜎2 VTEC′ HPF with the PLL phase jitter due only t th h i till ti d t i d i th C k t l [2003] f l th 2 b bt i d where r is the scaling factor derived from straight line ﬁtting of 𝜎2 VTEC′ HPF with the PLL phase jitter due only to the phase scintillation as determined using the Conker et al. [2003] formula, then 𝜎2 𝜙p can be obtained from equation (17). In this model, the estimated PLL variance is for high latitudes and hence rather weak amplitude scintillation can be assumed which is considered to be a valid assumption for this case commensurate with the observations presented in Aarons [1997], Coker et al. [2004], Tiwari et al. [2010], and many others. In Aquino and Sreeja [2013], the second-order polynomial was found to be the best ﬁt for jitter with just the phase jitter, although this phase scintillation would be correlated with other parameters such as amplitude scintillation, C/No, and oscillator noise. Thus, it may not be an optimal ﬁtting for determining a model for phase jitter for a range of these correlated parameters. where r is the scaling factor derived from straight line ﬁtting of 𝜎2 VTEC′ HPF with the PLL phase jitter due only to the phase scintillation as determined using the Conker et al. [2003] formula, then 𝜎2 𝜙p can be obtained from equation (17). In this model, the estimated PLL variance is for high latitudes and hence rather weak amplitude scintillation can be assumed which is considered to be a valid assumption for this case commensurate with the observations presented in Aarons [1997], Coker et al. [2004], Tiwari et al. [2010], and many others. In Aquino and Sreeja [2013], the second-order polynomial was found to be the best ﬁt for jitter with just the phase jitter, although this phase scintillation would be correlated with other parameters such as amplitude scintillation, C/No, and oscillator noise. Thus, it may not be an optimal ﬁtting for determining a model for phase jitter for a range of these correlated parameters. According to equation (12), the thermal phase jitter depends on S4 whereas in Van Dierendonck et al. Space Weather Space Weather 10.1002/2014SW001115 Vehicle (SV) elevation angle at two consecutive epochs, 𝜎VTEC′ HPF is the normalized standard deviation of the high-pass ﬁltered Rate of change of TEC (ROT), and vp is the radial Ionospheric Pierce Point (IPP) velocity. The estimated index is derived from EUREF network of receivers, the receiver biases are diﬀerent for diﬀerent receivers, and the index is used to alert the user of possible PLL error during the scintillation period. But for Precise Point Positioning or for surveying application where centimeter level of accuracy is needed in positioning, one can derive the S scaling constant for an individual receiver using the least squares ﬁtting technique for a few days of data. This new index uses a high-pass ﬁltered ROT collected at a 1 Hz rate which shows a good correlation with the standard phase scintillation observed during geomagnetic storms at high latitudes and was found to be well correlated with the spectral parameter p (slope of the phase Power Spectral Density (PSD)) for a range of values of the Kp index [Tiwari et al., 2013b]. The diurnal and seasonal variation of the analogous phase scintillation index also seemed to be well correlated with p for a 1 year data set collected at Trondheim, which is consistent with observations of Wernik et al. [2004]. In the phase jitter estimation using the Conker et al. [2003] formula, the spectral parameters (p, T) are required for the estimation of the PLL variance. These values can either be derived from the psd of the 50 Hz raw data (p the slope of phase psd when plotted on log-log axes and T its magnitude at 1 Hz) or using scintillation indices [Strangeways, 2009] which greatly reduce the processing time by eliminating the need for all the fast Fourier transforms necessary for determining the spectral parameters directly in the frequency domain. However, the processing time could be reduced even further if the need to determine the spectral parameters was obviated altogether. As an application of the analogous phase scintillation index, Tiwari et al. [2013b] used estimated analogous scintillation phase index data from a NovAtel GISTM receiver installed at Trondheim and derived a model for the expected PLL phase variance of a generic receiver during moderate to strong phase scintillation condition given by equation (17). 3. Experimental Setup and Methodology (14) 𝜎𝜙a = 𝜛(𝜒(M), vp ) × 𝜎VTEC′ HPF (14) 𝜙a (𝜒( ), p ) VTECHPF ( ) 𝜛(𝜒(M), vp ) = 2𝜋S40.3 cf (𝜒(M), vp ) (15) 𝜛(𝜒(M), vp ) = 2𝜋S40.3 cf (𝜒(M), vp ) (15) (15) 𝜎VTEC′ HPF = [ 1 n −1 n ∑ i=1 VTEC′ HPF −VTEC′ HPF ]1∕2 (16) (16) where 𝜛(𝜒(M), p) is the elevation weighted function given in equation (15), and S is a scaling constant of 0.003 (this is user deﬁned based on the application), 𝜒(M) is a mapping function based on Satellite TIWARI AND STRANGEWAYS ©2015. The Authors. 75 TIWARI AND STRANGEWAYS Space Weather 10.1002/2014SW001115 Figure 1. Standard deviation of PLL due to thermal noise with and without amplitude scintillation. Figure 1. Standard deviation of PLL due to thermal noise with and without amplitude scintillation. Figure 1. Standard deviation of PLL due to thermal noise with and without amplitude scintillation. employed by Hegarty [1997]. Using equation (13), the phase jitter due to amplitude scintillation, oscillator, and analogous phase scintillation (used to replace the standard phase scintillation index) can be obtained as employed by Hegarty [1997]. Using equation (13), the phase jitter due to amplitude scintillation, oscillato and analogous phase scintillation (used to replace the standard phase scintillation index) can be obtained 𝜎2 𝜙= 𝜎2 𝜙p + 𝜎2 𝜙T + 𝜎2 𝜙osc (20) (20) 𝜎2 𝜙= p × 𝜎2 VTEC′ HPF + Bn[1 + 1 2𝜂(C∕No)L1−C∕A ] (C∕No)L1−C∕A + 𝜎𝜙2 osc (21) (21) The above PLL phase variance (𝜎2 𝜙) can be used in a software receiver for updating the tracking loop parameter or by using the weighted covariance model discussed in Aquino et al. [2009]. The above PLL phase variance (𝜎2 𝜙) can be used in a software receiver for updating the tracking loop parameter or by using the weighted covariance model discussed in Aquino et al. [2009]. The regional alarm index 𝜎R𝜙is obtained for alerting GNSS users and is derived from the analogous phas scintillation index used as given in equation (22) [𝜎R𝜙 ]k ij = ∑nsat n ∑k t=1 𝜎𝜙a(n, t) nsat × k ; { [LatIPP ]t ij = (65 + (k × i) −k] ∶[65 + (k × (i + 1)) −k]) [LonIPP ]t ij = (−15 + (k × j) −k] ∶[−15 + (k × (j + 1)) −k]) (22) (22) where i and j deﬁne the region, k represents 5 min data samples observed from the SV, and the upper and lower latitudes are 65◦and 35◦, respectively, and similar for longitude. The epochs (k) and latitude and longitude grid can be user deﬁned; the smaller the grid size, the better the resolution although this of course also requires a suﬃciently dense spatial distribution of the GPS receivers in the given network. Space Weather [1993], the S4 parameter depends upon the WBP and NBP given in equations (4a) and (4b), respectively; and in Kaplan and Hegarty [2006], C/No only is considered suﬃcient for the estimation of thermal phase jitter. The C/No measures the degradation of signal intensity due to extra modulation in the received intensity and is alone a suﬃcient parameter for this estimation for high-latitude locations where only weak amplitude scintillation is observed. Figure 1 (left) shows the standard deviation of thermal phase jitter (third-order loop, Bn=15 Hz) obtained by using second term of equation (13) with S4=0.3 and without amplitude scintillation. The zoomed-in plot shows how close the values of phase jitter are with S4=0.3 and without amplitude scintillation for C/No values up to 30 dB, while Figure 1 (right) shows the diﬀerence between the Conker et al. [2003] and Kaplan and Hegarty [2006] models where closeness of phase jitter values with S4=0.3 level scintillation and without is quite apparent. This is the usual case for the high-latitude region. If the C/No measurements are not recorded, then the user can use an estimated C/No for a surveying antenna using equation (19) [Tiwari et al., 2013b]. 𝜎2 𝜙T = Bn [ 1 + 1 2𝜂(C∕No)L1−C∕A ] (C∕No)L1−C∕A (18) C∕No = asin(E × b + c) (19) 𝜎2 𝜙T = Bn [ 1 + 1 2𝜂(C∕No)L1−C∕A ] (C∕No)L1−C∕A (18) (18) C∕No = asin(E × b + c) (19) C∕No = asin(E × b + c) (19) C∕No = asin(E × b + c) (19) where a, b, c are constants (a=51.94, b=0.0093, c=0.7305) and E is the elevation angle. The constants are derived for diﬀerent loop orders. The oscillator noise, 𝜎2 𝜙osc =0.01 rad2, is taken to be the same as where a, b, c are constants (a=51.94, b=0.0093, c=0.7305) and E is the elevation angle. The constants are derived for diﬀerent loop orders. The oscillator noise, 𝜎2 𝜙osc =0.01 rad2, is taken to be the same as TIWARI AND STRANGEWAYS TIWARI AND STRANGEWAYS ©2015. The Authors. 76 Space Weather 4. Result and Discussion In order to validate the proposed model for phase jitter using TEC, two geomagnetic storms events observed on 24 April 2012 and 17 March 2013 were selected. Since the Kp index is the global index for representing the level of geomagnetic storm, it may not be optimum as a measure of localized geomagnetic activity. In this study, the Earth’s magnetic ﬁeld from Dombas (62.07◦N, 9.11◦E) is used which is close to the installed GISTM receiver at Trondheim (63.41◦N, 10.4◦E) and is used for measuring the storm intensity. On 24 April 2012, as can be seen in Figure 2, the Earth’s H and Z magnetic ﬁeld components show a strong ﬂuctuation between 00:00 and 04:00 UT which results from a geomagnetic storm near Dombas in the auroral region. During such a storm, the transionospheric GPS signal suﬀers a signiﬁcant amount of phase scintillation [Skone et al., 2008, 2009; Tiwari et al., 2010] and the resulting anticipated eﬀect on the carrier tracking loop of a GPS receiver. Thus, modeled phase jitter using the TEC derivative and phase jitter derived from the Conker et al. [2003] formula are investigated in the time domain for the same period of the storm. In Figure 3, the modeled phase jitter is indicated as a solid red line, while the Conker formula-derived phase jitter is shown as a solid blue line. These are shown for PRNs 03, 06, 11, 14, 18, 19, 20, and 21 with a 20◦ elevation cutoﬀbeing applied to all the visible PRNs. The eﬀect of storm can easily be seen in Figure 3. The storm was strong between 00:00 and 03:00 UT with a correspondingly large PLL error. The pattern in the ﬂuctuation of the modeled phase jitter and that determined using the Conker et al. [2003] formula is very TIWARI AND STRANGEWAYS ©2015. The Authors. 77 Space Weather 10.1002/2014SW001115 Figure 2. Earth magnetic ﬁeld measurements using the ﬂuxgate magnetometer at Dombas (62.07◦N, 09.11◦E) show the magnetic storm started at midnight on 24 April 2012. similar. The elevation angle to the satellite is shown as a solid green line in the ﬁgure and demonstrates that the PLL error is not just due to a low-elevation angle. The eﬀect of the storm can also be seen in the eﬀect on the paths to those satellites which were above 30◦elevation; and in this case, both modeled and Conker et al. TIWARI AND STRANGEWAYS 4. Result and Discussion [2003] PLL errors are well correlated although there are some cases where the modeled phase jitter is an overestimate compared with that determined from the Conker formula but would still be useful in an alarm indicator. Figure 2. Earth magnetic ﬁeld measurements using the ﬂuxgate magnetometer at Dombas (62.07◦N, 09.11◦E) show the magnetic storm started at midnight on 24 April 2012. A similar geomagnetic storm was recorded on 17 March 2013 and is employed for studying the eﬀectiveness of the modeled phase jitter using TEC. Figure 4 shows the Earth’s magnetic ﬁeld strength at Dombas, and it can be seen from the ﬁgure that a negative ﬂuctuation in the H component was recorded with a large value at 18:00 UT but with only small values after 20:00 UT until strong again around 22:00 UT. In order to study the eﬀect of the geomagnetic storm, the modeled and Conker et al. estimated phase jitters from 17:00 to 23:00 UT are presented in Figure 5. From 17:00 to 17:55 UT there was no evidence of a geomagnetic storm, but the modeled and Conker et al. [2003] phase jitters at 18:00 UT show a strong jump which is a clear indication of eﬀect of a geomagnetic storm on the carrier tracking loop. The pattern of the modeled phase jitter is similar to that derived using the Conker et al. [2003] formula, but, similar to the previous result, the Figure 3. The red lines are the estimated PLL error using the TEC derivative time series r for a GPS receiver (PLL Bn = 15 Hz, third order), the blue lines are the calculated PLL error obtained from the Conker et al. [2003] formula using Strangeways [2009] spectral parameter determination method and the appropriate elevation angle. Figure 3. The red lines are the estimated PLL error using the TEC derivative time series r for a GPS receiver (PLL Bn = 15 Hz, third order), the blue lines are the calculated PLL error obtained from the Conker et al. [2003] formula using Strangeways [2009] spectral parameter determination method and the appropriate elevation angle. TIWARI AND STRANGEWAYS TIWARI AND STRANGEWAYS ©2015. The Authors. 78 Space Weather 10.1002/2014SW001115 Figure 4. Earth magnetic ﬁeld measurements using the ﬂuxgate magnetometer at Dombas (62.07◦N, 09.11◦E), close to Trondheim, show the eﬀect of a magnetic storm which started at 18:00 UT on 17 March 2013. Figure 4. TIWARI AND STRANGEWAYS 4. Result and Discussion 79 Space Weather 10.1002/2014SW001115 Figure 6. Correlation of modeled and Conker et al. phase jitters for (left) strong geomagnetic storm conditions (Kp > 5) and (right) moderate geomagnetic storm conditions (Kp = 5) period. Figure 6. Correlation of modeled and Conker et al. phase jitters for (left) strong geomagnetic storm conditions (Kp > 5) and (right) moderate geomagnetic storm conditions (Kp = 5) period. is useful for measuring the level of eﬀect of geomagnetic storm on carrier tracking loops. The eﬀect of a second storm between 22:00 and 23:00 UT can also be seen in the modeled phase jitter. In addition to the case studies discussed above, a statistical study was also made for further validation. The modeled and Conker et al. [2003] calculated phase jitters are estimated for 1 year (October 2011 to September 2012) from Trondheim. To obtain these results, an elevation mask of 20◦and an SV lock time more than 120 s were employed; phase scintillation values above 2.5 rad were considered as outliers and ignored. The data points are further arranged into two sets of categories, a strong geomagnetic storm (Kp>5) data array and a moderate geomagnetic storm (Kp=5) data array. The modeled and Conker et al. [2003] phase jitters were correlated for both conditions. Figure 6 (left) shows the correlation when Kp>5 (considered strong geomagnetic storms) and the 95% conﬁdence interval of line ﬁtting is indicated as the red solid line with a correlation of 76.61%. Thus, the correlation between the phase jitters is very good, the best ﬁt is found in the region between 8◦and 12◦so that the probability of getting a false alarm is low. Similar results are seen for the data set when Kp=5, although the correlation coeﬃcient of 65.58% is smaller than that for the Kp>5 data arrays. The PLL error is susceptible to many sources of errors, the Conker method is an analytical method based on the spectral parameters T, p, and S4. In this work, T and p parameters are computed using phase and amplitude scintillation indices as input parameters required for the [Strangeways, 2009] model; in GISTM receivers, there are some phase scintillation (phi60 ≥2.5) outliers which are discarded while computing T and p parameters, but the new index can still be computed but shows an overestimated value of PLL error which is considered to be good for alerting the user. 4. Result and Discussion Earth magnetic ﬁeld measurements using the ﬂuxgate magnetometer at Dombas (62.07◦N, 09.11◦E), close Trondheim, show the eﬀect of a magnetic storm which started at 18:00 UT on 17 March 2013. modeled phase jitter seems to be an overestimate, and there are many cases where the modeled phase jitter had a high value. Especially for PRN 21, this could then cause a false alarm but this is diﬃcult to conclude because GISTM receiver may lose lock during strong scintillation, and in that situation, the GISTM receiver can indicate very high scintillation during the reacquisition period resulting in outlier which should be ignored. Also, during the same period, other locked PRNs show strong modeled phase jitter which is consistent with the Conker et al. [2003] phase jitter determination. This implies that the model phase jitter modeled phase jitter seems to be an overestimate, and there are many cases where the modeled phase jitter had a high value. Especially for PRN 21, this could then cause a false alarm but this is diﬃcult to conclude because GISTM receiver may lose lock during strong scintillation, and in that situation, the GISTM receiver can indicate very high scintillation during the reacquisition period resulting in outlier which should be ignored. Also, during the same period, other locked PRNs show strong modeled phase jitter which is consistent with the Conker et al. [2003] phase jitter determination. This implies that the model phase jitter Figure 5. The red lines represents the modeled PLL jitter using the TEC derivative time series for a GPS receiver (PLL Bn = 15 Hz, third order), the blue lines are the determined PLL error obtained from Conker et al. [2003] formula using the Strangeways [2009] method of spectral parameter determination and the appropriate elevation angle. Figure 5. The red lines represents the modeled PLL jitter using the TEC derivative time series for a GPS receiver (PLL Bn = 15 Hz, third order), the blue lines are the determined PLL error obtained from Conker et al. [2003] formula Figure 5. The red lines represents the modeled PLL jitter using the TEC derivative time series for a GPS receiver (PLL Bn = 15 Hz, third order), the blue lines are the determined PLL error obtained from Conker et al. [2003] formula using the Strangeways [2009] method of spectral parameter determination and the appropriate elevation angle. TIWARI AND STRANGEWAYS TIWARI AND STRANGEWAYS ©2015. The Authors. 4.1. Regional Alarming Index for Ionospheric Threat Figure 7 shows the standard deviation of the PLL error, for data collected from Trondheim (62.6◦N, 10.41◦E) on 17 March 2013 using a NovAtel GISTM receiver, for S4 and the standard scintillation index (Figure 7, left), and S4 and analogous phase scintillation index (Figure 7, right). According to the results in this ﬁgure, moderate (S4) and moderate phase scintillation can lead to 11◦phase jitter, while combined moderate (S4) and moderate to strong phase scintillation can lead to rather high values of the phase jitter that can exceed the 15◦(1𝜎) phase jitter threshold [Kaplan and Hegarty, 2006] which can lead to cycle slips. In Figure 7 (right), the dependence of the phase jitter is shown as determined using 𝜎𝜙a and S4, the obtained pattern of phase jitter variation seems to be consistent with the results observed in Figure 7 (left) based on (S4) and the analogous index. However, based on the similar distributions and consistent correlation of the modeled and analytical Conker et al. [2003] determined phase jitters, it is clear that the analogous phase scintillation index is a reliable phase scintillation indicator and so could be used as the basis of a regional alarm index. Thus, the analogous phase scintillation index derived from the TEC 1 Hz data is a promising tool to indicate the level of ionospheric threat. Similar to our high-pass ﬁlter TEC based index and its correlation with scintillation Alfonsi et al. [2011] also showed how rate of change of the ROT together with its RMS value can be used to indicate scintillation conditions. The expected correlation between both ROT and RMS of ROT with scintillation is presented: the study highlights (i) high ROT and high RMS of ROT shows predominate phase and amplitude scintillation, (ii) high ROT and low RMS of ROT also represents high scintillation, while low ROT and low RMS of ROT do not indicate scintillation. two cases: phase jitter variation determined using the phase scintillation 𝜎𝜙and amplitude scintillation (S4) indices, and phase jitter variation determined using the analogous phase scintillation index (𝜎𝜙a) and amplitude scintillation index (S4). 4.1. Regional Alarming Index for Ionospheric Threat In Figure 7 (left), the standard deviation of the PLL error is shown with respect to 𝜎𝜙a and S4; it is clear from the ﬁgure that the inﬂuence of phase scintillation on the PLL error is signiﬁcantly larger than that of the amplitude scintillation, and this result is also consistent with the ﬁnding in Skone et al. [2005]. Figure 7 shows the standard deviation of the PLL error, for data collected from Trondheim (62.6◦N, 10.41◦E) on 17 March 2013 using a NovAtel GISTM receiver, for S4 and the standard scintillation index (Figure 7, left), and S4 and analogous phase scintillation index (Figure 7, right). According to the results in this ﬁgure, moderate (S4) and moderate phase scintillation can lead to 11◦phase jitter, while combined moderate (S4) and moderate to strong phase scintillation can lead to rather high values of the phase jitter that can exceed the 15◦(1𝜎) phase jitter threshold [Kaplan and Hegarty, 2006] which can lead to cycle slips. In Figure 7 (right), the dependence of the phase jitter is shown as determined using 𝜎𝜙a and S4, the obtained pattern of phase jitter variation seems to be consistent with the results observed in Figure 7 (left) based on (S4) and the analogous index. However, based on the similar distributions and consistent correlation of the modeled and analytical Conker et al. [2003] determined phase jitters, it is clear that the analogous phase scintillation index is a reliable phase scintillation indicator and so could be used as the basis of a regional alarm index. Thus, the analogous phase scintillation index derived from the TEC 1 Hz data is a promising tool to indicate the level of ionospheric threat. Similar to our high-pass ﬁlter TEC based index and its correlation with scintillation Alfonsi et al. [2011] also showed how rate of change of the ROT together with its RMS value can be used to indicate scintillation conditions. The expected correlation between both ROT and RMS of ROT with scintillation is presented: the study highlights (i) high ROT and high RMS of ROT shows predominate phase and amplitude scintillation, (ii) high ROT and low RMS of ROT also represents high scintillation, while low ROT and low RMS of ROT do not indicate scintillation. 4.1. Regional Alarming Index for Ionospheric Threat The standard deviation of the PLL error of the installed NovAtel GPS receiver (technical speciﬁcation of carrier tracking loop: third order, 15 Hz bandwidth) at Trondheim (62.6◦N, 10.41◦E) is compared under Figure 7. Standard deviation of PLL error due to amplitude and phase scintillation for data collected from Trondheim (62.6◦N, 10.41◦E) on 17 March 2013 using a NovAtel GISTM receiver for (left) S4 and the standard scintillation index and (right) S4 and analogous phase scintillation index. Figure 7. Standard deviation of PLL error due to amplitude and phase scintillation for data collected from Trondheim (62.6◦N, 10.41◦E) on 17 March 2013 using a NovAtel GISTM receiver for (left) S4 and the standard scintillation index and (right) S4 and analogous phase scintillation index. TIWARI AND STRANGEWAYS ©2015. The Authors. 80 Space Weather 10.1002/2014SW001115 Figure 8. (top) The standard deviation of estimated PLL error (green solid circles) for the NovAtel GPS receiver observed when Kp was above 5 at Trondheim for 1 year, blue solid circles show the analogous phase scintillation index for the same time period. (bottom) The same set of observation at Kp = 5. Figure 8. (top) The standard deviation of estimated PLL error (green solid circles) for the NovAtel GPS receiver observed when Kp was above 5 at Trondheim for 1 year blue solid circles show the analogous phase scintillation index for the Figure 8. (top) The standard deviation of estimated PLL error (green solid circles) for the NovAtel GPS receiver observed when Kp was above 5 at Trondheim for 1 year, blue solid circles show the analogous phase scintillation index for the same time period. (bottom) The same set of observation at Kp = 5. two cases: phase jitter variation determined using the phase scintillation 𝜎𝜙and amplitude scintillation (S4) indices, and phase jitter variation determined using the analogous phase scintillation index (𝜎𝜙a) and amplitude scintillation index (S4). In Figure 7 (left), the standard deviation of the PLL error is shown with respect to 𝜎𝜙a and S4; it is clear from the ﬁgure that the inﬂuence of phase scintillation on the PLL error is signiﬁcantly larger than that of the amplitude scintillation, and this result is also consistent with the ﬁnding in Skone et al. [2005]. 4.1. Regional Alarming Index for Ionospheric Threat However, there are some points where the phase jitter appears to be rather high but on an average; the pattern of the variation of phase jitter is quite similar to that of the analogous phase scintillation index. Based on the investigation made, it is found that the analogous phase scintillation can be used for alerting probable ionospheric threat to a ground-based GPS receiver at a high latitude in near real time. For a regional alert in the European region, GPS observations from the EUREF network were used in estimating the analogous phase scintillation; the solid red triangles in Figure 9 indicates the geographic location of GPS stations capable of recording raw phase and code observations for both frequencies L1 and L2 at 1 Hz. The EUREF network has a dense network of dual-frequency GPS receivers; each receiver is capable of recording observations at 1 Hz. As discussed in the experimental setup methodology section, the mesh grid of 10◦× 5◦(longitude by latitude) producing 24 grid blocks delineated for the selected area map, i.e., 35◦to 65◦N and −15◦to 25◦E. The alarming regional analogous index is computed for every region by averaging the analogous phase scintillation indices for all the satellite paths existing for all the receivers in the region. Each region is selected to include a suﬃcient number of receiving stations to ensure good reliability of the determined regional analogous index. The alarming regional analogous phase index is the average of all the analogous phase indices observed within a 5 min period for the particular block. The time average of 5 min is employed based on the assumption that the ionosphere does not change signiﬁcantly in this time period. In Figure 9, the number given in each grid block represents the minimum number of GPS satellites visible on average at any location inside the grid block. The analogous phase index is derived every minute so that ﬁve such indices can be derived for every visible satellite in the averaging period. Then the total number of observations is 5 times the sum of the number of visible satellites at each receiver location in the respective grid block. The arithmetic mean of the analogous phase scintillation array indices in each region is then termed the regional analogous phase scintillation 𝜎R𝜙and is further used in deﬁn- ing the ionospheric threat level. 4.1. Regional Alarming Index for Ionospheric Threat Figure 8 presents two sets of observations for two diﬀerent Kp values, the analogous phase scintillation index and the determined phase jitter observed for a period of 1 year at Trondheim for Kp>5 in Figure 8 TIWARI AND STRANGEWAYS 81 ©2015. The Authors. Space Weather 10.1002/2014SW001115 Figure 9. EUREF network, red solid triangles are GPS stations capable of recording raw observations at 1 Hz. Grid of 10◦× 5◦ considered, the numeric value in each grid is the number of satellites viewed above 20◦elevation. Figure 9. EUREF network, red solid triangles are GPS stations capable of recording raw observations at 1 Hz. Grid of 10◦× 5◦ considered, the numeric value in each grid is the number of satellites viewed above 20◦elevation. (top) and for Kp=5 in Figure 8 (bottom). For Figure 8 (top) data, the modeled phase jitter determined (green solid circle in Figure 8) is large between 00:00 and 04:0 UT and also is the analogous phase scintillation index over this period. For the premidnight period, a similar kind of pattern is observed. This indicates the presence of auroral storms both premidnight and postmidnight which have their eﬀect on the PLL of the ground-based receiver. There are also some cases of high-phase jitter between 15:00 and 08:00 UT, (indicated by the blue solid circles) where the analogous phase scintillation index was also high. Data for moderate geomagnetic storm are also shown in Figure 8 (bottom), and the phase jitter and analogous phase scintillation index are similarly plotted. The analogous phase scintillation index appears weak most of the time as is the corresponding phase jitter as shown by the green solid circles. However, there are some points where the phase jitter appears to be rather high but on an average; the pattern of the variation of phase jitter is quite similar to that of the analogous phase scintillation index. Figure 9. EUREF network, red solid triangles are GPS stations capable of recording raw observations at 1 Hz. Grid of 10◦× 5◦ considered, the numeric value in each grid is the number of satellites viewed above 20◦elevation. (top) and for Kp=5 in Figure 8 (bottom). For Figure 8 (top) data, the modeled phase jitter determined (green solid circle in Figure 8) is large between 00:00 and 04:0 UT and also is the analogous phase scintillation index over this period. For the premidnight period, a similar kind of pattern is observed. 4.1. Regional Alarming Index for Ionospheric Threat This indicates the presence of auroral storms both premidnight and postmidnight which have their eﬀect on the PLL of the ground-based receiver. There are also some cases of high-phase jitter between 15:00 and 08:00 UT, (indicated by the blue solid circles) where the analogous phase scintillation index was also high. Data for moderate geomagnetic storm are also shown in Figure 8 (bottom), and the phase jitter and analogous phase scintillation index are similarly plotted. The analogous phase scintillation index appears weak most of the time as is the corresponding phase jitter as shown by the green solid circles. However, there are some points where the phase jitter appears to be rather high but on an average; the pattern of the variation of phase jitter is quite similar to that of the analogous phase scintillation index. (top) and for Kp=5 in Figure 8 (bottom). For Figure 8 (top) data, the modeled phase jitter determined (green solid circle in Figure 8) is large between 00:00 and 04:0 UT and also is the analogous phase scintillation index over this period. For the premidnight period, a similar kind of pattern is observed. This indicates the presence of auroral storms both premidnight and postmidnight which have their eﬀect on the PLL of the ground-based receiver. There are also some cases of high-phase jitter between 15:00 and 08:00 UT, (indicated by the blue solid circles) where the analogous phase scintillation index was also high. Data for moderate geomagnetic storm are also shown in Figure 8 (bottom), and the phase jitter and analogous phase scintillation index are similarly plotted. The analogous phase scintillation index appears weak most of the time as is the corresponding phase jitter as Figure 9. EUREF network, red solid triangles are GPS stations capable of recording raw observations at 1 Hz. Grid of 10◦× 5◦ considered, the numeric value in each grid is the number of satellites viewed above 20◦elevation. time as is the corresponding phase jitter as shown by the green solid circles. However, there are some points where the phase jitter appears to be rather high but on an average; the pattern of the variation of phase jitter is quite similar to that of the analogous phase scintillation index. time as is the corresponding phase jitter as shown by the green solid circles. 4.1. Regional Alarming Index for Ionospheric Threat The threat level is deﬁned based on three conditions, (i) 𝜎R𝜙< 0.2 rad (green) represents no ionospheric threat, (ii) 0.2 < 𝜎R𝜙< 0.4 rad (yellow to amber) represents a moder- ate level of threat, (iii)0.4 < 𝜎R𝜙rad (red) represents a severe ionospheric threat and probability of cycle slip occurrences. In order to validate the above index, a network of four GISTM receivers within Europe (solid red triangles in Figure 10) is used to measure the standard phase scintillation index for the L1 frequency. Figure 10 (top row) shows phase scintillation indices recorded from the GISTM receivers binned to a grid of 10◦× 5◦regions and averaged over 5 min. In order to have consistency with the derived regional analogous phase scintillation index, indices are averaged and binned in the same way; Figure 10 (bottom row) shows the regional TIWARI AND STRANGEWAYS ©2015. The Authors. 82 Space Weather 10.1002/2014SW001115 Figure 10. (top row) The phase scintillation index measurement from the GISTM receivers (red triangle is location of receivers) on 17 March 2013 from 18:00 to 18:25 during a geomagnetic storm period. (bottom row) The regional analogous phase scintillation index derived from EUREF GPS receiver for the same period. Figure 10. (top row) The phase scintillation index measurement from the GISTM receivers (red triangle is location of receivers) on 17 March 2013 from 18:00 to 18:25 during a geomagnetic storm period. (bottom row) The regional analogous phase scintillation index derived from EUREF GPS receiver for the same period. analogous phase scintillation index, 𝜎R𝜙derived from the EUREF GPS receiver data. In Figure 10 (top row), strong phase scintillation is observed at 18:00 UT, 18:05 UT, and 18:25 UT. This is due to a geomagnetic storm which can be identiﬁed from the geomagnetic ﬁeld variations in Figure 4 and lasts 25 min commencing at 18:00 UT and having a corresponding eﬀect on the L1 signal over this period seen from the strong phase scintillation indices shown in Figure 10. The regional analogous phase scintillation index (𝜎R𝜙) depicted in Figure 10 (bottom row) at 18:00 UT, 18:05 UT, and 18:25 UT are well correlated with the phase scintillation observed by the GISTM receivers. TIWARI AND STRANGEWAYS 5. Conclusion The variance of the dynamic phase error derived using high-pass ﬁltering of the TEC time derivative is used in the estimation of the phase jitter due to phase scintillation and further, the expected standard deviation of the PLL phase error estimated. The modeled PLL phase error variance was well correlated with the Conker et al. [2003] derived phase jitter for measurements made during geomagnetic storms. The two selected days were based on the storms recorded on 24 April 2012 and 17 March 2013, and the eﬀect of the auroral storms on the PLL can be identiﬁed using both models. The derived model was also veriﬁed statistically by considering 1 year of data from a high-latitude recording station; the following points are concluded based on this study: Strong geomagnetic storm (Kp>5): A linear correlation of modeled phase jitter was obtained using Conker’s formula; the correlation is 76.6% obtained based on a 95% conﬁdence of linear ﬁtting; for moderate geomagnetic storms (Kp=5): the correlation of 65.58% was obtained. The correlation for moderate storms is weaker than for the strong storms case (65.6%) but is still quite good. The time series of modeled and Conker phase jitters are also obtained, and the similarity in the pattern of the variation of both can be seen. A regional analogous phase scintillation index is derived using TEC at 1 Hz from the EUREF network enabling an expected variance of the phase error due to ionospheric phase scintillation. The regional analogous phase index is well correlated with the phase scintillation obtained from the GISTM receivers. Thus, this index could be useful in providing an alarm for high-scintillation receiving areas where there would be a threat to accurate GNSS positioning due to PLL phase errors or loss of lock. Considering the question of latency, ﬁrst, the obtained raw observations from EUREF passes through the quality check and is then used in the TEC estimation, all done in real time. The time required for ﬁltering the TEC derivatives is approximately 4 s. The estimation of the analogous phase index and the arithmetic mean of the analogous phase indices for one region take less than 1 s, and thus, the total latency of this determination for the whole region is less than 5 s. This could be reduced using parallel processing techniques. The TEC derived model has a number of advantages over the Conker et al. Space Weather Space Weather 10.1002/2014SW001115 A moderate-strong level of scintillation at Trondheim is observed at 21:10 UT, 21:20, and 21:25 UT as can be seen in Figure 5, while the variation in the Earth’s magnetic ﬁeld in Figure 4 is evidence of a moderate geomagnetic storm. Similar to the processes described in the previous paragraph, the measured scintillation index is obtained using the GISTM receivers and is shown in Figure 11 (top row). The regional analogous phase scintillation index is depicted in Figure 11 (bottom row) for the same time of 21:10 and 21:20 UT. The moderate level of the regional analogous phase index and scintillation at 21:25 UT can be seen to be well correlated comparing the right-hand ﬁgures in Figure 11 (top and bottom rows). The results obtained from the new index are consistent with the expected eﬀect of geomagnetic storms and also capable of measuring the corresponding level of threat. Considering potential applications, since these threats are produced in real time and the method based on a reliable GPS network, it can be used for alerting any GNSS users in the mapped high-latitude region to the threats. There can, however, be cases when not enough measurements are made in of the grid blocks, and so for that area the derived analogous phase scintillation index is not considered reliable. Insuﬃcient observations or lack of data in any mesh grid can lead to a false alarm and in order to avoid such a situation, we consider we need at least ﬁve observations (the threshold) in the 5 min period. In order to distinguish between a statistically reliable estimated regional analogous phase index and a nonreliable one, a grid boundary shown as a blue dotted line is included to denote this as shown in Figure 11. A minimum of ﬁve sets of observation are required for the index to be considered reliable; this helps users avoid a false alarm in that particular grid. 4.1. Regional Alarming Index for Ionospheric Threat Although, there is a slight diﬀerence in the modeled regional index compared with the measured scintillation, this is due to the fact that the number of observations used in the regional-based index are comparatively more than those from the GISTM network, but it is nevertheless considered to be good enough to alert the user in the given region. analogous phase scintillation index, 𝜎R𝜙derived from the EUREF GPS receiver data. In Figure 10 (top row), strong phase scintillation is observed at 18:00 UT, 18:05 UT, and 18:25 UT. This is due to a geomagnetic storm which can be identiﬁed from the geomagnetic ﬁeld variations in Figure 4 and lasts 25 min commencing at 18:00 UT and having a corresponding eﬀect on the L1 signal over this period seen from the strong phase scintillation indices shown in Figure 10. The regional analogous phase scintillation index (𝜎R𝜙) depicted in Figure 10 (bottom row) at 18:00 UT, 18:05 UT, and 18:25 UT are well correlated with the phase scintillation observed by the GISTM receivers. Although, there is a slight diﬀerence in the modeled regional index compared with the measured scintillation, this is due to the fact that the number of observations used in the regional-based index are comparatively more than those from the GISTM network, but it is nevertheless considered to be good enough to alert the user in the given region. Figure 11. (top row) The phase scintillation index measurement from the GISTM receivers (red triangle is location of receivers) on 17 March 2013 from 21:10 to 21:25 during a geomagnetic storm period. (bottom row) The regional analogous phase scintillation index derived from EUREF GPS receiver for the same period. Figure 11. (top row) The phase scintillation index measurement from the GISTM receivers (red triangle is location of receivers) on 17 March 2013 from 21:10 to 21:25 during a geomagnetic storm period. (bottom row) The regional analogous phase scintillation index derived from EUREF GPS receiver for the same period. TIWARI AND STRANGEWAYS TIWARI AND STRANGEWAYS ©2015. The Authors. 83 Acknowledgments Navig., 44(1), 25–34. Kaplan, E., and C. Hegarty (Eds.) (2006), Understanding GPS: Principles and Applications, 2nd ed., 184 pp., Artech House, Boston, London. Kintner, P. M., B. M. Ledvina, and E. R. de Paula (2007), GPS and ionospheric scintillations, Space Weather, 5, S09003, doi:10.1029/2006SW000260. Knight, M. F., and A. Finn (1998), The eﬀects of ionospheric scintillations on GPS, paper presented at 11th International Technical Meeting of the Satellite Division of The Institute of Navigation (ION GPS 1998), Nashville, Tenn., pp. 673–685. Krankowski, A., I. I. Shagimuratov, L. W. Baran, and I. I. 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https://openalex.org/W1984564835	https://bmcpsychiatry.biomedcentral.com/track/pdf/10.1186/1471-244X-7-S1-P2	English	null	Compulsory admission to a Portuguese psychiatric hospital: retrospective study of 497 involuntary admissions	BMC psychiatry	2,007	cc-by	578	BioMed Central BMC Psychiatry Open Access Poster presentation Compulsory admission to a Portuguese psychiatric hospital: retrospective study of 497 involuntary admissions Sofia Brissos1, Ana Carita2 and Fernando Vieira3 Address: 1Júlio de Matos' Psychiatric Hospital, Avenida do Brasil, 53, Lisbon, Portugal, 2Technical University of Lisbon, Faculty of Human Kinetics, Cruz Quebrada, Lisbon, Portugal and 3Department of Forensic Psychiatry, National Institute of Legal Medicine, Rua Bento Sousa, 3, Lisbon, Portugal BioMed Central BMC Psychiatry Open Access Poster presentation Compulsory admission to a Portuguese psychiatric hospital: retrospective study of 497 involuntary admissions Sofia Brissos1, Ana Carita2 and Fernando Vieira3 Address: 1Júlio de Matos' Psychiatric Hospital, Avenida do Brasil, 53, Lisbon, Portugal, 2Technical University of Lisbon, Faculty of Human Kinetics, Cruz Quebrada, Lisbon, Portugal and 3Department of Forensic Psychiatry, National Institute of Legal Medicine, Rua Bento Sousa, 3, Lisbon, Portugal BioMed Central Background respectively). The mean admission duration was 24 days, with patients being compulsorily detained for a mean of 12 days, and thereafter passing to voluntary treatment for another 12 days. Schizophrenia and psychosis not else- where specified constituted the most frequent diagnoses. g The Mental Health Act, implemented in 1998, establishes the principles of the Portuguese mental health policy and governs the compulsory admission of persons suffering from mental disorders. We examined factors concerning the civil commitment of patients in a Portuguese psychi- atric hospital, responsible for the admissions of a popula- tion of 1,202,000 inhabitants. Conclusion The results suggest a specific profile for the patient with high risk of psychiatric compulsory detention: a middle aged unmarried man, unemployed, with the diagnosis of schizophrenia without comorbid substance abuse, who is taken to hospital by the authorities due to aggressive or violent behavior, and needs treatment in hospital for about 3 weeks. Open A Poster presentation Compulsory admission to a Portuguese psychiatric hospital: retrospective study of 497 involuntary admissions Sofia Brissos1, Ana Carita2 and Fernando Vieira3 Address: 1Júlio de Matos' Psychiatric Hospital, Avenida do Brasil, 53, Lisbon, Portugal, 2Technical University of Lisbon, Faculty of Human Kinetics, Cruz Quebrada, Lisbon, Portugal and 3Department of Forensic Psychiatry, National Institute of Legal Medicine, Rua Bento Sousa, 3, Lisbon, Portugal Corresponding author from WPA Thematic Conference. Coercive Treatment in Psychiatry: A Comprehensive Review Dresden, Germany. 6–8 June 2007 Published: 19 December 2007 BMC Psychiatry 2007, 7(Suppl 1):P2 doi:10.1186/1471-244X-7-S1-P2 This abstract is available from: http://www.biomedcentral.com/1471-244X/7/S1/P2 © 2007 Brissos et al; licensee BioMed Central Ltd. Methods The case records of 497 from a total of 522 consecutive involuntary admissions to Júlio de Matos' Psychiatric Hospital between January 1, 2005 and December 31, 2006, were examined retrospectively to assess sociodemo- graphic, clinical and judicial characteristics. After exclud- ing 41 out of area admissions, 456 involuntary admissions of 412 patients were analyzed. BMC Psychiatry Open Access Results l Males accounted for 66% of the compulsory detentions, and the patient's age at the majority of admissions was between 35 and 44 years. The majority of admissions (56%) involved single persons, which were either unem- ployed (43%) or retired (30%). The majority of patients (63%) came from their home and were accompanied by the police (62%) and/or by their families (27%), due to aggressive or violent behavior (61%). In 14% of the cases there had been a suicide attempt, with men attempting suicide significantly more often than women. We found low levels of alcohol and drug abuse (19% and 14% Page 1 of 1 (page number not for citation purposes) Page 1 of 1 (page number not for citation purposes)
https://openalex.org/W4393060848	https://e-journal.uingusdur.ac.id/Muwazah/article/download/2140/1215	English	null	Stagnansi Keterpilihan Perempuan dalam Pemilu Indonesia: Sebuah Literature Review	Muwazah	2,023	cc-by	8,362	Article History Submitted: 2023-12-4 Reviewed: 2023-12-18 Approved: 2023-12-30 Abstract This study conducted a literature review study of articles on women's acceptability in elections in Indonesia, by analyzing around n = 299 articles from Scopus data base in the publication period of 2004 to 2022. The data obtained is then processed through the VOS Viewer software application as a data analysis tool, with some of the main menus used from VOS Viewer software (density visualization and overlay) so that visualization of features on the theme of the article related to women's acceptability in Indonesia is obtained. This bibliometric analysis Research shows that obtained articles that discuss women's electability in elections in Indonesia are quite varied although no one is so dominant, the dynamics of women's choice in a number of articles between 2004 to 2022 are not so specific about placing the issue of women's choice in the results of their research. However, this study can explain that women's electability in Indonesian elections is still strongly influenced by demands for women's representation policies, religious identity to Political liberalization through gender equality , gender gaps and women's social status. Therefore, the results of this study show that analysis through literature review will be very helpful and contribute to analyzing the achievements of women's political work in Indonesia. Keywords: Gender, Politics, Literature Review MUWA AH E-ISSN: 2502-5368 ISSN: 2085-8353 J U R N A L K A J I A N G E N D E R DOI: 10.28918/muwazah.v15i2.2140 Stagnancy of Women's Election in Indonesia: A Literature Review Fitriani Abbas1, Andy Arya Maulana Wijaya2, Herman Lawelai3, L.M Azh Sa’ban4, Hazel Jovita5 1,2,3,4 Universitas Muhammadiyah Buton, Indonesia 5 Mindanao State University-Iligan Institute of Technology, Philippines Correspondence: fitrianiabbas90@gmail.com MUWAZAH – Jurnal Kajian Gender (Volume 15, Nomor 2, Tahun 2023) MUWAZAH – Jurnal Kajian Gender (Volume 15, Nomor 2, Tahun 2023) political inclusiveness and gender equality (Amalia et al ., 2021; Bangun, 2018; Hillman, 2018) political inclusiveness and gender equality (Amalia et al ., 2021; Bangun, 2018; Hillman, 2018) political inclusiveness and gender equality (Amalia et al ., 2021; Bangun, 2018; Hillman, 2018) The democratic political foundation implemented in Indonesia can be seen through Law Number 7 of 2017 concerning Elections, which confirms the minimum representation of women in parliament at 30 percent. Meanwhile, in fact, women's participation in parliament is still very low (Bangun, 2018; Perdana & Hillman, 2020). According to the quota policy for women's participation in politics starting in the 2009 elections, the number of DPR RI legislative members a decreased by 101 members or a decrease from the previous election in 2004, then in the 2014 elections there were 97 female DPR RI members. Meanwhile, at the provincial level, the national average proportion of women only increased by 0.38% during the 2009 and 2014 elections, or if the percentage increase was from 15.94% to 16.32%. Figure 1 shows that from the 2004 to 2019 elections, the percentage of women elected was no more than 20 percent. Even though the women's electability rate is still below the minimum for women's representation in parliament, the trend of women's participation in elections tends to increase. Figure 1.1 Graph of Women's Electability from the 2004-2019 Election Sumber: kpu.go.id, 2023 Figure 1.1 Graph of Women's Electability from the 2004-2019 Election Sumber: kpu.go.id, 2023 This research seeks to fill the research gap in research related to politics and women through a literature review, in order to find out various information that can support studies related to the dynamics of women's representation, especially in Indonesia in the previous 4 election periods. From this, this research study was able to collect and find out very detailed information about women voters, including demographic information, voting behavior, and factors that influenced their vote. The importance of paying attention to the dynamics of quotas in party lists provided for women (Stokes, 2005 in Kurniawan, 2016) is a means for proportional gender mainstreaming. Therefore, the main idea behind establishing this quota is to ensure that women are included in politics (Dahlerup, 2007 in Umagapi, 2019). INTRODUCTION The relationship between women and politics has many unique interests in political research and its dynamics (Solihin, 2023). There are various studies that attempt to analyze it from various perspectives, including through surveys and the motives of women in politics. In addition, there are efforts to mainstream gender in politics in Indonesia, by achieving at least 30% representation of women in political activities at all levels. On the other hand, the significance of women's research through literature reviews has increased in the last few decades (Rahmat et al., 2022). Likewise, the facts influence research on women and politics along with changes in open and modern political culture (Fauzi et al., 2018), as part of the resistance to political monopoly in power which gives birth to agents of democratic change (Törnquist, 2006). In turn, equality in politics provides equal access for women and their participation in power structures (Graham Davies, 2005), which in several election periods in Indonesia has become a representation of the main issue that continues to increase both in terms of Stagnancy of Women's...251- 272, (Abbas, dkk) 251 MUWAZAH – Jurnal Kajian Gender (Volume 15, Nomor 2, Tahun 2023) However, this desire does not require a short period of time, as is currently happening with the quota for women's representation in parliament of 30 percent, which has not yet been realized to date (Parwati & Istiningdiah, 2020; Rasjidi, 2022) and a number of future studies still require more there is a lot of exploration, one of which is through literature reviews. This research seeks to accelerate a variety of information that is relevant to the discourse on women's electability in elections, on the other hand, as well as a developing discourse about the need for women in the world of politics. In this way, this research will provide an in-depth description of the discussion regarding the discourse on women's representation from various literature published in the reputable journal Scopus over a certain period of time, with the aim of bringing positive change in Indonesian politics (Aldary & Salamah, 2018). This type of research is a literature review research that uses big data as an open source, referring to large amounts of data volume (blade and baymap), speed (completed in real time) and variants. Full coverage but flexible enough to provide seamless insight into the population and /or certain relationships (Aldary & Salamah, 2018). In this way, this research can provide several benefits, including providing more detailed and accurate information about the dynamics of women's electability. In addition, this research can help identify factors that influence women's electability and how big data can help understand these dynamics in various study topics (Salahudin, 2019). There are several stages that must be completed before carrying out bibliometric analysis, including the data search process, bibliographic filtering, cross-checking bibliographic properties, and finally bibliometric analysis (Sadat & Lawelai, 2023). Several tools and websites help visualize the process. The data search technique uses an application or program known as Publish or Perish, or PoP, which is software that can retrieve data from Google Scholar Query articles to obtain citation information and convert it into various statistics. This program is used to search for data in the Scopus database and carry out journal searches. Scopus is a compilation of summaries of the world's major literature, with citations containing abstracts or summaries of academically reviewed scientific literature. MUWAZAH – Jurnal Kajian Gender (Volume 15, Nomor 2, Tahun 2023) If you look at the distribution of research in various countries (Chen, 2010 in Umagapi, 2019), it shows positive experiences and relationships between large women's quotas and increasing government spending on women's welfare (Pande and Ford, 2011 in Umagapi, 2019). Stagnancy of Women's...251- 272, (Abbas, dkk) 252 MUWAZAH – Jurnal Kajian Gender (Volume 15, Nomor 2, Tahun 2023) MUWAZAH – Jurnal Kajian Gender (Volume 15, Nomor 2, Tahun 2023) MUWAZAH – Jurnal Kajian Gender (Volume 15, Nomor 2, Tahun 2023) MUWAZAH – Jurnal Kajian Gender (Volume 15, Nomor 2, Tahun 2023) After obtaining the results and discussing them, the next step is to draw conclusions. VOSviewer software (http://www.VOSviewer.co m/) was used to display author co-occurrences of keywords, author contributions, international collaborations, and co-reference analysis citations. We also used the Bibliometrix R program (https://www.bibliometrix.org/) to calculate frequencies, percentages, and citations for each journal and country. “Biblioshiny” produces a global collaboration map as well as a number of other visualizations (related information, author collaboration) . The Scopus database was used as the basis for the research. A search query to find behavior change systems that had a technology component was applied. As a result, ( TITLE-ABS-KEY ( women's AND politics AND participation ) OR TITLE-ABS-KEY ( electoral AND dynamics ) OR TITLE-ABS-KEY ( gender AND elections ) OR TITLE-ABS-KEY ( women AND candidates ) OR TITLE -ABS-KEY (political AND representation) OR TITLE-ABS-KEY (gender AND gap AND in AND politics) OR TITLE-ABS-KEY (women AND in AND politics) OR TITLE-ABS-KEY (electoral AND gender AND quotas) OR TITLE-ABS-KEY ( political AND empowerment AND of AND women ) OR TITLE-ABS-KEY ( women's AND electoral AND success ) AND TITLE-ABS-KEY ( "Indonesia" ) ) AND PUBYEAR > 2003 AND PUBYEAR < 2023 AND ( LIMIT-TO ( PUBSTAGE , "final" ) ) AND ( LIMIT-TO ( LANGUAGE , "English" ) ) AND ( LIMIT- TO ( SRCTYPE , "j" ) OR LIMIT-TO ( SRCTYPE , "p" ) ). Articles published between 2004 and 2022. Procedures for integrating data are described further below. Scopus returned 299 articles, in response to search queries. The Electability of Women in Scientific Discussions: Between Policy and Political Policy This research begins its analysis by displaying ten subsections of data obtained using bibliometrics, by referring to the distribution of articles published and downloaded on the Scopus database in the publication years from 2004 to 2022 with n=299. The type of data obtained is classified into document type including year of publication, number of articles, author, number of references to the article, country coverage, and author affiliation. Then distribute the data, select keywords through analysis using Vos-Viewer analysis. MUWAZAH – Jurnal Kajian Gender (Volume 15, Nomor 2, Tahun 2023) Bibliometric analysis methodology is used to identify trends in publications released in 2020-2021 by utilizing a quantitative approach to collect data from internet databases to determine publications on modeling and artificial intelligence issues (Prihatini, 2018, 2019b, 2019c, 2019a, 2020a, 2020b). in management (Iriyani et al., 2023). Scientific output is currently evaluated using bibliometric analytical techniques based on the number of citations, keywords, geographic data, author collaboration, and disciplinary interactions (Istiana, 2022). Fundamental data include annual publication output, publishing country, research topics, number of citations, keyword co-occurrence, co-authoring, and collaboration networks between countries and institutions. This research data was collected through documentation, namely data from Scopus for 2004-2022. We used keywords related to women's electability and big data to extract bibliometric data from the Scopus database. We assessed and abstracted articles after extracting available literature to see whether they were eligible for bibliometric analysis. The search procedure was limited in 2020 and 2021 when the COVID-19 epidemic emerged in December 2019. The aim was to make data distribution and graphic display easier. In addition, the data is sorted into groups depending on the factors studied and presented in tabular form. 253 Stagnancy of Women's...251-272, (Abbas, dkk) Source: Analisis Dataset Vos Viewer "publication year", 2023 Source: Analisis Dataset Vos Viewer "publication year", 2023 Based on the data presented in the image above, it can be explained that over the last 2 (two) decades there has been a continuous increase in the number of article publications on Scopus with women and politics issues, although in some years there have been fluctuations, this illustrates a trend of increasing articles with women's issues. On the other hand, in the last few years (2019-2022) there has been a significant increase in the number of article publications on the issue of women's electability. The largest increase in this time period occurred in 2019 with 46 articles. However, in the following year or 2020 there was a decrease in the number of publications compared to the previous year. Therefore, the trend of published articles discussing women and politics issues in the four election periods in Indonesia continues to increase with the highest number in 2019 but gradually decreasing until 2022 with a total of 31 articles. y Article Publication Year This research is based on bibliometric analysis on Scopus article data with the issue of women's electability, based on the year the article was published in the period 2004 to 2022. From the data obtained, the year of publication was then classified according to four time sections over a 5 year period. The trend in the number of publications based on year of publication can be observed in Figure 1, as follows; Figure 1.2 Number of publications based on Publication Year Stagnancy of Women's...251-272, (Abbas, dkk) 254 Figure 1.2 Number of publications based on Publication Year Figure 1.2 Number of publications based on Publication Year Stagnancy of Women's...251-272, (Abbas, dkk) Stagnancy of Women's...251-272, (Abbas, dkk) 254 MUWAZAH – Jurnal Kajian Gender (Volume 15, Nomor 2, Tahun 2023) MUWAZAH – Jurnal Kajian Gender (Volume 15, Nomor 2, Tahun 2023) Profile article The next research stage was to create a distribution map of publishers based on the n=299 articles obtained. From the bibliometric search results based on publishers, 157 publishers were obtained with a varied distribution of the number of articles. This shows that, although the issue of women and politics is widely discussed in article s, this issue within the scope of journal publications has not yet become the main idea for accredited journals. The results of mapping the number of article publications based on publishers can be seen in table 1, as follows. Table 1. Number of Article Publications Based on Publisher Table 1. Number of Article Publications Based on Publisher Table 1. Number of Article Publications Based on Publisher Publisher Number of Article Percentage Journal of Current Southeast Asian Affairs 12 4% Indonesia And the Malay World 8 3% Journal Of International Women’s Studies 6 2% South East Asia Research 6 2% Asian Journal of Women S Studies 5 2% International Journal of Innovation Creativity and Change 5 2% Women’s Studies International Forum 5 2% Asian Studies Review 4 1% International Journal of Scientific and Technology Research 4 1% Iop Conference Series Earth and Environmental Science 4 1% Asia Pacific Journal of Anthropology 3 1% Asian Women 3 1% Austrian Journal of South East Asian Studies 3 1% Indonesian Journal of Applied Linguistics 3 1% Journal of Contemporary Asia 3 1% Source: Vos Viewer Analysis Results, 2023 Stagnancy of Women's...251- 272, (Abbas, dkk) 255 MUWAZAH – Jurnal Kajian Gender (Volume 15, Nomor 2, Tahun 2023) MUWAZAH – Jurnal Kajian Gender (Volume 15, Nomor 2, Tahun 2023) MUWAZAH – Jurnal Kajian Gender (Volume 15, Nomor 2, Tahun 2023) Regarding the data on the distribution of the number of articles and publishers that have been obtained, this research took the top 15 publishers with the highest number of published articles. Where the Journal of Current Southeast Asian Affairs has published 12 articles on the theme of women's electability in elections in the period 2004 to 2022, while Indonesia and the Malay World has published 8 articles, the Journal of International Women's Studies and South East Asia Research have 6 articles. Apart from that, the Asian Journal of Women's Studies, International Journal of Innovation Creativity and Change and Women's Studies International Forum published 5 articles. Meanwhile, 4 articles were published through the journals Asian Studies Review, International Journal of Scientific and Technology Research and Proceedings of the Iop Conference Series Earth and Environmental Science, and 3 articles were published respectively through the journals Asia Pacific Journal of Anthropology, Asian Women, Austrian Journal of South East Asian Studies, Indonesian Journal of Applied Linguistics and Journal of Contemporary Asia. Author Profile Based on the distribution of authors discussing the issue of women's electability in Scopus indexed articles, this research highlights the authors who published the most articles on relevant themes. Therefore, we obtained 11 authors who had published more than 1 article writing about the issue of women's electability in the period 2004 to 2022. An illustration of this data can be seen in the following figure; Figure 1.3 Author data based on number of publications Source: Vos Viewer Dataset Analysis "author", 2023 Source: Vos Viewer Dataset Analysis "author", 2023 The distribution of these authors includes Prihatini with 5 articles (Prihatini, 2018, 2019a, 2019b, 2020a, 2020b). Authors who published 3 articles include Aspinal, E (Aspinall et al., 2011, 2021), Törnquist, O., (Törnquist, 2006, 2007; Törnquist et al., 2017); McGregor, K (K. McGregor, 2012; K. E. McGregor & Hearman, 2007; K. McGregor & MacKie, 2018). Meanwhile, authors who only published 2 articles include Darwin, .I., (Darwin, 2017; Darwin & Haryanto, 2021); Dewi, K.H (Dewi, 2019, 2020); Hilman, B. (Hillman, 2017, 2018); Choi, N., (Choi, 2007, 2019); Drake, P. (Drake, 2013, 2018). And, authors who only published 1 article include Savirani, A. (Savirani et al., 2021) and Beta A.R (Beta, 2019). Document Profile Based on Publication Stagnancy of Women's...251-272, (Abbas, dkk) 256 MUWAZAH – Jurnal Kajian Gender (Volume 15, Nomor 2, Tahun 2023) A number of published articles on the theme of women's electability in elections, based on the bibliometric analysis carried out, can be classified as quite religious. This can be seen in the following picture; Figure 1.4 Percentage of Document Types Based on Article Publication Source: Analisis Dataset Vos Viewer "document type", 2023 yp Based on the figure above, this research classifies the types of documents that have been published with relevant themes, the most numerous being documents of the Article type, 260 documents, then 19 documents in Review form, then 15 documents published in Conference Paper form, Next, there are 2 documents in the form of notes, 2 erratums and 1 conference review document. In this way, through the distribution of documents that have been published on themes relevant to the issue of women's electability, namely articles, this indicates that the majority of researchers are interested in conducting academic studies on the theme of women's electability in their Scopus publications. Number of Article References Furthermore, from a number of articles obtained in the Scopus database with the issue of women's electability. This research organizes these articles based on articles that have the most citations through the top 10 articles. This can be noted in: Table 2. Number of Citations by Document Document title Source Cited by Direct Democracy and Local Public Goods: Evidence from A Field Experiment in Indonesia Author: (Olken, 2010) Transnational Domestication: State Power and American Political Science Review 163 Indonesian Migrant Women in Saudi Arabia. Author: (Silvey, 2004a) The familial technique for linking maternal death with poverty Author : (Graham et al., 2004) Tigers and gangsters: Masculinities and feminised migration in Indonesia Author:(Elmhirst, 2007) Political Geography 145 Lancet 91 Population, Space and 85 Place Table 2. Number of Citations by Document Table 2. Number of Citations by Document Stagnancy of Women's...251- 272, (Abbas, dkk) 257 MUWAZAH – Jurnal Kajian Gender (Volume 15, Nomor 2, Tahun 2023) MUWAZAH – Jurnal Kajian Gender (Volume 15, Nomor 2, Tahun 2023) Document title Source Cited by Development for whom? Social justice and the business of ecotourism Author:(Schellhorn, 2010) Representing world history in the 21st century: The impact of 9/11, the Iraq war, and the nation-state on dynamics of collective remembering. Author :(Liu et al., 2009) Coping by Colluding: Political Uncertainty and Promiscuous Powersharing in Indonesia and Bolivia Author: (Slater & Simmons, 2013) The management of chronic hepatitis B in Asian Americans. Author : (Tong et al., 2011) Transnational migration and the gender politics of scale: Indonesian domestic workers in Saudi Arabia Author: (Silvey, 2004b) Awkward encounters and ethnography Author :(Koning & Ooi, 2013) Journal of Sustainable 83 Tourism Journal of Cross- Cultural Psychology 78 Comparative Political Studies 71 Digestive Diseases and 66 Sciences Singapore Journal of 60 Tropical Geography Qualitative Research in Organizations and 58 Management: An International Journal Source: Dataset Analicis Vos Viewer "citation", 2023 Awkward encounters and ethnography Author :(Koning & Ooi, 2013) Author :(Koning & Ooi, 2013) Source: Dataset Analicis Vos Viewer "citation", 2023 The number of citations for an article indicates that the article has become a reference for researchers, as well as having an impact on other research. From the results of this research analysis, it was found that there were 10 articles that had a number of citations above 50 citations in one published article. Number of Article References Articles with more than 100 citations include articles entitled Direct Democracy and Local Public Goods: Evidence from A Field Experiment in Indonesia by Olken, B.A. (2010) has a total of 163 citations and Transnational Domestication: State Power and Indonesian Migrant Women in Saudi Arabia by Silvey, R. (2004) has a total of 145 citations. Meanwhile, articles that specifically discuss the issue of women in politics with the highest number of citations include articles with the title Coping by Colluding: Political Uncertainty and Promiscuous Powersharing in Indonesia and Bolivia by Author: Slater, D., Simmons, E. (2013) with the number of citations is 71 and the article with the title Transnational migration and the gender politics of scale: Indonesian domestic workers in Saudi Arabia, by Silvey, R. (2004) with the number of citations is 60. Thus, this indicates that the theme of women and politics is still has become an issue that has been discussed a lot by a number of studies as well as being a reference for various similar articles. Therefore, concerns about women and politics can be categorized as an arena that is still very possible for further study and discussion. Distribution of Documents by Region Figure 1.5 Distribution of Documents by Region Source: Dataset Analicis Vos Viewer "country", 2023 The facts that can be established in this figure show that there is sufficient intensity for the study of women's electoral characteristics to spread to several countries or regions. In particular, according to a number of studies in Indonesia, it has the largest percentage (48%), but this does not necessarily guarantee that women's electability can increase optimally. The facts that can be established in this figure show that there is sufficient intensity for the study of women's electoral characteristics to spread to several countries or regions. In particular, according to a number of studies in Indonesia, it has the largest percentage (48%), but this does not necessarily guarantee that women's electability can increase optimally. Distribution of Documents by Region Stagnancy of Women's...251-272, (Abbas, dkk) 258 Document Distribution by University y y Furthermore, this research highlights the distribution of documents based on the university of origin of the author. This is to describe the rhythm of the activities of university researchers in studying the issue of women's choice in elections in Indonesia. From the data obtained, the top 3 universities from which the article authors came from were Gajah Mada University (20 articles), University of Indonesia (16 articles) and The Australian National University (12 articles), which can be seen in the table; Table 3. Document Distribution by University MUWAZAH – Jurnal Kajian Gender (Volume 15, Nomor 2, Tahun 2023) Furthermore, documents based on the author's country of origin or study area in the article are categorized into document distribution based on country. From these results, it was found that Indonesia, with 143 articles, dominated the distribution of documents discussing the issue of women in politics. Furthermore, the regions that were also highlighted in this research included Australia (50 articles), United States (37 articles), Singapore (19 articles) and United Kingdom (18 Articles). In addition, other authors are located in Malaysia, Germany, Canada, the Netherlands and New Zealand. This can be seen in Table 3 as follows; Figure 1.5 Distribution of Documents by Region Source: Dataset Analicis Vos Viewer "country", 2023 Number Of Document 160 140 120 100 80 60 40 20 0 48% 143 Indonesia 18% 50 Australia 12% 37 United States New Zealand 8 2% Netherlands 8 2% Canada 8 2% Germ any 9 3% Malaysia 10 4% United Kingdom 18 5% Singapore 19 6% Number of Publication By Country Figure 1.5 Distribution of Documents by Region Table 3. Document Distribution by University Stagnancy of Women's...251- 272, (Abbas, dkk) 259 MUWAZAH – Jurnal Kajian Gender (Volume 15, Nomor 2, Tahun 2023) MUWAZAH – Jurnal Kajian Gender (Volume 15, Nomor 2, Tahun 2023) MUWAZAH – Jurnal Kajian Gender (Volume 15, Nomor 2, Tahun 2023) Source: Dataset Analicis Vos Viewer "university", 2023 Source: Dataset Analicis Vos Viewer "university", 2023 However, themes highlighting women and politics also come from a number of universities in the Asian region, Australia and the United States. This indicates that the issue of women and politics is an issue that is also an academic study for many academics in many regions. Overall, there are several articles from various universities. And the highest number of articles is at Gadjah Mada University with 20 articles. MUWAZAH – Jurnal Kajian Gender (Volume 15, Nomor 2, Tahun 2023) MUWAZAH – Jurnal Kajian Gender (Volume 15, Nomor 2, Tahun 2023) When using VOSviewer, the number of relationships between them is set to several terms. VOSviewer analysis, there are several groups, namely red, green, orange, blue, yellow and purple. Which shows the relationship between one topic and another topic. VOSviewer can display bibliometric mapping in three ways, namely: Network Visualization (Figure 5), Overlay Visualization (Figure 7), and density visualization (Figure 6). Colored circles are used to label authors. Circle size correlates well with the presence of keywords in the title and abstract. Consequently, the frequency of occurrence affects the size of letters and circles. The larger the letters and circles, the more often the keyword appears. In picture no. 5 illustrates that the author who discusses women's electability the most is Aspinal, E. Norris. P, Blackburn. S. Scope of Publication and Authors Publication coverage is intended as a form of network analysis of the number of researchers and research themes that are connected to each other, either with the same keywords or relevant journal study fields. From this analysis, it can provide a match between the number of publications, authors and themes that are connected, so that the network can guide research to visualize the most discussed keywords. Figure 1.6 Visualization of research topics using VOSViewer according to scope of number of publications Source: Data Processing using VOSviewer, 2023 Figure 1.6 Visualization of research topics using VOSViewer according to scope of number of publications Source: Data Processing using VOSviewer, 2023 Stagnancy of Women's...251-272, (Abbas, dkk) 260 Analysis Density Visualization The next step is to use Density Visualization Analysis or analysis unit density visualization, where this refers to the distribution of publishers/journals that have an interest (scope) of publications that are mutually relevant to the keywords that have been obtained previously. In this step, it will help the research to identify a number of journals or publishers that have a concentration on women's issues, as well as providing an overview of the area where women's issues are studied. Publisher No. of Articles Percentage Journal Of Current Southeast Asian Affairs 12 4% Indonesia And the Malay World 8 3% Journal Of International Womens Studies 6 2% Publisher No. of Articles Percentage Journal Of Current Southeast Asian Affairs 12 4% Indonesia And the Malay World 8 3% Journal Of International Womens Studies 6 2% Stagnancy of Women's...251- 272, (Abbas, dkk) 261 MUWAZAH – Jurnal Kajian Gender (Volume 15, Nomor 2, Tahun 2023) MUWAZAH – Jurnal Kajian Gender (Volume 15, Nomor 2, Tahun 2023) South East Asia Research 6 2% Asian Journal of Women S Studies International Journal of Innovation Creativity 5 5 2% 2% International Journal of Scientific and Technology Research Iop Conference Series Earth and Environmental Science 4 1% 4 1% Figure 1.7 Keyword visualization using VOSviewer with a density visualization approach to the number of publications in journals Source: Data Processing using Vos Viewer, 2023 and Change Women S Studies International Forum 5 2% Asian Studies Review 4 1% MUWAZAH – Jurnal Kajian Gender (Volume 15, Nomor 2, Tahun 2023) Meanwhile, the figure above illustrates the level of research from 2004 to 2022, where the most publications are currently in Southeast Asian journals. The frequency of keywords in (figure 1.7) was used to determine the most frequently found studies in this research. First, we ran keywords in VOSViewer that most emphasized the terms frequently cited in the research. And then arrange the words into a visualization overlay based on their frequency. As shown in the picture above, which shows the concentration of all the keywords in the article, overall, the most prominent points to be highlighted in certain issues in the study of women's electability in elections are found in the Journal of Current Southeast Asian Affairs, then Indonesia and The Malay World, and then Journal of International Women's Studies. Stagnancy of Women's...251-272, (Abbas, dkk) Figure 1.8 Visualization of keyword networks using VOSviewer according to Overlay Network Visualization Sumber: Data Processing Vos Viewer, 2023 Meanwhile, the image above illustrates the level of research in Indonesia, this shows that the darker the color appears, the more research is carried out. Based on analysis using VOSviewer as shown in the figure above, the keywords that often appear are women's status, political discourse, religious identity, women's representation, gender gap, and women's work. Therefore, researchers can help determine women's electability by conducting studies on the use of AI-based technology. A significant percentage of the news disseminated contains misinformation that has a negative impact on the cognitive and psychological health of readers. The analysis to be carried out on the problem of combining technology with AI can also be observed in various studies carried out by different scholars. Apart from that, the figure above shows that previous research with the keywords women's status, political discourse, religious identity, women's representation, gender gap, and women's employment has a fairly relative relationship. g y y Based on previous research, it can be concluded that the factor of women's electability in Indonesia is more about women's status, where there is still an old paradigm regarding women's position, women's work, and also political discourse where representation in carrying out roles and functions in politics is the responsibility of men. These keywords are most relevant to studies with relevant themes in this research conducted by Prihatini (Prihatini, 2018, 2019a, 2019b, 2020a, 2020b), examining a number of variables that show factors influencing the political role of women in Indonesia, starting from their relationship with understanding of religion to political liberalization through gender equality in politics in Indonesia from the colonial era until now. Overlay Network Visualization Visualization of a collection of article networks or Overlay Network Visualization is a description of a collection of keyword networks that represent the number of studies on a number of articles with certain themes. In analyzing this section, we can show response data from a number of studies on keywords with the theme of women's electability. This process provides a description of this research that the denser the colors and the more diverse the network colors that are visible indicate that the keyword has the strongest character in the analysis network. Stagnancy of Women's...251-272, (Abbas, dkk) 262 Stagnancy of Women's...251-272, (Abbas, dkk) 262 MUWAZAH – Jurnal Kajian Gender (Volume 15, Nomor 2, Tahun 2023) MUWAZAH – Jurnal Kajian Gender (Volume 15, Nomor 2, Tahun 2023) MUWAZAH – Jurnal Kajian Gender (Volume 15, Nomor 2, Tahun 2023) MUWAZAH – Jurnal Kajian Gender (Volume 15, Nomor 2, Tahun 2023) 2020; Sugiarto & Yuwanto, 2020) which continue to increase in strength but not substantially are an indicator that women are increasingly empowered in politics. 2020; Sugiarto & Yuwanto, 2020) which continue to increase in strength but not substantially are an indicator that women are increasingly empowered in politics. Cultural factors are also a concern in a number of articles regarding women's involvement in Indonesia, for example (Lestari et al., 2019) revealing the limitations of Javanese women in understanding politics, or in understanding post-reform political inclusiveness which does not have much influence on ethnic Chinese politics in Indonesia (Chen , 2022), which is ultimately narrated as patriarchal beliefs that continue to haunt the situation of low representation of women in politics in Indonesia (Vasandani et al., 2022; ) Even so, it needs to be acknowledged that the various articles on the theme of women and politics in a number of studies published on Scopus have their own interest, especially in terms of women's popularity. (Ichsan Kabullah & Fajri, 2021) introduced the term neo- motherism as a victory for women in regional head election contestations, resulting in the strengthening of clientelism at the grassroots which also won for women (Mahsun et al., 2021). Apart from that, intellectual circles pay more attention to the election of women as a representation of the voice of support for the struggle for issues of empowerment and resolving cases of violence against women (Budoyo & Hardiyanti, 2021). Based on the literature review analysis carried out, this research can provide an explanation that a number of studies in Scopus articles in the publication period 2004 to 2022, n=299, can explain that the issue of women's electability in Indonesian elections is still very much influenced by demands for women's representation policies, religious identity. , gender gap and women's social status. However, it is also necessary to pay attention in further studies or research regarding the motivation of women to get involved in politics which seems to continue to increase over time. On the other hand, clientelism is beginning to strengthen in grassroots communities, which is heavily influenced by the roles of women, which has led to an increase in the level of trust in lower society towards women in politics. MUWAZAH – Jurnal Kajian Gender (Volume 15, Nomor 2, Tahun 2023) Therefore, the results of this research show that analysis through a literature review will be very helpful and contribute to analyzing the political achievements of women in Indonesia. Figure 1.8 Visualization of keyword networks using VOSviewer according to Overlay Network Visualization Sumber: Data Processing Vos Viewer, 2023 In line with other research explaining findings related to the existence of obstacles to women's political participation in Indonesia, including understanding religion (Fattore et al., 2010), the strong patriarchal culture of society (Dzuhayatin, 2020; Prajuli et al., 2021), structural and institutional factors that confront with women's domestic duties (Bayo, 2021; Christoff et al., 2017), even the blurred understanding of all parties regarding gender bias which still often occurs both in government and society (Badruddin et al., 2019), lack of financial support and political support ( Adnan & Amri, 2021), where even political parties only view women's involvement as a formality in fulfilling the women's quota policy (Angin & Purnomo, 2022) as well as the lack of access to information, resources and political education received by women's groups (Yanti et al., n.d.; Yumasdaleni et al., 2022) On the other hand, in several articles it was also found that there are political parties that have not provided sufficient portions for women's involvement (Rofhani & Fuad, 2021; Savirani et al., 2021). Apart from that, it also needs to be acknowledged that there is still a gender gap in knowledge and preferences. women's politics in Indonesia (Prihatini, 2018). Although a number of studies show that the influence of kinship also strengthens women's participation in politics, accompanied by the shadow of being the wife, niece, or family member of a regional political leader (Aspinall et al., 2021b; Marw ah & Catur Pratiwi, 2022), or simply an actress in political celebrations celebrities (Ahmad, Stagnancy of Women's...251- 272, (Abbas, dkk) 263 Conclusion Based on the literature review analysis, an analysis of articles on the theme of women's acceptability in elections in Indonesia has been carried out, using around n=299 articles from the Scopus database in the publication period from 2004 to 2022. The results of the data review using VOSviewer, which shows several dominant keywords in each existing cluster. This condition is in accordance with a number of previous studies which assist researchers in finding information about the theme being studied. Software called VosViewer is capable of analyzing length metrics and statistics of scientific journals such as the Journal of Gender Studies. It contains several topics that have strong ties to women's electability. The most published articles with relevant themes were Prihatini with 6 articles, then Olken., et al. is the author with the highest number of citations n=163. At the same time, the article comes from a university explaining that academics have the most to contribute. In this research, there is a gap in the study of women's electability in elections in Indonesia, especially in the variety of keywords found, and there are no keywords that are dominant enough. This illustrates that the dynamics of women's electability in a number of articles in the period 2004 to 2022 do not specifically place the issue of women's electability in their research results. However, this study can explain that women's electability in Indonesian elections is still strongly influenced by demands for women's representation Stagnancy of Women's...251-272, (Abbas, dkk) 264 MUWAZAH – Jurnal Kajian Gender (Volume 15, Nomor 2, Tahun 2023) MUWAZAH – Jurnal Kajian Gender (Volume 15, Nomor 2, Tahun 2023) policies, religious identity, gender gaps and women's social status. Therefore, this study provides recommendations for future research to be able to explain more about women's motivational factors in political participation and the strengthening of women's social capital in lower level communities. Bibliography Adnan, G., & Amri, K. (2021). Do gender empowerment and democracy reduce poverty rate? A cross-provinces evidence from western indonesia. 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https://openalex.org/W2101934115	https://europepmc.org/articles/pmc2631540?pdf=render	English	null	Uterine tumours resembling ovarian sex cord tumours: a case report	Cases journal	2,009	cc-by	1,459	Background trabeculae, solid, or tubular structures. If this element pre- dominates, the tumour is considered to be a uterine tumour resembling ovarian sex cord tumour(UTROSCTs), and may cause diagnostic difficulties. g Endometrial stromal tumours with sex-cord-like elements are relatively rare (account for 0.25% of all uterine malig- nancies). These tumours were first described by Clement and Scully in 1976 who presented 4 cases of such tumours and classified them into groups I and II. The groups are defined by the amount of sex cordlike elements.[1] The recent World Health Organisation classification 2003 rec- ognises low grade stromal sarcoma and undifferentiated endometrial sarcoma. Low grade sarcomas may exhibit other forms of differentiation, including smooth muscles and sex cord differentiation. In the latter form, the tumour contains epithelial-like or sex cord like elements, often with epithelioid appearance, arranged in nests, cords, Cases Journal Open Access BioMed Central BioMed Central Received: 14 November 2008 Accepted: 14 January 2009 © 2009 Aziz 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. © 2009 Aziz 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. Abstract Background: Endometrial stromal tumours with sex-cord-like elements were first described by Clement and Scully in 1976. The recent World Health Organisation classification 2003 recognises low grade stromal sarcoma and undifferentiated endometrial sarcoma. Case presentation: A 62-year-old caucasian woman presented with recurrent postmenopausal bleeding. She underwent a diagnostic hysteroscopy which showed a large polyp on the posterior uterine wall. Polypectomy was performed and the histological report of the polyp showed a complex adenomatous lesion of uncertain malignant potential. She underwent a total abdominal hysterectomy and bilateral oophorectomy. The patient is asymptomatic at the time being, and is having annual check ups. Conclusion: Total abdominal hysterectomy and bilateral ovarian oophorectomy is the best approach at this stage. These tumours should be considered as having an uncertain but low likelihood of recurrence. It is acknowledged that no extra uterine spread or distant metastases have been reported thus far. Ope Case Report Uterine tumours resembling ovarian sex cord tumours: a case report Omar Aziz, John Giles and Simon Knowles Address: Gynaecology Department, Yeovil District Hospital, Higher Kingston, Yeovil, Somerset BA21 4AT, UK Email: Omar Aziz - omaraziz78@hotmail.com; John Giles - john.giles@ydh.nhs.uk; Simon Knowles - simon.knowles@ydh.nhs.uk * Corresponding author Published: 14 January 2009 Cases Journal 2009, 2:55 doi:10.1186/1757-1626-2-55 Received: 14 November 2008 Accepted: 14 January 2009 This article is available from: http://www.casesjournal.com/content/2/1/55 © 2009 Aziz 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. Page 1 of 3 (page number not for citation purposes) Abbreviations UTROSCTs: Uterine Tumours Resembling Sex Cord Tumours; CD: Cluster designation (cluster of differentia- tion). Case report We report this rare case of carcinoma of the endometrium in a 62-year-old woman who presented with recurrent postmenopausal bleeding. Her medical condition was unremarkable. She underwent a diagnostic hysteroscopy which showed a large polyp on the posterior uterine wall. Polypectomy was performed and the histological report of the polyp showed a complex adenomatous lesion of uncertain malignant potential, therefore she had a stand- Page 1 of 3 (page number not for citation purposes) Page 1 of 3 (page number not for citation purposes) http://www.casesjournal.com/content/2/1/55 Cases Journal 2009, 2:55 Cases Journal 2009, 2:55 http://www.casesjournal.com/content/2/1/55 Discussion ard pelvic MRI staging scan which revealed no evidence of pelvic lymphadenopathy, cystic changes in the cervix and a lesion on the left side of the fundus which appeared to be associated with some thinning of the endometrium which could represent invasion, however, it was not full thickness and the MRI excluded extra-uterine spread. There are 2 uterine neoplasms that contain endometrial stroma and are variants of the usual endometrial stromal tumour. One is termed as uterine neoplasm resembling an ovarian sex cord tumour(UTROSCTs), and the other one, a combined smooth muscle stromal tumour[2] Most papers in the literature consider UTROSCTs to be rare variants of endometrial stromal tumours. Since they were first described 30 years ago, the rarity and limited fol- low up does not provide robust evidence upon which to base a definite management strategy. In the most recent WHO publication, these tumours have been separated from the main group of endometrial stromal and related tumours and placed in the category of "miscellaneous tumours", however, there is no guidance as to predictive or prognostic factors for the behaviour of these tumours in the current WHO fascicle, the management of these tumours is therefore debatable. One case report states that there is a potential for recurrence or even a metastasis especially if the tumours are invasive [3] The patient underwent a total abdominal hysterectomy and bilateral oophorectomy. The pelvis looked clear; both ovaries looked healthy with no lymphadenopathy, and peritoneal washings were taken and sent for cytology. Macroscopically the uterus showed a large soft peach col- oured polyp in the endometrial cavity 20 × 9 × 20 mm and a small yellow deposit in the myometrium 3 mm in size 2 mm adjacent to the polyp. Microscopically, the polyp was composed of cord, nests, trabecular and occasional glan- dular structure that were formed by small tumour cells that resembled the granulosa cells of ovarian granulosa cell tumours (see Figure 1). Occasional luteinized like cells with abundant foamy cytoplasm were seen only focally, thought to reflect leydig cell differentiation. The tumour immunostained with antibodies that recognize sex cord elements(inhibin, calretinin and CD99), epithe- lial differentiation (AE1/3 and MNF1165, but not EMA) and myoid (a mesenchymal marker) and WT1, but not CD10, a marker of endometrial stromal cells. All tumour cells expressed oestrogen and progesterone strongly. Conclusion h l Due to the lack of evidence about the best management for this type of tumour, total abdominal hysterectomy and bilateral ovarian oophorectomy is the best approach at this stage. The long-term clinical behavior of UTROSCT remains to be established. Although favorable histological features including well-circumscribed borders and an absence of vascular invasion are usually present, these tumors may on occasion show infiltrative borders and focal vascular invasion, albeit not to the extent that is characteristic of endometrial stromal sarcomas[3]. In the same study four of the cases have had favourable outcomes, but the dura- tion of follow up was limited. The authors therefore advo- cate that these tumours be considered as having an uncertain but low likelihood of recurrence. It is acknowl- edged that no extra uterine spread or distant metastases have been reported thus far. At the present, the patient is asymptomatic and is under- going a yearly check up with pelvic examination and ultra- sound scanning which have been normal so far. Microscopic image of the polyp found in the endometrial cav- ity Figure 1 Microscopic image of the polyp found in the endome- trial cavity. Consent A written consent has been obtained from the patient. Competing interests Competing interests The authors declare that they have no competing interests. Microscopic image of the polyp found in the endometrial cav- ity Figure 1 Microscopic image of the polyp found in the endome- trial cavity. Page 2 of 3 (page number not for citation purposes) http://www.casesjournal.com/content/2/1/55 http://www.casesjournal.com/content/2/1/55 Cases Journal 2009, 2:55 Authors' contributions OA wrote the original paper. JG treated the patient, revised and edited the manuscript. SK analysed pathology slides, revised and edited the manuscript. References 1. Clement PB, Scully RE: Uterine tumors resembling ovarian sex- cord tumors. Am J Clin Pathol 1976, 66:512-525. J 2. Kuruvila Sarah, Samarasinghe Dushyanti, Thomas Sasomma: Uterine neoplasm resembling an ovarian sex cord tumor. Saudi Med J 2003, 24(2):221-223. ( ) 3. Irving JA, Carinelli S, Prat J: Uterine Tumour resembling ovarian sex cord tumours are polyphenotypic neoplasms with true sex cord differentiation. Mod Pathol 2006, 19:17-24. 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 3 of 3 (page number not for citation purposes) Publish with BioMed Central and every scientist can read your work free of charge
https://openalex.org/W4320405680	https://figshare.com/articles/thesis/The_effect_of_binge-like_ethanol_exposure_on_adolescent_performance_in_non-spatial_and_spatial_forms_of_the_novel_object_recognition_paradigm/17018819/1/files/31474886.pdf	English	null	The effect of binge-like ethanol exposure on adolescent performance in non-spatial and spatial forms of the novel object recognition paradigm	null	2,021	cc-by	17,959	THE EFFECT OF BINGE-LIKE ETHANOL EXPOSURE ON ADOLESCENT PERFORMANCE IN NON-SPATIAL AND SPATIAL FORMS OF THE NOVEL OBJECT RECOGNITION PARADIGM BY MIRIAM COLLINS Table of contents Page Acknowledgements .................................................................................................... 1 Abstract………………………………. ..................................................................... 2 List of figures and tables………………………………. ........................................... 3 Introduction…....…………………………………………………………………… 5 The current study…………………………………………………………………… 20 Method……………………………………………………………………………… 21 Results and discussion……………………………………………………………… 25 General discussion ..................................................................................................... 43 References…… .......................................................................................................... 50 1 1 Acknowledgements Acknowledgements This thesis is dedicated to my dad. I would like to thank my supervisor Susan Schenk for her guidance throughout my MSc, as well as Joyce Colussi-Mas, Sophie Muller, and Michael Roberts for their help with the technical side of things. I also want to thank all the people I did CBNS with for enrolling in the course at the same time as me and making the last two years a bit more colourful and exciting. 2 2 Abstract Previous research has demonstrated that ethanol produces differential effects on non-spatial or recognition memory and spatial memory; spatial memory deficits were consistently found to be more persistent than non-spatial memory deficits. Ethanol-produced deficits have also been found to be dependent on age at exposure, and exposure during adolescence produced more persistent deficits than when exposure was experienced by older subjects. The current study investigated the effects of a “binge-like’ 5 day episode of ethanol exposure (1.0g/kg x 5) on performance in non-spatial and spatial forms of the novel object recognition (NOR) task. Subjects were exposed either during adolescence or following maturity. Tests were conducted 2 or 9 days following exposure. NOR was tested following inter-trial intervals of 1, 3, or 5 minutes. Data from mature rats could not be obtained or analysed due to procedural issues that precluded NOR measurement. Control rats failed to demonstrate NOR at any of the time intervals. Reasons for these negative findings are discussed. 3 3 List of figures and tables List of figures and tables Table 1. Cycle 1 Novel Object Recognition (NOR) task items. e 2. Cycle 2 Novel Object Recognition (NOR) task items. Table 2. Cycle 2 Novel Object Recognition (NOR) task items. Table 2. Cycle 2 Novel Object Recognition (NOR) task items. Figure 1. Time spent on the old rota-rod following daily administration of saline (0.9%, IP) or ethanol (1g/kg, IP) at time zero, twenty, and forty. Figure 1. Time spent on the old rota-rod following daily administration of saline (0.9%, IP) or ethanol (1g/kg, IP) at time zero, twenty, and forty. Table 3. Analysis of variance following acute administration of saline or ethanol on initial rota-rod apparatus performance. Table 3. Analysis of variance following acute administration of saline or ethanol on initial rota-rod apparatus performance. Figure 2. Time spent on the second rota-rod apparatus following daily administration of saline (0.9%, IP) or ethanol (1g/kg, IP) at time zero, twenty, and forty. Table 4. Analysis of variance of rota-rod performance following acute administration of saline or ethanol. Figure 3. Number of approaches to each object in T1 (1) and T2 (2) for saline (A) and ethanol (B) treated animals across each cycle 1 non-spatial novel objection recognition trial. Figure 3. Number of approaches to each object in T1 (1) and T2 (2) for saline (A) and ethanol (B) treated animals across each cycle 1 non-spatial novel objection recognition trial. Figure 4. Proportion of number of approaches to duplicate objects in T1 and replacement novel objects in T2 for saline (A) and ethanol (B) treated animals across each cycle 1 non- spatial novel objection recognition trial. Figure 5. Time spent exploring both objects in T1 (1) and T2 (2) for saline (A) and ethanol (B) treated animals during the cycle 1 non-spatial objection recognition task. Figure 6. Proportion of time spent exploring duplicate objects in T1 and replacement novel objects in T2 for saline (A) and ethanol (B) treated animals during the cycle 1 non-spatial novel objection recognition task. 4 Figure 7. Number of approaches to each object in T1 (1) and T2 (2) for saline (A) and ethanol (B) treated animals across each cycle 1 spatial novel objection recognition trial. Figure 8. Proportion of number of approaches to duplicate objects in T1 and replacement novel objects in T2 for saline (A) and ethanol (B) treated animals across each cycle 1 spatial novel objection recognition trial. Figure 9. List of figures and tables Time spent exploring both objects in T1 (1) and T2 (2) for saline (A) and ethanol (B) treated animals during each trial of the cycle 1 spatial novel objection recognition task. Figure 10. Proportion of time spent exploring duplicate objects in T1 and replacement novel objects in T2 for saline (A) and ethanol (B) treated animals during the cycle 1 spatial novel objection recognition task. Table 5. Significant effects obtained from analysis of various measures of performance in Cycle 2 non-spatial NOR trials. Table 5. Significant effects obtained from analysis of various measures of performance in Cycle 2 non-spatial NOR trials. Table 5. Significant effects obtained from analysis of various measures of performance in Cycle 2 non-spatial NOR trials. Table 6. Significant effects obtained from analysis of various measures of performance in Cycle 2 spatial NOR trials. Table 6. Significant effects obtained from analysis of various measures of performance in Cycle 2 spatial NOR trials. 5 Alcohol is one of the most commonly abused drugs in the world (Centers for Disease Control and Prevention, 2014), and this abuse contributes to approximately 1/10 deaths in those aged 20-64, and 3.3 million deaths worldwide each year (World Health Organisation, 2015). Harmful use of alcohol can also play a causal role in over 200 diseases, such as mental illness (or the exacerbation of), heart disease, cancer, anaemia, liver cirrhosis, and pancreatitis, as well as various memory deficits (CDC, 2014; WHO, 2015). A binge drinking episode is defined as the consumption of a large quantity of alcohol in one session in order to become intoxicated. This is approximately 5 + drinks for men, and 4+ drinks for women (Foundation for a Drug-Free World, 2015). Multiple binge drinking episodes separated by withdrawal periods is defined as binge drinking in general, or chronic intermittent exposure (Hiller-Sturmhöfel and Swartzwelder, 2004). Binge drinking is a common activity in multiple western countries (Hiller-Sturmhöfel and Swartzwelder, 2004). In New Zealand adolescents on average have their first drink at 14.5 years of age, with 45% of secondary school students considering themselves to be regular drinkers, and 18% classifying as binge drinkers who drink to excess at least 3+ times a week (Adolescent Health Research Group, 2012). List of figures and tables Three types of elderly drinkers have been identified: early-onset drinkers, who have a continuing drinking problem that developed earlier in their lives, late-onset drinkers, who start drinking later in life, typically in response to negative experiences such as bereavement or retirement, and intermittent types, who drink to excess occasionally (Institute of Alcohol Studies, 2016). A longitudinal study on a group of mostly older-onset problem drinkers who had been assessed for treatment found that all subjects reported drinking most commonly 4 + drinks on a daily basis, similar to binge levels seen in adolescents (Christie, Bamber, Powell, Arrindell, and Pant, 2012). More research in this area is required to adequately address or the effect of later-onset drinking, as not much is known about the detrimental effects of alcohol on the elderly brain. List of figures and tables Adolescent binge drinking is also prevalent in the U.S.A., where underage drinkers consume more alcohol on average per drinking session than adults, and those aged 12-20 are responsible for the consumption of 11% of all alcohol consumed within the nation (Centers for Disease Control and Prevention, 2015). These figures are of concern as binge drinking can result in both short- and long-term impairments in cognition and memory due to persistent effects on the brain (Hiller- Sturmhöfel and Swartzwelder, 2004). This susceptibility of the human brain to the effects of alcohol abuse can differ depending on factors such as age and the developmental period of the brain. Adolescence is 6 one of the most critical periods of brain development. During this time there are alterations in neurochemistry, an increase in gray and white matter throughout the brain, increased development of different areas (e.g. prefrontal cortex) and development of circuity between different regions (e.g. fronto-subcortical circuitry development (Giedd, 2006). These processes can be interrupted by alcohol exposure, resulting in later life dysfunction of executive control, and disruption in the encoding, storage, and retrieval of memories due to shrinkage of the responsible areas such as the prefrontal cortex and hippocampus (Hiller- Sturmhöfel and Swartzwelder, 2004). At the other end of the developmental spectrum, alcohol consumption by retirees is also increasing (Institute of Alcohol Studies, 2016). The drastic change of post-employment life can result in increased alcohol consumption (Institute of Alcohol Studies, 2016), particularly if the individual was forced into retirement rather than choosing it for themselves (Gallo, Bradley, Siegel, and Kasl, 2001). Three types of elderly drinkers have been identified: early-onset drinkers, who have a continuing drinking problem that developed earlier in their lives, late-onset drinkers, who start drinking later in life, typically in response to negative experiences such as bereavement or retirement, and intermittent types, who drink to excess occasionally (Institute of Alcohol Studies, 2016). At the other end of the developmental spectrum, alcohol consumption by retirees is also increasing (Institute of Alcohol Studies, 2016). The drastic change of post-employment life can result in increased alcohol consumption (Institute of Alcohol Studies, 2016), particularly if the individual was forced into retirement rather than choosing it for themselves (Gallo, Bradley, Siegel, and Kasl, 2001). The hippocampus and spatial memory Hippocampal units differentially fire depending on the relative position of subjects within an environment (Best and Ranck, 1975; Hill, 1978; O'Keefe and Dostrovsky, 1971; Olton, Walker, and Gage, 1978; Thompson and Best, 1990). O'Keefe and Dostrovsky, 1971; Olton, Walker, and Gage, 1978; Thompson and Best, 1990). O'Keefe and Dostrovsky, 1971; Olton, Walker, and Gage, 1978; Thompson and Best, 1990). The hippocampus and spatial memory 7 Alcohol-related memory impairments may relate to drug-produced changes in the hippocampus (Ryabinin, 1998). Human studies have shown that those diagnosed with adolescent-onset alcohol use disorders have significantly smaller right and left hippocampal volumes compared to age-matched controls. These affects were negatively correlated with the disorder duration (De Bellis et al., 2000). On subjects with older onset alcoholism females demonstrated smaller bilateral hippocampus volumes, but males only had a significantly smaller right hippocampus (Agartz, Momenan, Rawlings, Kerich, and Hommer, 1999). Post- mortem analysis of human brain tissue also demonstrates that alcoholism results in a loss of white matter (Jensen and Pakkenberg, 1993), and decreased cortical gray matter density (Kril, Halliday, Svoboda, and Cartwright, 1997). On rodents both pre-natal (Barnes and Walker, 1981) and long-term chronic post- natal exposure (Walker, Barnes, Zornetzer, Hunter, and Kubanis, 1980) to ethanol results in a permanent reduction of CA1 and CA3 hippocampal pyramidal cells. A question remains as to whether these brain deficits are reflected in behavioural deficits. Spatial memory encodes information about spatial orientation within the surrounding environment (Burgess, Maguire, and O'Keefe, 2002) This type of memory permits navigation of familiar areas such as the route you take to drive to work (O'Keefe and Nadel, 1978), or in the case of animal studies the location of the food that was previously stored (Bird, Roberts, Abroms, Kit, and Cupri, 2003). This encoded spatial information can be stored as working, short and long term memories (Burgess, Maguire, and O'Keefe, 2002), and it is known that the hippocampus is involved in these processes (Burgess, Maguire, and O'Keefe, 2002). The hippocampus plays a role as the "cognitive map" of the brain, and the pyramidal hippocampal cells are the basic units of this map. These cells internally represent specific physical points in space and allow for referencing of the surrounding environment and ease of 8 8 navigation (O'Keefe and Nadel, 1978). Hippocampal units differentially fire depending on the relative position of subjects within an environment (Best and Ranck, 1975; Hill, 1978; navigation (O'Keefe and Nadel, 1978). Hippocampal units differentially fire depending on the relative position of subjects within an environment (Best and Ranck, 1975; Hill, 1978; O'K f d D t k 1971 Olt W lk d G 1978 Th d B t 1990) navigation (O'Keefe and Nadel, 1978). Human research assessing the effects of ethanol on spatial and non-spatial (recognition) memory Due to ethical constraints around the administration of alcohol to human subjects, alcoholism recovery status, and increased risk of injuries (NIAAA, 2015), research involving the effect of alcohol on memory in humans is often limited to studies with participants who already have an alcohol-related disorder. Studies often employ the Spatial Working Memory (SWM) task that presents the participant with a number of boxes on the screen which are opened by a process of elimination to locate tokens. Adolescents of both genders diagnosed with alcohol use disorder show a diminished blood oxygen level–dependent (BOLD) response on fMRI scans in the left precentral gyrus and bilateral cerebellar areas during the SWM task but performance on this task was not compromisd. These abnormalities in brain response were particularly enhanced in subjects who reported higher consumption levels of alcohol and/or hangover symptoms (Tapert et al., 2004). Those who qualified as "binge drinkers" (as defined by scores from the Alcohol Use Questionnaire) consistently displayed between-search errors in the Spatial Working Memory task (Townshend and Duka, 2005; Weissenborn and Duka, 2003; Scaife and Duka, 2009). Recognition memory comes under the umbrella of declarative or explicit memory, and refers to the ability to recall previous events, objects, and people encountered on a previous occasion. A second presentation of the stimulus is necessary in order for the subject to determine whether it is familiar, and does not require memory of the actual episode in 9 which the initial presentation occurred. This is unlike recall memory where the subject freely describes a previous encounter without any external stimuli to prompt them (Manns, Hopkins, Reed, Kitchener, and Squire, 2003). FAS children display a deficit in object recognition memory compared to age- matched controls when a delay is implemented, but not when recall is immediate (Uecker and Nadel, 1995). A case study on an amnesic individual with focal perinatal hippocampal damage revealed that when the subject was presented with a virtual town square scene and had to later recall object locations within the image from the same viewpoint, accuracy was similar to controls. However, when the subject had to recall object locations within the scene from a new viewpoint, performance was significantly impaired in contrast to controls (King, Trinkler, Hartley, Vargha-Khadem and Burgess, 2004). This indicates that different underlying processes are involved in recognition memory and memory involving spatial navigation. Human research assessing the effects of ethanol on spatial and non-spatial (recognition) memory Acute alcohol exposure has also been shown to have an effect on forced-choice picture recognition task performance in subjects who have not previously been diagnosed with an alcohol-related disorder. In a study by Parker, Birnbaum, and Noble (1976), subjects received either no alcohol, or a medium (0.5 mg/kg) or high (1 mg/kg) amount immediately prior to engaging in the task. Subjects were presented 40 images, at 4 second intervals. Acute alcohol exposure has also been shown to have an effect on forced-choice picture recognition task performance in subjects who have not previously been diagnosed with an alcohol-related disorder. In a study by Parker, Birnbaum, and Noble (1976), subjects received either no alcohol, or a medium (0.5 mg/kg) or high (1 mg/kg) amount immediately prior to engaging in the task. Subjects were presented 40 images, at 4 second intervals. Following this the familiar images were paired and presented with similar but novel distractor images, and subjects were asked to choose which of the images they had seen before. The results revealed a forgetting curve, with those who received the highest dose of alcohol correctly recognising the least amount of previously viewed images, followed by the medium dose group, and the group that received no alcohol. Following this the familiar images were paired and presented with similar but novel distractor images, and subjects were asked to choose which of the images they had seen before. The results revealed a forgetting curve, with those who received the highest dose of alcohol correctly recognising the least amount of previously viewed images, followed by the medium dose group, and the group that received no alcohol. 10 Studies with humans are limited due to ethical constraints, including the administration of an addictive substance, the potential danger of a subject hurting oneself when intoxicated due to weakened psychomotor functioning or inappropriate or dangerous behaviour due to lowered inhibitions (Tabakoff and Hoffman, 2000). The benefit of using animal models in alcohol research In animal models you can mimic both acute, chronic, and intermittent ethanol administration, while controlling for the one aspect of alcohol related behaviour you are interested in studying (such as memory functioning after a “binge-like” episode of drinking). This is done by minimising confounding variables, something which cannot be easily done in human research as you can never be certain about what subjects are doing outside of the time you see them. Researchers can also control both health and past substance use, important things which can inexplicably effect results when not mentioned or taken into consideration (Tabakoff and Hoffman, 2000). Rodents have similar biochemical and physiological systems to humans, so results produced by rodent models can be considered to have predictive validity, where by ethanol administration can be said to predict the same outcome in humans who have received an equivalent amount of the substance in an equivalent time period (e.g. intoxication, tolerance, dependence, organ damage). (Tabakoff and Hoffman, 2000). Past substance use can be controlled in animal models. This ensures that a change in cognition or behaviour is due to a treatment received within a study and not due to extraneous factors, something which could occur in a study with humans where a subject is not completely honest about their past or present medical history and/or substance use (Tabakoff and Hoffman, 2000). This is particularly beneficial for studying the effects of ethanol on cognitive and behavioural processes across ages. Though anatomical and physiological differences must be 11 taken into consideration, there are approximate equivalencies in ages that can be determined (Andreollo, Santos, Araujo, and Lopes, 2012). One month in the rat is approximately equal to 3 human years, with rats reaching the equivalence of 18 years at around 6 months of age. Earlier adolescence occurs at around 2-3 months, and 2.5 rat years equates to 65 human years (Andreollo et al., 2012). taken into consideration, there are approximate equivalencies in ages that can be determined (Andreollo, Santos, Araujo, and Lopes, 2012). One month in the rat is approximately equal to 3 human years, with rats reaching the equivalence of 18 years at around 6 months of age. Earlier adolescence occurs at around 2-3 months, and 2.5 rat years equates to 65 human years (Andreollo et al., 2012). Common paradigms used to assess spatial and non-spatial or recognition memory Numerous paradigms exist to measure different aspects of spatial memory in animals. The Morris Water Maze Task (MWMT) was originally designed to assess spatial working memory in particular, but has been adapted to also examine encoding and retrieval of short and long term spatial representations and recognition memory. In this paradigm a rodent is placed in an open field maze filled with opaque water with a hidden escape platform. The most common dependent variable measured is latency to escape the water maze. This can be measured across training sessions to analyse retention, depending on the amount of sessions and the time in between them (Morris, 1984). Successful performance in the MWMT is reliant on intact cognitive mapping abilities (Morris, 1984). Rats with hippocampal lesions displayed significantly poorer performance in the MWMT, as shown by an increase in latency to find the hidden platform, and a longer total distance travelled in the water. This deficit was persistent, and was still apparent following 28 trials (Morris, Garrud, Rawlins, and O'Keefe, 1982). Guinea pigs prenatally exposed to ethanol showed similar deficits (Richardson, Byrnes, Brien, Reynolds, Dringenberg, 2002). The MWMT has been modified so that it is a delayed matching to place (DMP) task, in that the subject must learn to locate a constantly changing hidden platform in opaque water. The task begins with a search trial where the animal discovers the hidden platform, followed by an inter-trial interval (ITI) or delay of alternating length and a testing phase 12 where the subject must remember the location of the hidden platform in order to escape. This assesses short-term spatial memory, and has been proved effective in revealing deficits produced by both hippocampal lesions (Steele and Morris, 1999) and ethanol exposure (Clements, Girard, Ellard, and Wainwright, 2005). However this version of the task requires at least one week of training (Steele and Morris, 1999). Object recognition memory specifically refers to the ability to differentiate between novel objects and familiar objects encountered on a previous occasion. Rodents can recognise objects through both visual, tactile, and olfactory cues (Manns and Eichenbaum, 2009).This form of memory is often assessed in animals using the delayed non-matching to samples (DNMS) task. Much like the DMP task, The DNMS task involves the presentation of a stimulus followed by an ITI and presentation of the old stimulus paired with a novel stimulus. Common paradigms used to assess spatial and non-spatial or recognition memory Subjects must visibly select the novel stimulus in the second presentation with many hours of training being required to reach criterion performance (Chudasama, 2010). The novel object recognition paradigm also assesses object recognition memory through presentation of familiar and novel stimuli. It is a one-trial task involving a familiarisation phase at time 1 (T1), during which the subject is placed in a chamber with two identical objects. The subject is then removed and one object is switched for a novel item or in the case of the spatial adaptation, one of the identical objects is moved to a new location within the arena. A delay or inter-trial interval (ITI) is then imposed, before the subject is returned to the chamber for the test phase at Time 2 (T2). The dependent measure is the amount of exploration of each object, which is recorded as both the amount of approaches and time spent with each object (Antunes and Biala, 2012). The primary purpose of the NOR task is to assess recognition memory after different delay times between T1 and T2 have been imposed. This paradigm is a useful and efficient 13 tool for testing memory as it requires no training. However, several aspects of the procedure have implications for unambiguous interpretation of the data. The habituation phase allows the subject to become familiar with the novel testing environment itself, reducing anxiety levels during the actual experimental trials and ensuring that the subjects will explore the objects rather than the environment itself (Silvers, Harrod, Mactutus, and Booze, 2007). It is important, therefore, to ensure a long enough habituation period. It is critical for subjects to explore both objects for a criterion amount of time during T1. Because some rats fail to explore, their data cannot be interpreted and so cannot be used. As a result, sample sizes can be substantially smaller than what one initially starts with. The minimum criterion for exploration time in the familiarisation phase varies widely between studies, with total exploration times as small as 10 seconds or more appearing sufficient enough to allow measurement of encoding and retention (Camarasa, Marimon, Rodrigo, Escubedo, and Pubil, 2008). Required times of up to 20 minutes have also been reported (Goulart et al., 2010). The NOR paradigm relies on the natural preference to explore novel items (Antunes and Biala, 2012). Common paradigms used to assess spatial and non-spatial or recognition memory In this respect, it does not depend on reinforcers, as do most other memory tasks. The procedure however is more taxing on cognitive load in comparison to tasks where the subject explores a single novel object or novel environment, as the subject must maintain the two identical T1 objects in their working memory in order to later discriminate between the familiar and novel objects in T2 (Silvers et al., 2007). A spatially modified version of this task that investigates novel location recognition memory also exists (Ennaceur and Meliani, 1992). This uses the same methodology in T1, with two identical objects being placed equidistantly apart from each other and the back wall of the testing arena, but in T2 both objects are placed back in the arena, with the sample 14 object in the same location as previously, and the duplicate object placed in a novel location (Ennaceur and Meliani, 1992). In both procedures memory is inferred when the subject explores the novel object/location more than the familiar object/location, due to rodents having an innate preference for novelty (Antunes and Biala, 2012). This preference for novelty occurs when memory of the familiar object/placement is easily accessible, shortly after exposure to the objects has occurred. Amount of contact with a novel object/placement is dependent on memory of the familiar object, thus allowing the subject to discriminate between the two objects. When the memory is in an intermediate phase and not as readily accessible both objects are explored equally, while if the memory is in a remote phase it is more difficult to access, resulting in forgetting of which object the subject has previously exposed to. The transition between these memory phases results in a forgetting curve, with the amount of contact with the novel object/placement decaying as the ITI time increases. (Antunes and Biala, 2012). There are several ways to measure and identify novel object recognition. The original measure as devised by Ennaceur and Delacour (1988) was time spent exploring both the novel and familiar objects in T2, with a longer length of time spent exploring the novel object being an indicator that the subject recognised the familiar object from previous encounters. Ennaceur (2010) later argued that the frequency of approaches to each object was a more appropriate measure of recognition memory, being more sensitive to both strain and gender differences. Common paradigms used to assess spatial and non-spatial or recognition memory Another measure is the recognition index, which can be a proportion of either the time spent exploring or approaches made to the novel object in T2, relative to total T2 exploration time or approaches (Antunes and Biala, 2012). This can be beneficial measure when there is a high amount of between subject variability in the amount of exploration time 15 15 or approaches made, as it allows the researcher to get a clearer picture of where the true preference lies across ITIs and groups. or approaches made, as it allows the researcher to get a clearer picture of where the true preference lies across ITIs and groups. The success of this differentiation between familiar and novel can be dependent on variables that alter memory encoding, consolidation, and/or retrieval, such as brain lesions or pharmacological substances (Antunes and Biala, 2012). Previous research shows that hippocampal lesions impair performance for object memory in the NOR paradigm. The success of this differentiation between familiar and novel can be dependent on variables that alter memory encoding, consolidation, and/or retrieval, such as brain lesions or pharmacological substances (Antunes and Biala, 2012). Previous research shows that hippocampal lesions impair performance for object memory in the NOR paradigm. Broadbent, Gaskin, Squire, and Clark (2010) investigated the effect of hippocampal lesions on NOR task performance. All subjects were allowed 12 five minute exploration sessions with two familiar objects followed by bilateral hippocampal lesions either 1 day, 4 weeks, or 8 weeks after the final exploration session. Two weeks post-surgery all subjects underwent a retention test. Both the 1 day and 4 week lesion groups displayed impaired object recognition memory, whereas the 8 week lesion group performed similarly to controls, displaying a novel object preference, suggesting that consolidation of memory had occurred. Broadbent, Gaskin, Squire, and Clark (2010) investigated the effect of hippocampal lesions on NOR task performance. All subjects were allowed 12 five minute exploration sessions with two familiar objects followed by bilateral hippocampal lesions either 1 day, 4 weeks, or 8 weeks after the final exploration session. Two weeks post-surgery all subjects underwent a retention test. Both the 1 day and 4 week lesion groups displayed impaired object recognition memory, whereas the 8 week lesion group performed similarly to controls, displaying a novel object preference, suggesting that consolidation of memory had occurred. Common paradigms used to assess spatial and non-spatial or recognition memory Studies assessing the effects of MDMA (Schenk, Harper, and Do, 2011), methamphetamine (Schröder, O'Dell, and Marshall, 2003), cocaine (Schindler, Li, and Chavkin, 2010), and ketamine (Goulart et al., 2010) on NOR performance also show that the negative impact drugs of abuse have on memory can be successfully measured in this paradigm. A meta-analysis of papers that investigated the role of the hippocampus in NOR performance, indicated that a forgetting effect is most consistently found following delays of 10 minutes (Cohen and Stackman, 2015). Normal rats aged post-natal day (PND) 29 and above displayed typical recognition memory profiles in the NOR paradigm following delays of up to 24 hours, but not following a delay of 48 hours (Reger, Hovda, and Giza, 2009). 16 There is limited research on the effect of ethanol on NOR paradigm performance, but one study by Stragier et al. (2015) found that C57BL/6J mice exposed to a chronic free- choice ethanol treatment for 21 days impaired NOR performance after both a 1 hour and 24 hour delay, an effect which was particularly pronounced after the 24 hour delay. However it is important to note that ITIs shorter than an hour in length were not included in this study. This might be critical since other studies (Schenk et al. 2011), have found MDMA caused memory deficits that are observed following shorter ITIs. The authors of the original novel object recognition paradigm paper have stated that a minimum time of 20 seconds exploration during T1 is adequate (Ennaceur and Delacour, 1988). Even if subjects meet or exceed the minimum exploration time, they can also be eliminated from further analyses if they fail to explore both objects in the testing phase, because if only one object is explored for the duration of the second phase it cannot truly be determined whether it is due to preference or another extraneous variable (Ennaceur and Delacour, 1988). Animal research assessing the effects of ethanol on spatial and non-spatial (recognition) memory A wide range of studies have linked both acute and chronic alcohol exposure to impairments in spatial memory in animals. There is evidence that both ethanol exposure and hippocampal damage also have a detrimental effect on object recognition memory, but contradictory findings exist as well. A dose-dependent memory impairment, as measured by NOR, was demonstrated in mice exposed to ethanol depending on when the ethanol was administered (Ryanbinin, Miller, and Durrant, 2002). Additionally, most subjects that were administered saline or the lower dose of ethanol spent more time exploring the novel object, whereas the majority of subjects that received the high ethanol dose spent a similar amount of 17 time with both objects. The interpretation of the results is difficult, however, because there was no habituation phase. Adolescent and adult rats given an aqueous ethanol solution for their sole source of fluid starting from PND 21 until either PND 90 or PND 180 showed significantly impaired DNMS performance when tested following a delay of an hour (but not delays of 1 or 15 minutes) (García-Moreno et al., 2002). Similar DNMS results were produced in monkeys with hippocampal lesions when delays were longer than 8 seconds (Zola et al., 2000), but no deficits were produced in monkeys with neonatal hippocampal lesions (Pascalis and Bachevalier, 1999). Animal models of FAS have also been shown to result in spatial memory deficits in later life. Matthews and Simson (1998) found that adult rats prenatally exposed to ethanol demonstrated longer latencies to find the platform in the spatial version of the MWMT, but only when a 3-day delay between training and testing was imposed rather than a 1 day delay. The same effect has also been found when tested without a delay (Kim et al., 1997). Rats that were prenatally exposed to ethanol were tested first in the DNMS followed by the spatial version of the MWMT. No significant difference between groups was found for DNMS performance, but for the MWMT prenatally exposed rats took significantly longer to learn find the submerged platform across all 10 daily sessions. Similar results have been found in animal studies with exposure to ethanol in adolescence and adulthood. Acute ethanol exposure greater than or equal to 1.5 g/kg resulted in longer latencies for locating the submerged platform in the spatial version of the MWMT for both age groups. Animal research assessing the effects of ethanol on spatial and non-spatial (recognition) memory However, in the non-spatial version of the paradigm, no significant differences were found between groups, demonstrating a dissociation in the effect of ethanol exposure on the two types of memory (Matthews, Morrow, Tokunaga, and McDaniel, 2002) 18 Similar patterns of performance in the spatial version of the MWMT were produced in mice that received ethanol (1.75 or 2.25 g/kg). There was no difference for the non-spatial version of the task (Berry and Matthews, 2004). Adult rats were given either a vehicle or binge alcohol treatment followed by additional vehicle or ethanol doses for four consecutive days. Rats were tested five days (after ethanol withdrawal symptoms ceased), and ten weeks following this exposure. Within ten consecutive sessions (at both testing periods), rats underwent an object exploration task (OET) for both spatial memory and object recognition. The treatment produced an impairment in detecting spatial change. The ethanol treatment produced deficits in the non- spatial memory OET sessions when tested five days after exposure, but not at 10 weeks post- exposure (Cippitelli et al., 2010). The impact of both acute and chronic ethanol exposure on spatial and non-spatial memory was further compared in adult rats from PND 21-90 (García-Moreno and Cimadevilla, 2012). Those in the chronic exposure group received free access to an aqueous ethanol solution as their only liquid source, while those in the acute exposure group received one 2g/kg injection (I.P) per week. Chronic ethanol treatment produced deficits in both non- spatial and spatial versions of the DNMS task. In the non-spatial DNMS task both ethanol treated groups displayed similar levels of novel object preference compared to the control subjects following delays of 1 and 15 minutes, but performance was significantly worse following a 60 minute delay. In the spatial DNMS task both ethanol treated groups displayed memory deficits following all delays imposed. However, the acute ethanol treatment group still outperformed the chronic ethanol treatment group at the 1 and 15 minute delays. 19 In summary, spatial memory impairments are more consistently present after both acute and chronic ethanol exposure, and are more persistent than non-spatial memory impairments. Effects of ethanol on memory as a function of age Numerous studies indicate that alcohol causes more severe memory impairments in spatial memory domains relative to object memory. However, it appears that the severity of this spatial memory impairment is age dependent. Several studies have found that adolescent rats were more sensitive than adults to acute ethanol exposure prior to MWMT and sand box maze task testing (Markwiese, Acheson, Levin, Wilson, & Swartzwelder, 1998; Obernier et al., 2002; Rajendran and Spear, 2004), and with chronic exposure adolescent rats displayed an increased suppression of hippocampal long term potentiation (Swartzwelder, Wilson, and Tayyeb, 1995; Pyapali, Turner, Wilson, and Swartzwelder, 1999). Adolescent rats also took longer to recover from spatial memory deficits produced by long-term ethanol exposure (Sircar and Sircar, 2005; Rajendran and Spear, 2004). Adult and aged C57Bl/6 mice provided with ethanol (12% v/v) as their only fluid source for 5 months were then tested in the 8-arm radial arm maze discrimination task (Krazem et al., 2003). Adult mice began receiving this treatment in adolescence (2-3 months old) whereas aged mice began receiving this treatment at 16-18 months of age. After this time period all subjects took part in the discrimination task, which was then repeated 21 days later. The aged control mice displayed deficits in relational memory (but not procedural) and retention across the 21 day interval between the two testing sessions. These effects were reversed in aged rats that received ethanol. In contrast adult rats that received ethanol displayed a deficit in relational memory, but no deficit in retention. These results indicate that ethanol affect memory domains in very different ways at different stages of development. 20 A similar effect was found in adult and aged C57Bl/6 mice that received long-term ethanol exposure. Aged mice that received ethanol for 5 months performed significantly better in an 8-arm radial arm maze discrimination task than mice that started receiving the ethanol treatment in adolescence (Krazem, Marighetto, Higueret, and Jaffard, 2003). These findings suggest that long-term ethanol exposure is more damaging to developing adolescent brains, while potentially alleviating some cognitive deficits that arise with aging (Krazem, Marighetto, Higueret, and Jaffard, 2003). These two studies suggest that older brains can recover more easily from chronic ethanol exposure, but more research in this area is required before it can be determined what the true extent of the harmful nature of ethanol to the older brain is. The current study To determine whether ethanol administered at different stages of neurological development effects spatial and recognition or non-spatial memory differently, this study aims to compare the effect of ethanol on spatial and non-spatial memory, in rats aged approximately 8-12 weeks or 9-18 months. The NOR task trials will be used to test both non- spatial and spatial memory. It is predicted that saline subjects’ performance may improve over time within and across days, while ethanol subjects will not due to ethanol-produced forgetting (Uzbay, and Kayaalp, 1995). For cycle 1 of NOR testing it is predicted that as ITI time increases, a forgetting curve will present itself for ethanol-treated subjects in both non-spatial and spatial NOR task trials. Similarly, to subjects with hippocampal lesions that undergo NOR trial testing (Broadbent et al., 2010; Cohen and Stackman, 2015), no preference for either objects (equal approaches and/or time spent with both objects) or a preference for the familiar object that is no longer recognised from T1 could indicate hippocampal impairment from previous ethanol exposure. 21 Due to the shorter ITIs implemented in this study, it is predicted that control subjects will still have intact memory processes (displayed as a preference for the novel object) even after the longest delay between phases, as seen in previous research with normal NOR task performance (Ennaceur and Delacour, 1988; Reger, Hovda, and Giza, 2009; Broadbent et al., 2010; Schenk et al., 2011). Performance is expected to be impaired for younger ethanol treated rats in comparison to older ethanol treated rats, due to heightened susceptibility of the adolescent brain (particularly the hippocampus) to the detrimental effects of ethanol (Swartzwelder, Wilson, and Tayyeb, 1995; Pyapali, Turner, Wilson, and Swartzwelder, 1999 Sircar and Sircar, 2005) and apparent lack of susceptibility of the older, previously un- exposed brain/hippocampus (Krazem et al., 2003). In addition, it is predicted that spatial memory deficits exhibited by younger ethanol treated subjects will be sustained one week later, whereas memory deficits in non-spatial trials will no longer be displayed due to recovery of recognition memory capabilities, as evidenced in previous research (Sircar and Sircar, 2005; Cippitelli et al., 2010). Older ethanol treated rats are predicted to display no remaining memory impairments for either type of trial. Subjects The sample consisted of 76 Male Sprague-Dawley rats aged approximately 8-12 weeks (n = 56) and 9-18 months (n = 20). However an insufficient amount of the rats aged 9- 18 months met the initial criteria of a minimum of 20 seconds exploration time in T1, so only younger rats were used for further analyses. Not all the younger rats met this criteria either resulting in varied numbers of ethanol and control rats for each trial type (3-9 ethanol rats, 10-16 controls). Additionally, several rats that met the initial criteria but either explored only one or none of the objects in T2 were eliminated from further analyses in order to gain a fair 22 assessment of discriminatory capabilities. Assignment to groups (ethanol or control) was at random. The young rats weighed between 170-257g. Prior to testing, the rats were housed in groups of four in standard polycarbonate cages. These were located in a temperature (21°C) and humidity (55%) controlled colony which was maintained on a 12h light/dark cycle with lights on at 07:00. The subjects were handled regularly, and had free access to food and water (outside of the testing phase time). All subjects were bred in the vivarium located at Victoria University of Wellington. Treatment The ethanol/vehicle was provided over a 5 day period consisting of one 1.0g/kg injection per day. This amount was chosen as it has been proven to be effective in producing memory impairments (Ryabinin, Miller, & Durrant, 2002; Hayes, Deeny, Shaner, & Nixon, 2013). Testing began 2 days following the last injection. Rota-rod. Rota-rod. The first rota-rod model used consisted of a 70 mm diameter rotating plastic rod covered in sandpaper (to assist with grip). This was one lane in total semi-enclosed by white plastic walls and suspended above a plastic base. The second rota-rod model used was a Panlab/Harvard Apparatus model (GmbH, Germany) with four lanes measuring 75 mm each in width. The rod itself was 60 mm in diameter. This apparatus was fully composed of plastic. Nor arena. The NOR arena is a 42cm x 42cm x 30cm rectangle chamber with transparent Pyrex walls enclosed in a sound-proof cupboard (Medical Associates, ENV-001). A video camera 23 was positioned above the chamber to record all activity. Two different sets of thirteen different pairs of objects were used for cycle 1 and 2. See tables 1 and 2 below for objects used Cycle 1 Novel Object Recognition (NOR) task items. Object Description Beer bottle A brown glass bottle with long narrow neck Can A 355 ml metal can Graduated cylinder A short, clear plastic graduated cylinder Shot glass A short, transparent shot glass Pink hole punch A pink metal hole puncher, pressed down with squared edges Beaker A tall glass beaker Glass A short, purple round glass Mug A tall white ceramic mug with handle Egg cup A small white ceramic egg cup Funnel A white plastic funnel placed upside down during testing Tuna can A metal tuna can with no label Plastic bottle A clear plastic bottle 700ml Decorative dish A small black ceramic plate Procedure On each day of the 5 ethanol/saline pre-treatment motor coordination was assessed using a rota-rod. Initially an older model of the rotor rod was used to assess motor co- ordination, with the rotor rod was set at 30 revolutions per minute (RPM). This was replaced by a newer model which was used for subsequent groups of rats, with the speed set at 20 RPM. Each rat was placed upon the rod 0, 20, and 40 minutes after saline or ethanol exposure and timed for endurance. If the rat remained on the rod for longer than 10 minutes the duration time was recorded as 600 seconds. In both of the two days following the last injection subjects explored the NOR arena sans-objects for 5 minutes per day. After this the experimental trials commenced. For the procedure testing non-spatial memory, at time 1 the subject was placed in a chamber which included two identical objects. They were given 3 minutes to explore the objects. Following this, the subject was returned to the home cage while an inter-trial intervals (ITI) of 1, 3, or 5 minutes was implemented, in which time one object was replaced with a novel object (for non-spatial trials) or moved to a new location within the experimental chamber (for spatial trials) Subsequently the subject was placed back in the chamber for another 3 minutes. Video recordings of each trial were later viewed and amount of approaches and length of time of each approach was recorded. Table 2 Cycle 2 Novel Object Recognition (NOR) task items. Cycle 2 Novel Object Recognition (NOR) task items. Object Description Tea-light holder A small, round, purple shallow tea-light holder made of glass Milk bottle A tall, glass milk bottle Palette A small, plastic painting palette with six wells Baking mould A round, metal baking mould, placed upside down during testing Round container A small, round, plastic container with a white screw-on cap Glass holder A short, red, square glass holder Teacup A dark blue ceramic teacup with no handle Salt shaker A short glass salt shaker with a metal cap Rice mould A plastic, pink triangular rice mould Glass bottle A short, round glass container with an orange cap Pencil sharpener A small, pink, rectangular pencil sharpener 24 24 Exploratory behaviour Exploratory behaviour was defined when the rats’ nose came within one centimetre of the object. This excluded rearing behaviour and/or climbing on top of the object. In order for the data from each NOR trial to be considered reliable, the rat was required to explore both objects in T1 for a minimum of 20 seconds, and to explore both objects for any given amount of time in T2. All tests were conducted during the light portion of the cycle (between 0700 25 and 1900). All procedures were approved by the Animal Ethics committee at Victoria University of Wellington. Data analysis Data were analysed using SPSS statistics package (SPSS Inc.; version 19.0 for Windows 7). To examine the effects of treatment on rota-rod endurance lever responding a 3 3 (time) x 5 (day) x group (2) repeated measures analysis of variance (ANOVA) was used. Time and day were used as within-subjects factors, while treatment (ethanol or saline) was used as a between subject factor. Performance on the two rota-rod apparatuses used were analysed separately To examine the effects of treatment on approaches to or time spent exploring each object in T1 and T2 of NOR task trials, a 2 (time 1 or 2) x 4 (object novelty- sample, duplicate, familiar or novel) x 3 (ITI length of 1, 3 , or 5 minutes) x 2 (group) repeated measures ANOVA was used. Time, novelty and ITI were used as within-subjects factors, while treatment (ethanol or saline) was used as a between subject factor. Non-spatial and spatial data were separately analysed, as were data from cycle 1 and cycle 2. To examine the effects of treatment on the proportion of approaches or proportion of time spent exploring the duplicate object in T1 and the novel object in T2 of NOR task trials, a 2 (object novelty) x 3 (ITI) x 2 (group) repeated measures ANOVA was used. Novelty and ITI were used as within-subjects factors, while treatment (ethanol or saline) was used as a between subject factor. Once again non-spatial and spatial data were separately analysed, as were data from cycle 1 and cycle 2. Rota-rod performance 26 Initial rota-rod model. Initial rota-rod model. Figure 1 shows the amount of time spent on the rota-rod for ethanol and vehicle- treated rats. Saline-treated animals remained on the rota-rod for a longer duration at all time points compared to ethanol treated animals. While there is slight improvement for the saline group on days 1 and 2, the existence of practice effects is unclear, and performance otherwise remained relatively stable across time for both groups. Figure 1. Time spent on the old rota-rod following daily administration of saline (0.9%, IP) or Ethanol (1g/kg, IP) at time zero, twenty, and forty. Error bars represent standard error of the mean. 0 10 20 30 40 50 60 70 80 90 100 0 min 20 min 40 min 0 min 20 min 40 min 0 min 20 min 40 min 0 min 20 min 40 min 0 min 20 min 40 min Day 1 Day 2 Day 3 Day 4 Day 5 Time spent on rotorod (s) at 30 RPM Ethanol Saline Figure 1. Time spent on the old rota-rod following daily administration of saline (0.9%, IP) or Ethanol (1g/kg, IP) at time zero, twenty, and forty. Error bars represent standard error of the mean. As shown in table 1, a repeated measures ANOVA (time x day x group) revealed a significant main effect of day and time as shown in Table 1. This may indicate improvement over time, within and between sessions. A significant interaction between day and group, and time and group suggests that treatment altered performance. Post-hoc independent samples t- 27 tests revealed that compared to ethanol treated subjects, control subjects remained on the rota-rod for a significantly longer duration at 0 minutes (control: M = 33.23, SD = 33.83; ethanol: M = 7.89, SD = 2.21) t (126) = 5.91, p < .001, 20 minutes (control: M = 41.20, SD = 33.28; ethanol: M = 8.85, SD = 13.01) t (126) = 7.58, p < .001, and 40 minutes post- injection (control: M = 44.84, SD = 35.74; ethanol: M = 8.45, SD = 12.06) t (126) = 7.90, p < .001. Table 3. Analysis of variance following acute administration of saline or ethanol on initial rota-rod apparatus performance. * Indicates significance at the .05 level. Table 3. Analysis of variance following acute administration of saline or ethanol on initial rota-rod apparatus performance. * Indicates significance at the .05 level. rota-rod apparatus performance. Initial rota-rod model. * Indicates significance at the .05 level. Main effects Day F(4, 84) = 4.497, p = .002* Time F(2, 42) = 5.392, p = .008* Day x time F(3.88, 81.43) = 1.040, p = .390 Day x group F(4, 84) = 3.197, p = .017* Time x group F(2, 42) = 3.919, p = .028* Figure 2 shows performance on the new rota-rod at 20 RPM at the 3 time points across the 5 days of testing. Saline treated animals remained on the rota-rod for a longer duration at all time points compared to ethanol treated animals, with performance across time being stable for the ethanol group, while the saline groups’ performance varied within and between days. 28 Figure 2. Time spent on the second rota-rod apparatus following daily administration of saline (0.9%, IP) or Ethanol (1g/kg, IP) at time zero, twenty, and forty. Error bars represent standard error of the mean. 0 50 100 150 200 250 300 0 20 40 0 20 40 0 20 40 0 20 40 0 20 40 Day 1 Day 2 Day 3 Day 4 Day 5 Time spent on the rotorod (s) Ethanol Saline 0 50 100 150 200 250 300 0 20 40 0 20 40 0 20 40 0 20 40 0 20 40 Day 1 Day 2 Day 3 Day 4 Day 5 Time spent on the rotorod (s) Ethanol Saline Figure 2. Time spent on the second rota-rod apparatus following daily administration of saline (0.9%, IP) or Ethanol (1g/kg, IP) at time zero, twenty, and forty. Error bars represent standard error of the mean. Figure 2. Time spent on the second rota-rod apparatus following daily administration of saline (0.9%, IP) or Ethanol (1g/kg, IP) at time zero, twenty, and forty. Error bars represent standard error of the mean. Table 4 shows the results of a repeated measures ANOVA (time x day x group), which revealed a significant main effect of day and time. This may indicate improvement over time, within and between sessions. A significant interaction between day and group, time and group, and day time and group suggests that treatment altered performance. Initial rota-rod model. Post- hoc independent samples t-tests revealed that compared to ethanol treated subjects, control subjects remained on the rota-rod for a significantly longer duration at 0 minutes (control: M = 47.87, SD = 49.99; ethanol: M = 9.11, SD = 13.21) t (87) = 5.75, p < .001, 20 minutes (control: M = 73.60, SD = 96.00; ethanol: M = 10.03, SD = 11.37) t (87) = 5.25, p < .001, and 29 40 minutes post- injection (control: M = 144.99, SD = 387.78; ethanol: M = 10.77, SD = 10.55) t (2.79) = 7.90, p = .006. 40 minutes post- injection (control: M = 144.99, SD = 387.78; ethanol: M = 10.77, SD = 10.55) t (2.79) = 7.90, p = .006. Table 4. Analysis of variance of rota-rod performance following acute administration of saline or ethanol. * Indicates significance at the .05 level, whereas ^ indicates a Greenhouse-Geisser correction of degrees of freedom following a violation of sphericity. Main effects Day F(1.69, 25.28) = 3.54, p = .051 ^ Time F(1.29, 19.35) = 4.40, p = .041* Day x time F(2.04, 30.61) = 3.60, p = .039* Day x group F(4, 60) = 2.67, p = .041* Time x group Day x time x group F(2, 30) = 3.51, p = .043* F(8, 120) = 3.28, p = .002* Greenhouse-Geisser correction of degrees of freedom following a violation of sphericity Cycle 1 novel object recognition testing Cycle 1 novel object recognition testing Cycle 1 of NOR testing occurred 2 days after the final dose of saline or ethanol had been administered to each subject. Non-spatial and spatial trials were analysed separately, however for both trial types the dependent measures of approaches, proportion of approaches, exploration times, and proportion of exploration times were examined to determine whether one measure was more appropriate as a reflection of object preference than another. Non-spatial NOR trials Non-spatial NOR trials Approaches. Approaches. 30 Figure 3. Number of approaches to each object in T1 (1) and T2 (2) for saline (A) and Fi 3 N b f h h bj i T1 (1) d T2 (2) f li (A) d Figure 3. Number of approaches to each object in T1 (1) and T2 (2) for saline (A) and ethanol (B) treated animals across each cycle 1 non-spatial novel objection recognition trial. Error bars represent standard error means. Error bars represent standard error means. Figure 3 shows average number of approaches to the objects presented in time 1 and time 2 by both saline and ethanol-treated subjects. There was no obvious difference in the number of approaches to the sample and duplicate objects for saline-treated rats between trials (1A), however for ethanol treated rats there was an observable decrease in the amount of approaches made to both sample and duplicate objects between NS1 and NS3 trials (1B). It is also evident from figure 3 that approaches to the novel object increased when longer ITIs beyond 1 minute were implemented for saline-treated animals (1B), and decreased with increasing ITIs for ethanol-treated subjects (2B). 31 A group x ITI x time x novelty repeated measures ANOVA was conducted to assess between and within-subject differences in amount of approaches to objects in both T1 and T2 across all 3 ITIs for non-spatial trials. As depicted in table 3, a significant effect of ITI was found, F(1, 44) = 3.45, p = .041, as well as a significant effect of time, F(1, 44) = 8.28, p = .006. A significant interaction between time and ITI was also revealed, with post-hoc comparisons indicating that more approaches were made in total in T1 (p = .006) of NS1 trials (p = .041) compared to T1 and/or T2 of all other non-spatial trials. An additional significant interaction between group and ITI, F(2, 44) = 7.03, p = .002, indicates that this increase in approaches in T1 of NS1 trials is due to ethanol-treated subjects, as presented in figure 3. Figure 4. Proportion of number of approaches to duplicate objects in T1 and replacement Figure 4. Proportion of number of approaches to duplicate objects in T1 and replacement Figure 4. Approaches. Proportion of number of approaches to duplicate objects in T1 and replacement novel objects in T2 for saline (A) and ethanol (B) treated animals across each cycle 1 non- spatial novel objection recognition trial. Error bars represent standard error means. novel objects in T2 for saline (A) and ethanol (B) treated animals across each cycle 1 non- spatial novel objection recognition trial. Error bars represent standard error means. Figure 4 shows the average proportion of approaches made by both saline and ethanol treated subjects towards the duplicate object in T1 and the novel object which replaced it in T2. This allows for a direct comparison of approach data between T1 and T2. Saline-treated subjects allocated close to 0.5 of their approaches to the duplicate object in T1 of all non- spatial trials, while making a proportion of approaches slightly higher than chance to the novel object in T2 of NS3 and NS5 trials (A). There was minimal difference in the proportion 32 of approaches made by ethanol treated subjects to either the duplicate or novel objects for any trial, with the exception of NS3 trials where the proportion of approaches made to the novel object in T2 was less than 0.5. From this figure there was is no apparent position bias from subjects affecting where approaches were made, nor an effect of ITI on the proportion of approaches made to the novel object for either group. A further group x novelty x ITI repeated measures ANOVA was conducted to assess differences in proportion of approaches to the duplicate object in T1 and the novel object in T2 of all non-spatial trials. Results revealed an effect of novelty F(1, 44) = 4.46, p = .040 with a higher proportion of approaches made towards the novel object compared to the duplicate (p = .040) and an effect of group F(1, 44) = 4.62, p = .037, with the saline group having a higher proportion of approaches towards the novel object compared to the ethanol group (p = .037). Exploration time. 33 Figure 5. Time spent exploring both objects in T1 (1) and T2 (2) for saline (A) and ethanol (B) treated animals during the cycle 1 non-spatial objection recognition task. Error bars represent standard error means. Figure 5. Time spent exploring both objects in T1 (1) and T2 (2) for saline (A) and ethanol (B) treated animals during the cycle 1 non-spatial objection recognition task. Error bars represent standard error means. (B) treated animals during the cycle 1 non-spatial objection recognition task. Error bar d d Figure 5 shows average time spent exploring each object presented in time 1 and time 2 by both saline and ethanol-treated subjects. Total exploration time was no more than 45 seconds for either T1 or T2, however ethanol treated subjects spent less time exploring objects compared to saline treated subjects at both times. For saline-treated subjects there was a slight difference in time spent exploring sample and duplicate objects for NS3 trials in T1 (1A), whereas time spent exploring both objects in T1 appeared relatively equal for NS1 and NS5 trials. At T2 saline-treated subjects spent more time exploring the novel object in comparison to the familiar object, which was most prominent in NS3 trials. This pattern suggests that saline treated subjects were displaying a novelty preference across all ITIs. For ethanol-treated subjects there was a visible difference in exploration time of each object at T1 (1B), and T2 (2B), but in no discernible pattern. It is once again difficult to ascertain whether 34 this is a result of forgetting or simple lack of motivation to explore, as suggested by lower exploration times in general. A repeated measures ANOVA revealed a significant effect of novelty, F(1, 44) = 5.32, p = .026, which post-hoc comparisons indicated to be due to an increased amount of time spent exploring the duplicate object in T1 and the replacement novel object in T2 in comparison to the sample object at both T1 and T2. A significant effect was also found for time F(1, 44) = 15.98, p < .001 due to an increased amount of total time spent exploring objects in T1 compared to T2 (p < .001). A significant interaction between novelty and group was revealed F(1, 44) = 5.28, p = .026, in addition to a significant interaction between novelty and time, F(1, 44) = 7.83, p = .008. Figure 6. Exploration time. Proportion of time spent exploring duplicate objects in T1 and replacement novel re 6. Proportion of time spent exploring duplicate objects in T1 and replacement novel objects in T2 for saline (A) and ethanol (B) treated animals during the cycle 1 non-spatial novel objection recognition task. Error bars represent standard error means. objects in T2 for saline (A) and ethanol (B) treated animals during the cycle 1 non-spatial novel objection recognition task. Error bars represent standard error means. Figure 6 shows the average proportion of time spent exploring the duplicate object in T1 and the novel object which replaced it in T2 for all non-spatial NOR trials for both saline (A) and ethanol-treated subjects (B) for the duplicate object in T1 and the novel object which replaced it in T2. Saline-treated subjects allocated almost equal time with each object in T1 of each trial (A), whereas the proportion of time spent with the duplicate object in T1 varied 35 across all 3 trials for ethanol-treated subjects (B). Saline-treated subjects consistently spent a larger proportion of time with the novel object across all trials in T2, while ethanol-treated subjects spent only slightly over 0.5 of their total time with the novel object. From this figure there it can be established that there was no apparent position bias from subjects affecting exploration, nor an effect of increasing ITI on the proportion of time spent exploring to the novel object for either group. A further repeated measures ANOVA was conducted to assess differences in proportion of time spent exploring the duplicate object in T1 and the novel object in T2 for all non-spatial trials. A significant effect of novelty was revealed F(1, 44) = 20.11, p < .001, with a higher proportion of time spent exploring the novel object compared to the duplicate for both groups, and an effect of group F(1, 44) = 6.81, p = .037, with the saline group spending a greater proportion of time exploring the duplicate and novel objects compared to the ethanol group. It is shown from these results that when performance in the non-spatial NOR task was measured by total approaches towards objects, no group differences in approaches to each object were found after any ITI. Exploration time. However, when performance was measured by proportion of approaches, results showed that saline treated subjects allocated a significantly larger proportion of approaches to the novel object in T2 of cycle 1 non-spatial trials compared to ethanol treated subjects. When performance in the non-spatial NOR task was measured by total time and proportion of time spent exploring each object, saline treated subjects explored objects more in general compared to ethanol treated subjects. However, both groups spent a larger proportion of time exploring the novel object in T2 in comparison to the duplicate in T1 which indicates some preference for the novel object. 36 As there was no overall effect of ITI, it is difficult to determine whether these differences are due to impaired recognition memory across all trials on part of the ethanol group or a lack of motivation to explore objects altogether, thus it cannot be concluded whether these results support the hypotheses. Spatial NOR trials. Approaches. Figure 7. Number of approaches to each object in T1 (1) and T2 (2) for saline (A) and ethanol (B) treated animals across each cycle 1 spatial novel objection recognition trial. Error bars represent standard error means. Figure 7. Number of approaches to each object in T1 (1) and T2 (2) for saline (A) and ethanol (B) treated animals across each cycle 1 spatial novel objection recognition trial. Error bars represent standard error means. ethanol (B) treated animals across each cycle 1 spatial novel objection recognition trial. Error bars represent standard error means. Figure 7 shows the amount of approaches made by saline (A) and ethanol-treated subjects (B) to the sample and duplicate objects in T1 (1) and the familiar and novel objects in T2 (2) of spatial NOR task trials. For saline-treated subjects there were minimal differences in the amount of approaches made to each object in both T1 and T2, with the exception of S5 trials where there was a more pronounced difference between approaches 37 made to the familiar and novel objects in T2. Ethanol treated subjects had slight differences between approaches to each object in both T1 and T2, although interestingly made slightly more approaches to the novel object over the familiar object in T2 of both S1 and S3 trials, a trend not seen in the saline treated subjects. A repeated measures ANOVA was conducted to assess between and within-subject differences in amount of approaches to objects in both T1 and T2 across all 3 spatial NOR task trials. A significant effect of time was revealed F(1, 55) = 7.99, p = .007, with pairwise comparisons indicating that more approaches were made to both objects in T1 compared to T2. Figure 8. Proportion of number of approaches to duplicate objects in T1 and replacement Figure 8. Proportion of number of approaches to duplicate objects in T1 and replacement novel objects in T2 for saline (A) and ethanol (B) treated animals across each cycle 1 spatial novel objection recognition trial. Error bars represent standard error means. novel objects in T2 for saline (A) and ethanol (B) treated animals across each cycle 1 spatial novel objection recognition trial. Error bars represent standard error means. novel objects in T2 for saline (A) and ethanol (B) treated animals across each cycle 1 spatial novel objection recognition trial. Error bars represent standard error means. Approaches. Figure 8 shows the average proportion of time spent exploring the duplicate object in T1 and the novel object which replaced it in T2 for all spatial NOR trials. Saline-treated subjects allocated approximately equal time with each object in T1 of S3 and 5 trials, Figure 8 shows the average proportion of time spent exploring the duplicate object in T1 and the novel object which replaced it in T2 for all spatial NOR trials. Saline-treated subjects allocated approximately equal time with each object in T1 of S3 and 5 trials, however only 40% of approaches were made towards the duplicate object in S1 trials. Ethanol-treated subjects were close to 50% in the proportion of approaches they made to however only 40% of approaches were made towards the duplicate object in S1 trials. Ethanol-treated subjects were close to 50% in the proportion of approaches they made 38 objects in both T1 and T2 of spatial NOR trials. No evidence of location bias or a forgetting curve in T2 due to increasing ITIs is evident. objects in both T1 and T2 of spatial NOR trials. No evidence of location bias or a forgetting curve in T2 due to increasing ITIs is evident. A further repeated measures ANOVA was conducted to assess differences in proportion of approaches to the duplicate object in T1 and the novel object in T2 for all spatial trials. No significant effects or any interactions between effects were found. Exploration times. Exploration times. Figure 9. Time spent exploring both objects in T1 (1) and T2 (2) for saline (A) and ethanol (B) treated animals during each trial of the cycle 1 spatial novel objection recognition task. Figure 9. Time spent exploring both objects in T1 (1) and T2 (2) for saline (A) and ethanol B) treated animals during each trial of the cycle 1 spatial novel objection recognition task Figure 9 shows average time spent exploring each object presented in time 1 and time 2 by both saline and ethanol-treated subjects. For saline-treated subjects the most noticeable difference in T1 is between times spent exploring the sample and duplicate objects in S1 39 trials. Ethanol-treated subjects spent relatively equal time exploring both objects in T1 across all 3 trials, although less time was spent exploring objects overall in S5 trials. Both groups had lower overall exploration times in T2 compared to T1. Approaches. Saline-treated subjects had a noticeable increase in time spent exploring the novel object in S1 trials, an effect which reversed with increasing ITIs. Ethanol-treated subjects had an increase in time spent exploring the novel object in comparison to the familiar object in S3 trials, and a contrasting decrease in S5 trials, indicating a possible forgetting effect due to a longer ITI. A repeated measures ANOVA assessing differences in time spent exploring objects for spatial NOR trials revealed a main effect of time F(1, 55) = 29.83, p < .001. Pairwise comparisons indicated that subjects spent significantly more time exploring objects in T1 in comparison to T2 (p < .001). Figure 10. Proportion of time spent exploring duplicate objects in T1 and replacement novel Figure 10. Proportion of time spent exploring duplicate objects in T1 and replacement novel Figure 10 shows the average proportion of time spent exploring the duplicate object in T1 and the novel object which replaced it in T2 for all spatial NOR trials for both saline (A) and ethanol-treated subjects (B) for the duplicate object in T1 and the novel object which replaced it in T2. Both groups allocated a relatively equal proportion of time to exploration of 40 both objects in T1 and T2 with the except of T2 of S5 trials for ethanol subjects, where they spent less than 40% of their time with the novel object, again indicating a potential forgetting effect due to the implementation of a longer ITI. A repeated measures ANOVA assessing difference in proportion of time spent exploring the duplicate object in T1 and the novel object in T2 revealed no significant main effects, or any interaction between effects. These findings demonstrate that any measure of performance in cycle 1 of the spatial NOR task resulted in no significant between or within group differences in object preference after any ITI. This indicates that 5 consecutive days of ethanol administration followed by a 2 day recovery period did not lead to an impairment in memory in a capacity that would alter spatial NOR task performance. Cycle 2 novel object recognition testing Cycle 2 of NOR testing occurred 9 days after the final dose of saline or ethanol had been administered to each subject, and 7 days after cycle 1 of NOR testing had been completed. Once again non-spatial and spatial trials were analysed separately, with the dependent measures of approaches, proportion of approaches, exploration times, and proportion of exploration times all being examined to determine whether one measure is more appropriate as a reflection of object preference than another. Non-spatial NOR trials. Table 5. Significant effects obtained from analysis of various measures of performance in Cycle 2 non-spatial NOR trials. 41 Approaches Exploration time Proportion of exploration time Group F(1, 25) = 6.74, p = .016 Time F(1, 25) = 6.27, p = .016 Time F(1, 25) = 16.71, p < .001 Novelty x time F(1, 25) = 15.92, p = .001 Novelty F(1, 25) = 20.03, p < .001 Table 5 displays all significant effects found when cycle 2 non-spatial NOR trial performance across all three ITIs was measured by approaches to each object, proportion of approaches, time spent exploring each object, and proportion of exploration time. For approaches, a repeated measures ANOVA revealed a significant effect of group, with pairwise comparisons indicating that more approaches were made as a whole by the ethanol group, as well as a significant effect of time, due to more approaches as a whole being made in T1. When performance was analysed by the proportion of approaches to the duplicate object in T1 and the novel object in T2, a repeated measures ANOVA revealed no significant main effects, or any interaction between effects. For exploration time, a repeated measures ANOVA revealed a significant effect of time which pairwise comparisons indicated to be due to an increased amount of time spent exploring both of the objects in T1 compared to T2. A significant interaction was also found between novelty and time due to more time being spent with the sample object in T1 vs. T2 for both groups. 42 A further repeated measures ANOVA was conducted to assess differences in proportion of time spent exploring the duplicate object in T1 and the novel object in T2 for all non-spatial trials. A significant effect of novelty was revealed with a higher proportion of time spent exploring the novel object compared to the duplicate for both groups. These results show that when performance in cycle 2 of the non-spatial NOR task was measured by approaches, proportion of approaches, total time and proportion of time spent exploring objects, no between or within group differences were found after the implementation of any ITI. Although these similarities in performance between groups could reflect our prediction that the non-spatial memory of ethanol treated subjects would recover by the time of cycle 2 testing, it cannot be determined whether these results support our hypothesis as there is no indication that non-spatial memory was ever impaired in cycle 1 testing in the first place. Spatial NOR trials. Table 6. Significant effects obtained from analysis of various measures of performance in Cycle 2 spatial NOR trials. Table 6. Significant effects obtained from analysis of various measures of performance in Cycle 2 spatial NOR trials. Approaches Exploration time Time F(1, 38) = 17.03, p < .001 Time F(1, 38) = 15.91, p < .001 Table 6 displays all significant effects found when cycle 2 spatial NOR trial performance was measured by approaches to each object, proportion of approaches, time spent exploring each object, and proportion of exploration time. 43 A repeated measures ANOVA assessing between and within-subject differences in amount of approaches to objects in both T1 and T2 across all 3 spatial NOR task trials revealed a significant effect of time, with pairwise comparisons indicating that more approaches were made to both objects in T1 compared to T2. The same effect was found when exploration time was analysed, with pairwise comparisons indicating that subjects spent significantly more time exploring objects in T1 in comparison to T2. Repeated measures ANOVAs assessing differences in both the proportion of approaches to the duplicate object in T1 and the novel object in T2 and proportion of exploration time revealed no significant main effects, or any interaction between effects. From these results it is shown that any measure of performance in cycle 2 of the spatial NOR task revealed no significant group differences in approaches to each object were found after any ITI. This indicates that contrary to our hypothesis, treatment did not affect memory in a prolonged capacity that would alter spatial NOR task performance 9 days after administration of ethanol had ceased. General discussion This study aimed to investigate the effect of acute ethanol exposure on spatial and non-spatial memory at two stages of development. However due to a lack of subjects meeting the initial experimental criteria from the older ethanol treated group this became a study of the effect of a “binge-like” period of ethanol exposure on spatial and non-spatial memory in young rats aged 2-3 months only. We firstly hypothesised that ethanol treated subjects would remain on the rota-rod for a shorter period of time at all 3 time points across the 5 days of treatment compared to saline treated control subjects. Our hypothesis was supported when subjects were placed on rota-rods running at both 20 and 30 RPM, indicating that the ethanol treatment was effective to the point of producing motor co-ordination impairments. 44 The effects of this pre-treatment on NOR performance was measured at two time periods. The first assessment took place 2 days following treatment in order to determine whether there was an effect on spatial and/or non-spatial memory. The second test occurred one week later in order to assess whether the hypothesised initial effects would dissipate over time. NOR testing was conducted at various time intervals (ITIs) in order to generate a “forgetting curve”. It was expected that preference for the novel object would decrease as ITI increased, as has been observed in other studies (Ennaceur and Delacour, 1988; Reger, Hovda, and Giza, 2009; Broadbent et al., 2010; Schenk et al., 2011). We also expected that the ethanol exposed rats would demonstrate compromised performance. For cycle 2 trials we hypothesised that memory deficits predicted to be exhibited by the young ethanol-treated subjects in cycle 1 spatial NOR trials would also be present one week later in cycle 2 of the testing phase, whereas memory deficits in non-spatial trials would no longer be displayed, with normal preference for the novel object being demonstrated. This was expected due to findings from previous literature where non-spatial memory was demonstrated to recover at a faster rate than spatial memory (Sircar and Sircar, 2005; Cippitelli et al., 2010). From the results we can conclude that all of our hypotheses for performance in the NOR task across both cycles were not supported. General discussion When performance in cycle 1 non-spatial trials was measured by the proportion of approaches to the novel object in T2, saline treated subjects had a significantly larger proportion of approaches to the novel object compared to ethanol treated subjects. However, when this was measured as a proportion of time spent exploring the novel object, there were no differences between groups, with both saline and ethanol subjects showing a preference for the novel object across all ITIs that were implemented. 45 These results contradict the findings of Cippitelli et al. (2010), where ethanol treated subjects displayed an impairment in novel object recognition and the ability to detect spatial change in an object exploration task when delays of only four minutes were imposed, something which occurred five days after the ethanol treatment had desisted. This pattern was found when performance was measured by both approaches and exploration time, suggesting that there is some overlap between the two. These results also contrast with effects of 2g/kg of ethanol once a week for 10 weeks. These rats displayed significantly worse performance in a spatial version of the DNMS task compared to controls at delays as small as 1 minute (García-Moreno and Cimadevilla, 2012). This was measured by both exploration times and proportion of time spent exploring the novel object/ novel placement. These results contradict the findings of Cippitelli et al. (2010), where ethanol treated subjects displayed an impairment in novel object recognition and the ability to detect spatial change in an object exploration task when delays of only four minutes were imposed, Though other research has found that rodents that received acute exposure to ethanol display intact object recognition memory after short ITIs (< 15 minutes) (García-Moreno et al., 2002; Matthews et al., 2002; Berry and Matthews, 2004; García-Moreno and Cimadevilla, 2012), whether the current findings support the previous literature is dubious as novel object preference was only found for both groups in cycle 1 of non-spatial trials when measured as the proportion of time spent exploring the novel object. As this aforementioned previous research typically used multiple measures of performance for the same task, it is likely that the difference in the current study between measures of cycle 1 non-spatial NOR task novelty preference is a spurious result. General discussion This lack of support is only further increased when the cycle 2 non-spatial results are considered, as neither group displayed a novel object preference after any ITI is imposed, nor a forgetting effect as ITI increased. This also contradicts Cippitelli et al. (2010), as ethanol treated subjects still displayed impairments in spatial memory in the object exploration task 10 weeks after treatment had desisted, showing an ability to detect spatial change after delays of four minutes. Results found by Matthews et al. (2002) with adult rats, and Berry and 46 Matthews (2004) with adolescent mice also showed a clear disparity between non-spatial and spatial MWMT performance after acute administration of ethanol, although only in doses of 1.5 g/kg and above. While ethanol treated subjects failed to display a forgetting curve across increasing ITIs despite previous literature suggesting an effect would occur, it is even more curious that saline treated controls subjects did not display consistent novelty preference in the testing phase of any NOR task trial. Novelty preference in normal subjects has been found consistently across a wide range of both non-spatial (Ennaceur and Delacour, 1988; Reger et al., 2008; Hovda, and Giza, 2009; Antunes and Biala, 2012) and spatial studies (Ennaceur and Meliani, 1992; Ennaceur, Neave, and Aggleton, 1997; Weible, Rowland, Pang, and Kentros, 2009) involving the NOR paradigm. This makes it difficult to interpret the results of the ethanol-treated group in comparison, and could mean that one or more extraneous variables are influencing the current results. One of these potential extraneous variables could be the handling of the subjects that occurred between T1 and T2, something which occurred frequently in a short period of time due to ITI lengths, and may have distressed the animals. The fact that no forgetting curve was found for ethanol treated subjects despite a wide breadth of literature demonstrating the detrimental effect of ethanol on spatial memory and even non-spatial memory could be due to a number of factors. In this study we narrowed down the delay in which a forgetting curve could occur to a window of between 1 and 5 minutes. As multiple studies have found memory deficits when longer delays were implemented, perhaps a forgetting effect would have appeared in the current study had this also been the case. General discussion García-Moreno and Cimadevilla (2012) found that both control and alcohol-treated rats that received both acute and chronic treatment performed best in memory tasks with a 15 minute delay, in comparison to delays of 1 minute or 1 hour. However, Schenk et al. (2011) found that control rats that self-administered saline prior to undergoing 47 the NOR task displayed a forgetting curve as a function of ITIs 1-15 minutes long, something which was only revealed when performance was measured by the proportion of approaches to the novel object. This discrepancy could be due to differing habituation times between studies, or the amount of handling subjects received between the familiarisation and testing phases. A further possible explanation is a general decrease in movement or hypolocomotion as a result of ethanol withdrawal. Logan, Seggio, Robinson, Richard, and Rosenwasser (2010) found this effect when they administered C57BL/6J mice a short-term 4 day CIE treatment (16 hours of ethanol vapour alternating with 8 hour withdrawal periods), which resulted in a temporary albeit significant reduction in both running-wheel activity and free- running circadian rhythms compared to controls, lasting approximately one week after the final ethanol vapour exposure. This could potentially explain the lower amounts of ethanol subjects meeting the minimum exploration criteria in T1 in both cycle 1 and 2 trials, as cycle 1 trials commenced two days after the final dose of ethanol was administered, while cycle 2 trials occurred only slightly over one week following this. Limitations Sample sizes in the current study were substantially decreased from initial amounts due to lack of exploration in T1, particularly ethanol subjects where the proportion of rats that met the criteria was very small. It known that intraperitoneal ethanol injections can result in inflammation to the administration site for up to 3 weeks (In, Kim, Park, and Kim, 2014), which may result in a restriction of movement due to associated pain. Considering that the methodology of the current study involved administering 5 consecutive daily injections, the effect may have been aggravated even more so, even when the side of injection site was alternated each day. 48 As the cycle one NOR testing took place two days after the last dose of ethanol, withdrawal may have also played a part in the performance of the ethanol-treated rats during the trials. In the chronic intermittent ethanol (CIE) model of ethanol exposure repeated withdrawal is a key component, and during this period rats often display increased susceptibility to seizures, anxiety, and behavioural excitability (Cagetti et al., 2003). Cagetti et al. (2003) observed a significant increase in anxiety in CIE exposed rats compared to control subjects in the elevated plus maze, an effect that lasted for at least two days after ethanol cessation. Future directions Future investigation in this area could potentially solve the issue of lack of sufficient T1 exploration times by modifying the length of time for the T1 period to allow more opportunity for exploration. When Ennaceur and Delacour (1988) found that their initial NOR experiment was not sensitive enough to retention duration, they modified the test so that instead of T1 being a fixed amount of time, the phase was only over when the minimum 20 second exploration criteria were met. If this modification was used in the future with the NOR paradigm it could allow for increased familiarisation of objects, and more subjects could potentially be included in further analyses, yielding a different pattern of results. The effect of multiple schedules of administration (acute exposure, chronic exposure, chronic intermittent exposure) on NOR task performance could also be assessed. This would allow researchers to determine if one model of binge alcohol consumption is more appropriate than another in demonstrating memory deficits in this paradigm, since there is so little previous research related to alcohol and NOR. Multiple schedules of administration could also aide researchers in identifying confounding variables that impact on results such as dosage, periods of withdrawal, and irritation caused by injecting substances. 49 Conclusions Conclusions We measured the effect of a binge-like episode of ethanol exposure on performance in both non-spatial and spatial forms of the novel object recognition paradigm in adolescent rats only. This was assessed both 2 days after the final dose of ethanol was administered, and then 1 week later to determine if there was a recovery effect of memory. Rota-rod results during treatment revealed that ethanol treated subjects remained on the rota-rod for significantly less time at two different RPMs compared to saline treated subjects. This indicated that intoxication to the point of impairing motor co-ordination had occurred each day of the five day treatment. In the NOR task trials, the inter-trial intervals that were implemented between the familiarisation phase (T1) and the testing phase (T2) were limited to a range of between 1 and 5 minutes. This resulted in no significant difference between groups for performance in T2 of non-spatial and spatial NOR trials in both cycle 1 and cycle 2 of testing. The lack of In the NOR task trials, the inter-trial intervals that were implemented between the familiarisation phase (T1) and the testing phase (T2) were limited to a range of between 1 and 5 minutes. This resulted in no significant difference between groups for performance in T2 of non-spatial and spatial NOR trials in both cycle 1 and cycle 2 of testing. The lack of difference remained consistent when performance was measured by amount of approaches to each object, proportion of approaches to the novel object, exploration time with each object, and proportion of time spent exploring the novel object. As the current results are inconclusive, further investigation is required to determine how appropriate the NOR paradigm is as a measure of ethanol-produced memory impairments. This can potentially be assessed through allowing longer periods of time to explore objects in T1, and by using multiple forms and durations of ethanol administration and comparing and contrasting the subsequent effects. 50 References References Administering Alcohol in Human Studies (2015). 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https://openalex.org/W2752471650	https://europepmc.org/articles/pmc5615536?pdf=render	English	null	Stress and Depressive Symptoms in Cancer Survivors and Their Family Members: Korea Community Health Survey, 2012	International journal of environmental research and public health/International journal of environmental research and public health	2,017	cc-by	4,959	Mi Ah Han Department of Preventive Medicine, College of Medicine, Chosun University, Gwangju 61452, Korea; mahan@chosun.ac.kr; Tel.: +82-62-230-6481 Academic Editor: Sharon Lawn Received: 25 July 2017; Accepted: 31 August 2017; Published: 1 September 2017 Abstract: This study examined the prevalence of perceived stress and depressive symptoms in cancer survivors and their family members compared with subjects without cancer and without family members with cancer. The subjects of this cross-sectional study were adults ≥19 years old who participated in the 2012 Korea Community Health Survey. Stress and depressive symptoms in cancer survivors and their family members were assessed and compared to symptoms in control groups by chi-square tests and multiple logistic regression analyses. Of the 6783 cancer survivors, 26.9% and 8.7% reported having stress and depressive symptoms, respectively, and 27.7% and 5.9% of family members of cancer survivors reported having stress and depressive symptoms, respectively. Cancer survivors showed higher adjusted odds ratio (aOR) for stress (aOR = 1.26, 95% conﬁdence interval (CI) = 1.16–1.37) and depressive symptoms (aOR = 1.82, 95% CI = 1.57–2.11) than subjects without cancer history. Family members of cancer survivors showed a higher OR for stress and depressive symptoms than subjects without a family member who survived cancer. Cancer survivors and family members of cancer survivors had more stress and depressive symptoms than controls. Careful management for cancer patients and their family members should include screening for stress and depression to improve mental health associated with cancer survivorship. Keywords: chronic disease; depression; family; neoplasms; stress; survivors Keywords: chronic disease; depression; family; neoplasms; stress; survivors Article Stress and Depressive Symptoms in Cancer Survivors and Their Family Members: Korea Community Health Survey, 2012 Mi Ah Han International Journal of Environmental Research and Public Health International Journal of Environmental Research and Public Health Int. J. Environ. Res. Public Health 2017, 14, 999; doi:10.3390/ijerph14090999 1. Introduction About 14.1 million new cancer cases and 8.2 million cancer-associated deaths occurred worldwide in 2012, and the occurrence of cancer is steadily increasing [1]. In Korea, the number of cancer patients is continuously increasing, and the probability of survival is simultaneously increasing [2]. Despite signiﬁcant advances, cancer and cancer treatments can result in signiﬁcant physical and emotional morbidity, and several epidemiological studies indicate that the risk of poor mental health is elevated in cancer patients compared with the general population [3–7]. Cancer patients experience several stressors and suffer from depression [8,9]. Depression is the most important risk factor for suicide [10–12]. Subjects with a history of cancer had a higher odds ratio (OR) for attempted suicide in both genders using the Third US National Health and Nutrition Examination Survey [4]. Thus, understanding the prevalence and correlation of stress and depression with cancer is crucial for the management and control of mental health. g Family members of cancer patients also are at risk for psychological problems and are more susceptible to depression [13]. Family caregivers with anxiety or depression also have a high risk of suicide [14]. This study aimed to describe stress and depressive symptoms among cancer survivors and their family members in Korea. We compared stress and depressive symptoms of cancer survivors and their Int. J. Environ. Res. Public Health 2017, 14, 999; doi:10.3390/ijerph14090999 www.mdpi.com/journal/ijerph www.mdpi.com/journal/ijerph 2 of 8 Int. J. Environ. Res. Public Health 2017, 14, 999 family members with two control populations (non-cancer controls, non-cancer non-family member controls) using the 2012 Korea Community Health Survey (KCHS, 2012). family members with two control populations (non-cancer controls, non-cancer non-family member controls) using the 2012 Korea Community Health Survey (KCHS, 2012). 2.3. Covariates Covariates included sex, age, marital status (with spouse, without spouse), education (uneducated, elementary school, middle school, high school, at least some college), smoking status (never, ex, current), drinking frequency (none, ≤1/month, ≥2/month), number of chronic disease (none, 1, 2 or more), unmet needs for health care (no, yes), and utilization of community health service (no, yes). The chronic diseases included were hypertension, diabetes, dyslipidemia, stroke, myocardial infarction, osteoarthritis, osteoporosis, and tuberculosis. Having unmet needs for health care was deﬁned as a “yes” response to the following question: “During the past 12 months, was there any time when you did not get the medical care you needed?” When subjects reported “yes” response, the reasons for unmet needs were asked: (1) cost; (2) reservation problems; (3) transportation problems; (4) not available at the required time; (5) waiting time too long; (6) mild symptoms; (7) other. 2.2. Stress and Depressive Symptoms Perceived stress, consultation experience due to stress, depressive symptoms, and consultation experience due to depressive symptoms were collected. Stress was deﬁned as the responses “very much” or “much” to the following question: “How much do you feel stress in your usual life?” Depressive symptoms was deﬁned as a “yes” response to the following question: “During the past 12 months, did you feel so much sadness/hopelessness for two or more weeks that you stopped doing some usual activities?” Consultation experience was deﬁned as professional consultation (medical institute, professional consultation ofﬁce, community health center, etc.) due to stress or depressive symptoms. 2.1. Data Source and Study Subjects The 2012 KCHS is a cross-sectional survey conducted by the Korea Centers for Disease Control and Prevention that collected information related to health status, health care utilization, and health determinants for Korean individuals living in private occupied dwellings in 254 regional sites covering all provinces and territories. Using a multistage stratiﬁed cluster-sampling procedure, the 2012 KCHS surveyed 228,921 household residents 19 years of age and over. Detailed information about the KCHS has been documented elsewhere [15]. Participants were classiﬁed as cancer survivors if they reported having ever been told by a doctor that they had any kind of cancer. Family members of cancer survivors were deﬁned as subjects who lived with cancer survivors during the survey time. Non-cancer and non-family member controls were selected among subjects without cancer and without family members with cancer with 1:1 individual matching on sex and age using matched simple random sampling methods. Subjects who were living alone were excluded. Therefore, the ﬁnal study subjects included 6783 cancer survivors, 6783 non-cancer controls, 8585 family members of cancer survivors, and 8585 non-cancer, non-family controls. 3. Results Cancer survivors and non-cancer controls showed signiﬁcant differences in general characteristics. The proportions of current smokers and individuals drinking ≥2/month were lower in cancer survivors than in non-cancer controls. There were signiﬁcant differences between family members and controls in the number of chronic illnesses (Table 1). Table 1. Characteristics of cancer survivors, family members, and controls. 2.4. Statistical Analysis Descriptive statistics and chi-square tests were used to examine differences in demographic characteristics between cancer survivors, their family members, and the controls. Chi-square tests and logistic regression models were used to investigate whether stress and depressive symptoms differed between cancer survivors, family members, and controls. Finally, the associated factors for stress and depressive symptoms in cancer survivors and family members were investigated using multiple Int. J. Environ. Res. Public Health 2017, 14, 999 3 of 8 logistic regression analysis. All data analyses were performed using SAS 9.2 software (SAS Institute, Cary, NC, USA). Differences were considered statistically signiﬁcant at p < 0.05. logistic regression analysis. All data analyses were performed using SAS 9.2 software (SAS Institute, Cary, NC, USA). Differences were considered statistically signiﬁcant at p < 0.05. 3. Results Characteristics Cancer Survivors (n = 6783) Non-Cancer Controls (n = 6783) p Family Members of Cancer Survivors (n = 8585) Non-Cancer Non-Family Controls (n = 8585) p Gender 1.000 1.000 Male 3157 (46.5) 3157 (46.5) 4148 (48.3) 4148 (48.3) Female 3626 (53.5) 3626 (53.5) 4437 (51.7) 4437 (51.7) Age (years) 1.000 1.000 18–39 698 (10.3) 698 (10.3) 2864 (33.4) 2864 (33.4) 40–64 2952 (43.5) 2952 (43.5) 3139 (36.6) 3139 (36.6) ≥65 3133 (46.2) 3133 (46.2) 2582 (30.1) 2582 (30.1) Marital status 0.258 0.145 Without spouse 894 (13.2) 939 (13.8) 2371 (27.6) 2286 (26.6) With spouse 5889 (86.8) 5844 (86.2) 6214 (72.4) 6299 (73.4) Education 0.259 0.654 Uneducated 1002 (14.8) 1072 (15.8) 1080 (12.6) 1144 (13.3) Elementary school 1770 (26.1) 1818 (26.8) 1626 (18.9) 1594 (18.6) Middle school 1180 (17.4) 1125 (16.6) 1000 (11.7) 1006 (11.7) High school 1773 (26.1) 1752 (25.8) 2715 (31.6) 2711 (31.6) ≥College 1058 (15.6) 1016 (15.0) 2164 (25.2) 2130 (24.8) Smoking <0.001 0.945 Never 4137 (61.0) 4196 (61.9) 5324 (62.0) 5338 (62.2) Ex 2041 (30.1) 1546 (22.8) 1448 (16.9) 1452 (16.9) Current 605 (8.9) 1041 (15.4) 1813 (21.1) 1795 (20.9) Drinking frequency <0.001 0.202 None 3892 (57.4) 2891 (42.6) 3068 (35.7) 3119 (36.3) ≤1/month 1264 (18.6) 1524 (22.5) 2105 (24.5) 2005 (23.4) ≥2/month 1627 (24.0) 2368 (34.9) 3412 (39.7) 3461 (40.3) Number of diseases 0.007 0.044 None 2853 (42.1) 3008 (44.4) 4714 (54.9) 4818 (56.1) 1 1928 (28.4) 1922 (28.3) 1863 (21.7) 1896 (22.1) ≥2 2002 (29.5) 1853 (27.3) 2008 (23.4) 1871 (21.8) Unmet needs for health care 0.007 0.150 No 6120 (90.2) 6023 (88.8) 7588 (88.4) 7525 (87.7) Yes 663 (9.8) 760 (11.2) 997 (11.6) 1058 (12.3) Reasons for unmet needs a Cost 223 (33.6) 208 (27.4) 232 (23.3) 238 (22.5) Reservation problems 25 (3.8) 15 (2.0) 24 (2.4) 22 (2.1) Transportaion problems 75 (11.3) 81 (10.7) 72 (7.2) 92 (8.7) Not available at time required 162 (24.4) 229 (30.1) 385 (38.7) 364 (34.4) Waiting time too long 30 (4.5) 29 (3.8) 39 (3.9) 39 (3.7) Mild symptoms 72 (10.9) 112 (14.7) 150 (15.1) 170 (16.1) Other 76 (11.5) 86 (11.3) 94 (9.4) 133 (12.6) Community health service utilization 0.882 0.276 No 6302 (55.8) 3563 (55.7) 5272 (64.5) 5304 (65.3) Yes 2850 (44.2) 2834 (44.3) 2904 (35.5) 2819 (34.7) Data are expressed as number (%). a Limited to subjects who have the unmet health care needs. Table 1. 3. Results Characteristics of cancer survivors, family members, and controls. Among 6783 cancer survivors, 26.9% and 8.7% reported stress and depressive symptoms, respectively. Cancer survivors were more likely to have stress (adjusted odds ratio (aOR) = 1.26, 95% conﬁdence interval (CI) = 1.16–1.37), consultation experience due to stress (aOR = 1.57, 95% CI = 1.21–2.03), depressive symptoms (aOR = 1.82, 95% CI = 1.57–2.11), and consultation experience due to depressive symptoms (aOR = 1.86, 95% CI = 1.32–2.64). In addition, 27.7% and 5.9% of cancer 4 of 8 Int. J. Environ. Res. Public Health 2017, 14, 999 survivor family members reported stress and depressive symptoms, respectively. The risks for stress (aOR = 1.08, 95% CI = 1.01–1.16) and depressive symptoms (aOR = 1.31, 95% CI = 1.14–1.51) were signiﬁcantly higher in family members of cancer survivors than controls without cancer and without family members with cancer (Table 2). Table 2. Stress and depressive symptoms in cancer survivors, family members, and controls. Characteristics Cancer Survivors Controls p Family Members of Cancer Survivors Non-Cancer Non-Family Controls p Stress <0.001 0.015 No 4957 (73.1) 5245 (77.3) 6206 (72.3) 6348 (73.9) Yes 1826 (26.9) 1538 (22.7) 2379 (27.7) 2237 (26.1) cOR (95% CI) 1.26 (1.16–1.36) 1.00 1.09 (1.02–1.17) 1.00 aOR (95% CI) a 1.26 (1.16–1.37) 1.00 1.08 (1.01–1.16) 1.00 Consultation due to stress <0.001 0.603 No 6625 (97.7) 6682 (98.5) 8428 (98.2) 8437 (98.3) Yes 158 (2.3) 101 (1.5) 157 (1.8) 148 (1.7) cOR (95% CI) 1.58 (1.23–2.04) 1.00 1.06 (0.85–1.33) 1.00 aOR (95% CI) a 1.57 (1.21–2.03) 1.00 1.05 (0.84–1.32) 1.00 Depressive symptoms <0.001 <0.001 No 6194 (91.3) 6471 (95.4) 8082 (94.1) 8203 (95.6) Yes 589 (8.7) 312 (4.6) 503 (5.9) 382 (4.5) cOR (95% CI) 1.98 (1.72–2.28) 1.00 1.34 (1.17–1.54) 1.00 aOR (95% CI) b 1.82 (1.57–2.11) 1.00 1.31 (1.14–1.51) 1.00 Consultation due to depressive symptoms <0.001 0.378 No 6676 (98.4) 6732 (99.3) 8501 (99.0) 8512 (99.2) Yes 107 (1.6) 51 (0.8) 84 (1.0) 73 (0.9) cOR (95% CI) 2.12 (1.52–2.97) 1.00 1.15 (0.84–1.58) 1.00 aOR (95% CI) b 1.86 (1.32–2.64) 1.00 1.10 (0.80–1.52) 1.00 Data are expressed as numbers (%). aOR, adjusted odds ratio; cOR, crude odds ratio; CI, conﬁdence interval. a Adjusted for marital status, education, smoking, alcohol drinking, and number of chronic diseases; b Adjusted for marital status, education, smoking, alcohol drinking, number of chronic diseases, and perceived stress. Table 2. 3. Results Stress and depressive symptoms in cancer survivors, family members, and controls. Stress was associated with gender, age, education, smoking, number of chronic disease, unmet needs for health care, and utilization of community health service in cancer survivors. Depressive symptoms were associated with gender, marital status, smoking, alcohol drinking, number of chronic diseases, and unmet needs for health care in cancer survivors. Similar associations were shown in family members of cancer survivors (Table 3). Table 3. Stress and depressive symptoms by characteristics in cancer survivors and family members. Table 3. Stress and depressive symptoms by characteristics in cancer survivors and family members. Characteristics Cancer Survivors Family Members of Cancer Survivors Stress Depressive Symptoms Stress Depressive Symptoms % aOR (95% CI) % aOR (95% CI) % aOR (95% CI) % aOR (95% CI) Total 26.9 8.7 27.7 5.9 Gender Male 24.4 1.00 7.2 1.00 24.3 1.00 3.8 1.00 Female 29.1 1.47 (1.20–1.79) 10.0 1.42 (1.03–1.96) 30.9 1.77 (1.50–2.08) 7.8 2.56 (1.81–3.61) Age (years) 18–39 29.7 1.65 (1.29–2.11) 8.5 1.22 (0.82–1.82) 31.2 2.43 (1.96–3.00) 5.1 1.33 (0.88–1.99) 40–64 27.4 1.28 (1.11–1.48) 8.4 1.05 (0.84–1.32) 27.8 1.53 (1.31–1.79) 6.3 1.27 (0.96–1.68) ≥65 25.9 1.00 9.0 1.00 23.9 1.00 6.2 1.00 Marital status Without spouse 29.1 0.94 (0.79–1.12) 13.7 1.53 (1.20–1.95) 27.6 0.75 (0.66–0.86) 5.7 1.11 (0.85–1.44) With spouse 26.6 1.00 7.9 1.00 27.8 1.00 5.9 1.00 Int. J. Environ. Res. Public Health 2017, 14, 999 5 of 8 Table 3. Stress and depressive symptoms by characteristics in cancer survivors and family members. 3. Results Characteristics Cancer Survivors Family Members of Cancer Survivors Stress Depressive Symptoms Stress Depressive Symptoms % aOR (95% CI) % aOR (95% CI) % aOR (95% CI) % aOR (95% CI) Education Uneducated 32.2 1.31 (1.04–1.65) 9.9 0.87 (0.59–1.28) 29.8 1.20 (0.96–1.50) 7.9 1.06 (0.70–1.61) Elementary school 28.4 1.15 (0.95–1.40) 9.8 1.17 (0.85–1.61) 28.7 1.07 (0.89–1.29) 6.8 0.90 (0.63–1.30) Middle school 25.2 0.92 (0.74–1.13) 9.5 1.17 (0.84–1.64) 22.4 0.72 (0.59–0.89) 6.4 1.03 (0.71–1.50) High school 23.6 0.81 (0.67–0.97) 7.2 0.84 (0.62–1.14) 26.5 0.84 (0.74–0.96) 5.1 0.86 (0.65–1.14) ≥College 26.8 1.00 7.3 1.00 29.9 1.00 4.9 1.00 Smoking Never 26.8 1.00 9.1 1.00 28.3 1.00 6.7 1.00 Ex 24.7 1.44 (1.17–1.76) 7.4 1.28 (0.92–1.78) 22.2 1.25 (1.02–1.52) 4.4 1.55 (1.04–2.31) Current 34.9 2.13 (1.67–2.72) 10.4 1.75 (1.19–2.57) 30.5 1.81 (1.52–2.16) 4.7 1.63 (1.12–2.37) Drinking frequency None 28.2 1.10 (0.95–1.27) 10.3 1.68 (1.30–2.17) 27.2 0.94 (0.82–1.08) 7.1 1.12 (0.86–1.46) ≤1/month 24.0 0.85 (0.71–1.03) 7.0 1.11 (0.80–1.53) 28.7 0.98 (0.86–1.13) 5.5 0.96 (0.73–1.26) ≥2/month 26.2 1.00 6.0 1.00 27.6 1.00 4.9 1.00 Number of diseases None 24.0 1.00 7.1 1.00 26.9 1.00 4.5 1.00 1 25.9 1.16 (1.01–1.34) 8.1 1.10 (0.87–1.40) 25.6 1.18 (1.02–1.36) 6.2 1.48 (1.12–1.96) ≥2 32.1 1.48 (1.28–1.72) 11.5 1.51 (1.19–1.91) 31.7 1.67 (1.43–1.94) 8.7 2.13 (1.60–2.82) Unmet needs for health care No 24.5 1.00 7.4 1.00 25.1 1.00 4.9 1.00 Yes 49.5 3.00 (2.38–3.79) 21.0 3.21 (2.38–4.34) 47.6 2.44 (2.04–2.92) 13.0 3.03 (2.30–3.98) Community health service utilization No 27.8 1.14 (1.01–1.29) 8.6 1.05 (0.86–1.29) 28.5 1.09 (0.97–1.23) 5.7 1.04 (0.83–1.31) Yes 23.5 1.00 7.4 1.00 23.5 1.00 5.3 1.00 aOR, adjusted odds ratio; CI, conﬁdence interval. Table 3. Stress and depressive symptoms by characteristics in cancer survivors and family members. 4. Discussion In this study, we described the stress and depressive symptoms of cancer survivors and their family members, and compared these symptoms with controls. Among 6783 cancer survivors, 26.9% of survivors were stressed, and 8.7% of survivors had depressive symptoms. Cancer survivors were more likely to have stress and depressive symptoms compared with controls. Similar results were found in family members of survivors. Cancer survivors have a high incidence of stress and depressive symptoms compared with controls, which are risk factors for suicide in cancer survivors [16]. Unfortunately, depression and psychosocial stresses associated with cancer often go unrecognized or underestimated, leaving many patients untreated. Previous studies reported that among 144 patients with gynecologic or breast cancer who were diagnosed with major depression, only 12% reported receiving antidepressants, and only 5% reported seeing a counselor or participating in a cancer support group [17]. Compared with healthy people and those with benign diseases, cancer patients report signiﬁcantly depressed mood that persists up to four months after diagnosis and during anticipation of treatment [18]. These results suggest that careful monitoring of psychotic aspects of cancer patients is as important as their cancer management. Family members of cancer survivors were more likely to have stress and depressive symptoms than controls. In this study, the prevalence of depressive symptoms among family members of cancer survivors was 5.9%, which was lower than that among caregivers of stroke survivors (40.2%) [19] and Alzheimer disease (34.0%) [20]. There are several possible explanations for these results. First, stroke survivors and patients with dementia are more likely to exhibit cognitive impairments and behavioral problems. As a result, their caregivers are more likely to develop depression. Second, the mortality rate of cancer patients may be higher than that of stroke survivors and dementia. The duration and burden of caregiving for cancer patients may be shorter and less than those of looking after stroke survivors and patients with dementia. Diagnosis of cancer is a stressful event for family members as well as Int. J. Environ. Res. Public Health 2017, 14, 999 6 of 8 cancer patients [21]. In previous studies, the prevalence of stress and depression among family member of cancer patients was high [22], and the perceived burden is the best predictor of depression [23]. Recognition of cancer diagnosis, caring for patients, medical expenses, and the threat of death can lead to psychological problems in family members [24]. 4. Discussion These psychological effects of cancer can be a threat to the physical health of family members, thus efforts to reduce the psychological effects of cancer should focus not only on the patient but also on the family members. Smartphone applications can be applied to improve after-care of cancer survivors [25] and provides psychoeducation to caregivers [26] based on previous applications on stroke survivors and dementia. Women cancer survivors and family members were more likely to report stress and depressive symptoms. This was consistent with previous study in Korea which showed that women cancer survivors were more likely to experience depression than men [27]. Higher psychological problems were reported among women cancer patients. They had a tendency to be more depressed and anxious than men with cancer [28] and were more likely to express their feelings and report more symptoms than men [29]. Although the unmet needs for health care in cancer survivors and family member were not higher than those of controls, they were signiﬁcantly associated with stress and depressive symptoms in this study. Previous studies revealed that unmet needs were associated with low quality of life in cancer patients [30] and were positively associated with depression among caregivers of cancer patients [31]. These results suggested that monitoring and offering support for adequate health care needs of cancer survivors and family member are important for reducing stress and depressive symptoms. There are several limitations to using KCHS data to examine stress and depressive symptoms in cancer survivors and their family members. First, because of the cross-sectional nature of this data, we were unable to examine stress and depressive symptoms before cancer diagnosis. Stress and depression have been considered as risk factors for cancer development [32]. Second, the KCHS does not include information about cancer type or current cancer status. Thus, we were unable to determine the proportion of cancer survivors who were actively dealing with treatment or recurrent/advanced disease versus those living disease- and/or symptom-free. Third, cancer survivors who lived in nursing homes, long-term care facilities, or hospitals were not included in the survey, so the results of this study might not accurately reﬂect the status of all cancer patients in Korea. Fourth, reliability and validity for the single-item surveys on stress and depressive symptoms were not investigated. 4. Discussion However, the single-item measure of stress or depression was suggested to be a valid measure for group level data or screening for psychological status [33,34]. 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https://openalex.org/W2610338273	http://www.scielo.br/pdf/sausoc/v26n1/1984-0470-sausoc-26-01-00229.pdf	Portuguese	null	Diplomacia da saúde global: proposta de modelo conceitual	Saúde e Sociedade	2,017	cc-by	7,280	Diplomacia da saúde global: proposta de modelo conceitual1 2 Global health diplomacy: conceptual framework proposal 229 Pollyanna Martins Secretaria de Saúde do Estado do Ceará. Fortaleza, CE, Brasil. E-mail: pollysobral@yahoo.com.br Andréa Silvia Walter de Aguiar Universidade Federal do Ceará. Fortaleza, CE, Brasil. E-mail: aswaguiar@pq.cnpq.br Caroline Antero Machado Mesquita Faculdade Maurício de Nassau. Fortaleza, CE, Brasil. E-mail: carolantero41@gmail.com Francisca Jamila Ricarte Alexandrino Universidade Federal do Ceará. Fortaleza, CE, Brasil. E-mail: jamilaricartealexandrino@gmail.com Nayane Cavalcante Ferreira da Silva Faculdade Maurício de Nassau. Fortaleza, CE, Brasil. E-mail: nayanecferreira@gmail.com Melinna dos Santos Moreno Universidade Federal do Ceará. Programa de Pós-Graduação em Odontologia. Fortaleza, CE, Brasil. E-mail: melinnamoreno@hotmail.com Pollyanna Martins Secretaria de Saúde do Estado do Ceará. Fortaleza, CE, Brasil. E-mail: pollysobral@yahoo.com.br Andréa Silvia Walter de Aguiar Universidade Federal do Ceará. Fortaleza, CE, Brasil. E-mail: aswaguiar@pq.cnpq.br Caroline Antero Machado Mesquita Faculdade Maurício de Nassau. Fortaleza, CE, Brasil. E-mail: carolantero41@gmail.com Francisca Jamila Ricarte Alexandrino Universidade Federal do Ceará. Fortaleza, CE, Brasil. E-mail: jamilaricartealexandrino@gmail.com Nayane Cavalcante Ferreira da Silva Faculdade Maurício de Nassau. Fortaleza, CE, Brasil. E-mail: nayanecferreira@gmail.com Melinna dos Santos Moreno Universidade Federal do Ceará. Programa de Pós-Graduação em Odontologia. Fortaleza, CE, Brasil. E-mail: melinnamoreno@hotmail.com 1 Este artigo é resultado de pesquisa denominada “Diplomacia da saúde nas ações e serviços de saúde bucal na faixa de fronteira brasi- leira”, financiada pelo CNPq, por meio do Edital Universal (14/2014). Resumo Este estudo busca construir, a partir das publicações sobre diplomacia da saúde global (DSG), representa- ção deste campo de conhecimento, seus principais conceitos e características. A partir de uma pesquisa bibliográfica na base de dados PubMed, fez-se uma busca integrada das produções científicas publica- das até agosto de 2016. Os descritores identificados e critérios de inclusão adotados selecionaram 54 publicações que compuseram a amostra final. Os dados quantitativos foram analisados por estatística descritiva, e as informações qualitativas, pela análise de conteúdo temática. As publicações sobre o tema ti- veram início em 1970, mas 85,2% abrangem o período de 2007 a 2014. A partir da análise de conteúdo dos resultados das produções científicas, foi elaborado um modelo teórico conceitual que abrange os seguin- tes macrotemas da DSG: conceito de diplomacia da saúde; uso da diplomacia na cooperação internacional em pesquisa; diplomacia da saúde como ferramenta para fortalecer a política externa; estratégias dos paí- ses para fortalecer a DSG e cooperação internacional entre os países; diplomacia da saúde fortalecendo a saúde global; saúde como um bem comum entre as nações em um período de globalização e desafios da diplomacia da saúde para fortalecer a saúde global. A DSG compreende estratégias dos países e coope- ração internacional para fortalecer a saúde global e é uma ferramenta para fortalecer a política externa das nações. Promover a cooperação internacional em pesquisa, capacitar recursos humanos nessa área e emancipar países emergentes com base nas interven- ções realizadas constituem grandes desafios da DSG. Palavras-chave: Saúde Mundial; Cooperação Inter- nacional; Relações Internacionais. Abstract The aim of this article is to build, from the pub- lications on global health diplomacy (GHD), a representation of this field of knowledge, its main concepts and features. We searched the literature in PubMed database for publications on the subject until August 2016. The keywords and inclusion criteria selected 54 publications that composed the final sample. Quantitative data were ana- lyzed using descriptive statistics, and qualitative information, by the thematic content analysis. Publications on the subject began in 1970, but 85.2% of the studies cover the period from 2007 to 2014. With the content analysis of the results of this scientific production, we produced a con- ceptual theoretical model covering the following subjects of GHD: health diplomacy concept; use of diplomacy in international cooperation with research; health diplomacy as a tool for strength- ening foreign policy; strategies for strengthening the diplomacy of global health and international cooperation among countries; health diplomacy strengthening global health; health as a common good among the nations in globalization; health diplomacy challenges to strengthen global health. GHD comprises country strategies and interna- tional cooperation to strengthen global health and is a tool for strengthening the foreign policy of na- tions. The major challenges of GHD are to promote international cooperation in research, train human resources from this area and emancipate emerging countries based on ongoing interventions. Ministros e autoridades sanitárias de 194 países participaram da 68ª Assembleia Mundial da Saúde, da Organização Mundial da Saúde (OMS)2, realizada de 18 a 26 de maio de 2015, em Genebra, Suíça. A incorporação da saúde na agenda para o desenvol- vimento pós-2015 e o impacto da contaminação am- biental na saúde estavam entre os temas debatidos. Esse conjunto de negociações desenvolvidas em diversos níveis, que delineia e gerencia o ambiente das políticas globais em saúde e que, idealmente, produz melhores resultados para a saúde da popula- ção de cada país envolvido e reforça o compromisso de um amplo arco de atores em prol do empreendi- mento comum de assegurar a saúde como direito humano e bem público, tem sido denominado “di- plomacia da saúde global” (DSG) (Kicksbusch et al., 2007). Trata-se de um campo novo de conhecimento e prática, cujo objeto é a saúde e as negociações in- ternacionais em torno desta, envolvendo diferentes disciplinas e profissionais de diversos perfis, como diplomatas e profissionais de saúde (Buss, 2013). Abstract A DSG é uma disciplina emergente em que a saúde é incorporada à diplomacia tradicional como uma ferramenta de política externa (Kicksbusch et al., 2007). A DSG é usada por muitos países para melhorar as relações de confiança entre os estados, a saúde nacional e mundial. Nessa perspectiva, os ministérios da saúde passaram a desempenhar um papel duplo: promover a saúde do país e a saúde global (Fourie, 2013). Objetiva-se propor, a partir das publicações sobre DSG, representação deste campo de conheci- mento, seus principais conceitos e características, assim como apontar novas questões que carecem de pesquisas. Keywords: World Health; International Coopera- tion; International Relations. Correspondência Andréa Sílvia Walter de Aguiar Rua Monsenhor Furtado, s/nº, Rodolfo Teófilo. Fortaleza, CE, Brasil. CEP 60430-355. 1 Este artigo é resultado de pesquisa denominada “Diplomacia da saúde nas ações e serviços de saúde bucal na faixa de fronteira brasi- leira”, financiada pelo CNPq, por meio do Edital Universal (14/2014). Saúde Soc. São Paulo, v.26, n.1, p.229-239, 2017 229 DOI 10.1590/S0104-12902017168881 230 Saúde Soc. São Paulo, v.26, n.1, p.229-239, 2017 Resultados 1. Saúde Mundial/World Health/Salud Mun- dial. Sinônimos: Problemas Internacionais de Saúde, Saúde Global; As publicações sobre o tema iniciaram em 1970. Todavia, a partir de 2007 observou-se maior frequência (85,2% abrangem o período de 2007 a 2014). A maioria das instituições ou universidades às quais estão vinculados os primeiros autores dos artigos localiza-se nos Estados Unidos da América (61%), na Suíça (14,8%) e no Brasil (5,6%). Na Suíça destacaram-se a World Health Organization, de Genebra, e o Graduate Institute for International Development Studies, da mesma cidade; nos EUA, organizações não governamentais, departamentos estatais, Columbia University, Indiana University e University of California; no Brasil, a Fundação Oswaldo Cruz. Entretanto, não se observou seme- lhante tendência quando se considerou o país de pu- blicação do periódico; neste caso, os EUA (53,7%) e o Reino Unido (25,9%) lideraram o total de publicações. 2. Cooperação Internacional/International Co- operation/Cooperación Internacional. Sinô- nimos: Auxílio Internacional, Ajuda Externa; 3. Internacionalidade/Internationality/Interna- cionalidad. Sinônimos: Relações Internacionais. A estratégia de busca adotou os seguintes des- critores no campo de busca avançada da PubMed: “world health” [MeSH Terms] or “world” [All Fields] and “health” [All Fields] or “world health” [All Fields] and “international cooperation” [MeSH Terms] or “international” [All Fields] and “cooperation” [All Fields] or “international cooperation” [All Fields] and “internationality” [MeSH Terms] or “internationality” [All Fields] and “health” [MeSH Terms] or “health” [All Fields] and “diplomacy” [All Fields]. Os critérios de inclusão para a seleção da amostra em relação à dispo- nibilidade de texto foram: “Free full text”, “Full text”. Foram encontradas 54 publicações que compu- seram a amostra final. O material foi submetido à pré-análise, e, pela leitura integral do texto, foram categorizados segundo as seguintes variáveis: 1º autor, ano de publicação, periódico, título, obje- tivo, resultado principal relativo ao tema, tipo de publicação, universidade/instituição do 1º autor e país de publicação do periódico. Na variável “tipo de publicação” foram considerados como editoriais os comentários, editoriais, debates e artigos de opinião publicados no período. Os resultados foram transcritos em uma planilha eletrônica no Microsoft Excel, os dados quantitativos foram analisados por estatística descritiva simples, e os dados qualitati- vos, pela análise temática de conteúdo. Metodologia Para construir o modelo conceitual, optou-se por uma pesquisa bibliométrica embasada em dados publicados e usada para mensurar as características de determinados temas, o que de certa forma indica sua produção científica (Vanti, 2002). 2 WHO – WORLD HEALTH ORGANIZATION. 68th World Health Assembly. Disponível em: <https://goo.gl/Lbuk71>. Acesso em: 2 maio 2015. 230 Saúde Soc. São Paulo, v.26, n.1, p.229-239, 2017 A análise de conteúdo temática foi feita para descobrir os “núcleos de sentido” que compõem a comunicação e cuja presença ou frequência pode significar alguma coisa para o tema em estudo (Bar- din, 2011). Na coleta dos dados qualitativos (título, objetivos, resultados principais) extraídos da leitura dos textos, foram usadas frases e parágrafos como unidades de registro, codificadas em temas reagru- padas em categorias ou subcategorias. A base de dados consultada foi a U.S. National Li- brary of Medicine (PubMed), em que se fez uma bus- ca integrada das produções científicas publicadas até agosto de 2016. Os descritores adotados foram identificados pelo Medical Subject Headings (MeSH) dessa base de dados. Na busca, foram encontrados descritores que se apresentaram como sinônimos do tema pesquisado, visto que o termo “diplomacia da saúde” não foi encontrado no MeSH. Os descritores identificados e seus respectivos sinônimos foram: Saúde Soc. São Paulo, v.26, n.1, p.229-239, 2017 231 Resultados A estratégia de busca adotou os seguintes des- critores no campo de busca avançada da PubMed: “world health” [MeSH Terms] or “world” [All Fields] and “health” [All Fields] or “world health” [All Fields] and “international cooperation” [MeSH Terms] or “international” [All Fields] and “cooperation” [All Fields] or “international cooperation” [All Fields] and “internationality” [MeSH Terms] or “internationality” [All Fields] and “health” [MeSH Terms] or “health” [All Fields] and “diplomacy” [All Fields]. Os critérios de inclusão para a seleção da amostra em relação à dispo- nibilidade de texto foram: “Free full text”, “Full text”. 5 9 p ç Quanto ao tipo de publicação, destacaram-se os editoriais (38,9%), seguidos de artigos de revisão/ reflexão (29,6%) e artigos originais/estudo de caso (22,2%). Artigos teóricos conceituais totalizaram 9,3% das publicações. Os resultados principais das pesquisas sobre o tema foram submetidos à análise de conteúdo temática e, depois do processo de codificação e categorização, emergiram sete macrotemas: conceito de diplomacia da saúde; uso da diplomacia na cooperação internacional em pesquisa; diplomacia da saúde como ferramenta para fortalecer a política externa; estratégias dos países para fortalecer a DSG e cooperação interna- cional entre os países; diplomacia da saúde forta- lece a saúde global; a saúde como um bem comum entre as nações em um período de globalização e desafios da diplomacia da saúde para fortalecer a saúde global. f Foram encontradas 54 publicações que compu- seram a amostra final. O material foi submetido à pré-análise, e, pela leitura integral do texto, foram categorizados segundo as seguintes variáveis: 1º autor, ano de publicação, periódico, título, obje- tivo, resultado principal relativo ao tema, tipo de publicação, universidade/instituição do 1º autor e país de publicação do periódico. Na variável “tipo de publicação” foram considerados como editoriais os comentários, editoriais, debates e artigos de opinião publicados no período. Os resultados foram transcritos em uma planilha eletrônica no Microsoft Excel, os dados quantitativos foram analisados por estatística descritiva simples, e os dados qualitati- vos, pela análise temática de conteúdo. Saúde Soc. São Paulo, v.26, n.1, p.229-239, 2017 231 Discussão Suíça, o que pode ser explicado pelo fato de esses dois países serem os líderes nas questões diplomá- ticas na área da saúde, pois sediam a Organização das Nações Unidas (ONU) e OMS, respectivamente. A produção científica sobre DSG é recente, e o tema ainda é pouco conhecido nas comunidades aca- dêmicas, o que foi confirmado pela predominância de editoriais entre as produções científicas seleciona- das. A função de um editorial em um periódico é dis- cutir temas atuais e controversos e publicar análises ou opiniões sobre um tema importante. A abordagem qualitativa com base em estudos de caso e os artigos de revisão que refletem sobre o tema ou têm a função de introduzir conceitos sobre determinado campo de conhecimento endossam essa premissa. Com base na análise dos resultados das pes- quisas sobre o tema, elaborou-se uma árvore da DSG, e suas raízes representam os conceitos e as teorias sobre o tema; os galhos, as consequências da diplomacia; e os frutos, os produtos gerados (Figura 1). A respeito dos principais resultados das pesqui- sas sobre o tema, discutem-se a seguir os macrote- mas que emergiram do processo de codificação e ca- tegorização dos dados qualitativos e que compõem a árvore da DSG. A inserção dos autores que pesquisam e escre- vem sobre o tema está concentrada nos EUA e na Evolução conceitual da Diplomacia da Saúde Global Evolução conceitual da Diplomacia da Saúde Global o que contribuiu para a inclusão como uma ferra- menta de política externa dos países. French (1992) introduziu o termo “diplomacia internacional” no que diz respeito aos esforços e à preocupação das nações com as questões ambientais. Sharpless (1995) considerou que as relações diplomáticas funcionam como agentes de modificação comportamental nos países em de- senvolvimento. Ao analisar os primeiros cem anos de diplomacia da saúde internacional, mostrou-se a vulnerabilidade da governança global e os riscos que a globalização causou à saúde pública, refletin- do sobre a necessidade da cooperação internacional para controlá-los. Dentro da tipologia da diplomacia pública, a DSG é um conceito autônomo, um instrumento de política externa que não recorre aos tradicionais meios e canais diplomáticos para atingir seus objetivos definidos. Ela assume características próprias que a distingue de outros conceitos que lhe são próximos, como a diplomacia tradicional, e seu conceito evoluiu ao longo do tempo, conforme o quadro a seguir. Embora a emergência do termo “DSG” seja recen- te, a interação entre diplomacia e saúde não é uma prática contemporânea entre as nações. “Os regis- tros diplomáticos do Egito antigo que datam de 29 séculos atrás identificam os médico-sacerdotes do Nilo que acompanhavam as missões egípcias para países vizinhos” (Badeau, 1970, p. 303). A partir das premissas anteriores e consideran- do que a saúde internacional depende do melhor controle de doenças em nível nacional ou local (Hardiman, 2003), nasce o termo “DSG”, definido como um conjunto de negociações desenvolvidas em diversos níveis que delineia e gerencia o ambiente das políticas globais em saúde e que, idealmente, produz melhores resultados para a saúde da popu- lação de cada país envolvido, bem como reforça o compromisso de um amplo arco de atores em prol da garantia da saúde como direito humano e bem público (Kickbusch et al., 2007). Entretanto, Drager e Fidler (2007) assinalaram o desafio de alinhar os interesses nacionais, a política externa e a DSG em um mundo globalizado, com distintas realidades epidemiológicas e éticas. Nos períodos pós-guerra, a saúde constituiu-se em um objetivo comum entre as nações, e isso pas- sou a se refletir nas ações diplomáticas. A expansão da comunicação pública (rádio, televisão, imprensa etc.) favoreceu a difusão de assuntos nacionais e internacionais. Evolução conceitual da Diplomacia da Saúde Global A libertação de colônias, a educação popular e a conscientização política aumentaram a pressão por acesso à educação e à saúde nos países emergentes e gerou novas demandas para as ações diplomáticas (Badeau, 1970). Figura 1 – Modelo conceitual: árvore da diplomacia da saúde global a 1 Modelo conceitual: árvore da diplomacia da saúde global Saúde global e desenvolvimento Relação de confança entre nações Saúde bucal integra a saúde global Redefnição do papel do Estado no comércio mundial Acesso equitativo a vacinas e medicamentos Formação de recursos humanos Adaptação à cultura, segurança da equipe Resolução de problemas comuns Uso de metadados Melhoria da equidade e diminuição dos gastos em saúde Distribuição global da carga de doenças Oportunidades de comércio Liderança dos países em ONGs Resultados de saúde sustentáveis Promoção da saúde global Redes de vigilância em saúde global Controle do tabaco, acesso a água e serviços de saúde Saúde como um bem comum entre as nações Cooperação internacional em pesquisa Desafos Infuência na economia do país Infuência na economia do país Fortalecimento da saúde global Diplomacia da Saúde Global Ferramenta para fortalecer a política externa Construção de novos conceitos Sistema de organização dos processos de comunicação e negociação da saúde global Uso de metadados Sistema de organização dos processos de comunicação e negociação da saúde global Ferramenta para fortalecer a política externa Ferramenta para fortalecer a política externa Construção de novos conceitos 232 Saúde Soc. São Paulo, v.26, n.1, p.229-239, 2017 232 Saúde Soc. São Paulo, v.26, n.1, p.229-239, 2017 Evolução conceitual da Diplomacia da Saúde Global Intensificaram-se as ações humanitárias diplomáticas na área da saúde, Quadro 1 – Evolução conceitual de Diplomacia da Saúde Global ao longo do tempo Ano Macrotema 1970 Diplomacia médica – ações humanitárias (Badeau, 1970) 1992 Diplomacia internacional – questões ambientais (French, 1992) 2001 Efeitos da globalização na saúde (Fidler, 2001) 2003 Saúde global dependente da saúde de cada nação (Hardiman, 2003) 2007 DSG – negociações entre nações para promover a saúde global (Kickbusch et al., 2007) DSG – ferramenta para fortalecer a política externa (Drager; Fidler, 2007) 2008 DSG – participação das ONGs em negociações na promoção da saúde global (Adams; Novotny; Leslie, 2008) 2010 DSG – parcerias entre ONGs e governos (Kosaraju et al., 2010) 2013 DSG – melhora da saúde global, equidade, relações de confiança entre estados (Kickbusch; Kökény, 2013) DSG – políticas e programas que geram resultados de saúde sustentáveis (Ratzan, 2013) Quadro 1 – Evolução conceitual de Diplomacia da Saúde Global ao longo do tempo Ano Macrotema 1970 Diplomacia médica – ações humanitárias (Badeau, 1970) 1992 Diplomacia internacional – questões ambientais (French, 1992) 2001 Efeitos da globalização na saúde (Fidler, 2001) 2003 Saúde global dependente da saúde de cada nação (Hardiman, 2003) 2007 DSG – negociações entre nações para promover a saúde global (Kickbusch et al., 2007) DSG – ferramenta para fortalecer a política externa (Drager; Fidler, 2007) 2008 DSG – participação das ONGs em negociações na promoção da saúde global (Adams; Novotny; Leslie, 2008) 2010 DSG – parcerias entre ONGs e governos (Kosaraju et al., 2010) 2013 DSG – melhora da saúde global, equidade, relações de confiança entre estados (Kickbusch; Kökény, 2013) DSG – políticas e programas que geram resultados de saúde sustentáveis (Ratzan, 2013) Quadro 1 – Evolução conceitual de Diplomacia da Saúde Global ao longo do tempo Ano Macrotema 1970 Diplomacia médica – ações humanitárias (Badeau, 1970) 1992 Diplomacia internacional – questões ambientais (French, 1992) 2001 Efeitos da globalização na saúde (Fidler, 2001) 2003 Saúde global dependente da saúde de cada nação (Hardiman, 2003) 2007 DSG – negociações entre nações para promover a saúde global (Kickbusch et al., 2007) DSG – ferramenta para fortalecer a política externa (Drager; Fidler, 2007) 2008 DSG – participação das ONGs em negociações na promoção da saúde global (Adams; Novotny; Leslie, 2008) 2010 DSG – parcerias entre ONGs e governos (Kosaraju et al., 2010) 2013 DSG – melhora da saúde global, equidade, relações de confiança entre estados (Kickbusch; Kökény, 2013) DSG – políticas e programas que geram resultados de saúde sustentáveis (Ratzan, 2013) Saúde Soc. Evolução conceitual da Diplomacia da Saúde Global São Paulo, v.26, n.1, p.229-239, 2017 233 Autores acrescentaram que a DSG consiste na participação de organizações não governamentais (ONGs) em programas de saúde internacionais que abrangem a globalização da ciência e da pesquisa farmacêutica, bem como o uso de línguas militari- zadas de biossegurança para reformular programas de saúde pública (Adams; Novotny; Leslie, 2008). Kosaraju et al. (2010) complementaram esse con- ceito ao afirmarem que a parceria entre governos e ONG cria infraestrutura sustentável para ações de saúde global. Assim, a saúde pode interagir com a política externa como instrumento para atingir outras metas, podendo ser empregada para atingi- -las na diplomacia da saúde e influenciando a arena multilateral da saúde (Kickbusch, 2011). doenças crônicas, os acordos entre indústrias para diminuírem teores de açúcar e sal nos alimentos, a inserção e contribuição de subáreas da saúde na saúde global, como a saúde bucal, e o papel desses atores na DSG ainda são pouco discutidos na biblio- grafia acadêmica. No caso da saúde bucal, Garcia e Tabak (2011) consideraram que a saúde oral integra a saúde global em macrotemas, como o controle do taba- co, a água potável, a saúde materna e infantil e a pesquisa sobre os sistemas de saúde. Entretanto, a efetividade dessas intervenções e acordos bem como as responsabilidades dos países e governos com suas implementações permanecem questionáveis. Para enfrentar essa complexidade de inter-relações, que afeta globalmente a saúde das populações em diversas partes do mundo, são necessárias interven- ções, políticas específicas e suficiente produção de conhecimentos e capacitação de recursos humanos nessa área (Almeida, 2010). Complementou-se esse conceito ao apontar a in- fluência da saúde na economia dos países. As inter- -relações na saúde global podem se tornar um con- flito para as relações externas dos países-membros de uma região ou bloco econômico, comercial ou político. No contexto da globalização, a saúde está intimamente ligada ao meio ambiente, ao comércio, ao crescimento econômico, ao desenvolvimento so- cial, à segurança nacional e aos direitos humanos (Bernardini-Zambrini; Pedrosa; Sotelo, 2011). 234 Saúde Soc. São Paulo, v.26, n.1, p.229-239, 2017 Estratégias dos países para fortalecer a DSG e a cooperação internacional Barraclough e Phua (2007) apontaram que as ameaças das doenças infecciosas à saúde humana e à atividade econômica causaram intensificação e formalização organizacional da DSG tanto em nível nacional quanto internacional. Cahill (1997) postulou que a saúde e as situações de conflito e desastre são as bases para a interven- ção internacional, devendo-se adotar medidas diplo- máticas, sobretudo nos países menos desenvolvidos, à época denominados “de terceiro mundo”. Nessa perspectiva, o Brasil tem um papel ativo na pres- tação de ajuda para o desenvolvimento de outros países na administração pública, saúde, educação, agricultura, meio ambiente, energia e pequenas empresas (Kickbusch; Buss, 2011). O estreitamento das relações pode gerar benefícios mútuos em ne- gociações pacíficas e a construção de alianças para além da saúde. A DSG tem sido uma pedra angular da política externa cubana desde o início da revolução, há cin- quenta anos. Cuba começou a conduzir a diplomacia da saúde em 1960, mediante o envio de uma equipe médica para o Chile, a fim de fornecer socorro depois de um grande terremoto. Doravante, Cuba prestou assistência médica a mais de cem países em todo o mundo, tanto em situações de emergência a cur- to prazo quanto a longo prazo (Feinsilver, 2010). Exemplos recentes dessas iniciativas são a Missión Barrio Adentro, na Venezuela, e o Programa Mais Médicos, no Brasil. Collins et al. (2012) descreveram que o plano de emergência do ex-presidente dos EUA Barack Obama para combater a epidemia de aids é um dos carros-chefe das ações diplomáticas norte-america- nas. Em um cenário econômico mundial adverso, o desenvolvimento de ações diplomáticas em saúde tem se configurado como uma boa oportunidade para melhorar as relações entre países com uma boa relação custo-benefício, além de contribuir para a segurança nacional em saúde global (Boe- tig, 2012). Entretanto, as ações são voltadas para resolver problemas de curto prazo e não melhoram a capacidade de resposta local a médio e longo prazos (Reaves; Termini; Burkle, 2014). Os investimentos em programas de saúde globais têm função diplo- mática, atenuando a tensão em áreas de conflito ou pós-conflito com uma boa relação custo-benefício (Kevany et al., 2014). Além de ser utilizada para promover a saúde global, os países menos desenvolvidos utilizam a DSG para fortalecer sua liderança em organismos internacionais, como a ONU, gerar oportunidades comerciais e melhorar a relação de confiança com outras nações (Kickbusch; Kökény, 2013). Diplomacia da saúde na cooperação internacional em pesquisa Fortaleceu-se o uso de metadados na análise de distribuição global da doença, que podem ser basicamente definidos como “dados que descrevem dados”, ou seja, informações úteis para identificar, localizar, compreender e gerenciá-los. Eles podem ajudar na agenda pós-2015 a partir de um espaço de intervenção para integrar a ciência com seu contex- to sociotécnico (Özdemir et al., 2014). A DSG resulta em melhoria da saúde global, equidade e relações de confiança entre estados. Os elementos que contribuíram para a ascensão da di- plomacia foram os acordos comerciais, pandemias, maior coerência entre a política externa e interna de saúde (Kickbusch; Kökény, 2013). Esses autores postularam que a DSG refere-se a um sistema de organização dos processos de comunicação e ne- gociação que moldam o ambiente político global em matéria de saúde e seus determinantes.Nessa perspectiva, a saúde faz parte das ações diplomá- ticas da ONU, do G8, do G20, do BRICS, da União Europeia e dos ministérios da saúde que passaram a desempenhar um papel duplo: promover a saúde do país e a saúde global. Em consonância, Fedoroff (2009) destacou que colaborações científicas entre as nações para en- frentar problemas comuns e as interações científi- cas internacionais podem contribuir para diminuir o fosso digital (acesso desigual à ciência e à tecno- logia) e o acesso desigual a comida, água, energia, saúde e desenvolvimento econômico. A cooperação científica internacional é um fator determinante para superar as desigualdades entre os países, geradas pela transição epidemiológica, pela carga de doença desigual entre os países e pela pobreza, podendo colaborar para a governança da saúde global colaborativa e resolver problemas de saúde comuns das nações, por meio de metadados para priorizar intervenções com base no conheci- A DSG é o processo de se engajar, motivar e co- municar múltiplas partes para desenvolver políticas e programas que promovam resultados de saúde sustentáveis (Ratzan, 2013). O principal objetivo da DSG é promover a saúde global, entretanto, al- gumas questões que são comuns às nações, como os acordos internacionais e metas para atenção às 234 Saúde Soc. São Paulo, v.26, n.1, p.229-239, 2017 mento da distribuição global da carga de doenças (Novotny, 2006). Diplomacia da saúde na cooperação internacional em pesquisa A DSG, adotada como ferramenta para fortalecer a política externa das nações, parte da premissa de que a saúde influencia a economia dos países e que a saúde global é parte do bem-estar e do de- senvolvimento, em particular dos países menos desenvolvidos, além de colaborar para a liderança de países em organismos internacionais, gerando oportunidades comerciais e melhorando a relação de confiança entre as nações. Um dos desafios para fortalecer a cooperação internacional no desenvolvimento de pesquisas em saúde global é melhorar o alinhamento e a har- monização do financiamento das investigações de acordo com as necessidades dos países beneficiários (Røttingen et al., 2009). Saúde Soc. São Paulo, v.26, n.1, p.229-239, 2017 235 Diplomacia da saúde e o fortalecimento da saúde global Essa nova abordagem de cooperação internacio- nal em saúde tem sido denominada de “cooperação estruturante em saúde”, tendo como características a horizontalidade dos acordos e intervenções, cons- trução de parcerias, intercâmbio de experiências, aprendizado conjunto, compartilhamento de resul- tados e responsabilidades. Esse novo paradigma inova em dois aspectos em comparação ao modelo tradicional de cooperação internacional em saúde: integra formação de recursos humanos, promove o fortalecimento organizacional e desenvolvimento institucional e rompe com a tradicional transfe- rência passiva de conhecimentos e tecnologias (Almeida et al., 2010). Esforços multilaterais para desenvolver progra- mas estratégicos que reduzam a probabilidade de futuras ameaças criam a capacidade de informações integradas entre os países (Ratzen, 2005). Nessa perspectiva, a visão de DSG dos EUA mostra que é necessário elaborar políticas globais para enfrentar os problemas de saúde, incluindo o HIV/aids e a pandemia do H1N1. Data de 1995 a primeira estratégia interna- cional dos EUA sobre HIV/aids. Em 5 de maio de 2009, o então presidente Barack Obama anunciou a Iniciativa de Saúde Global para resolver alguns dos problemas mais graves de saúde que o mundo enfrenta. A experiência em lidar com o HIV/aids e o H1N1 mostra, claramente, que enfrentar os desa- fios da saúde global com estratégias de diplomacia e de desenvolvimento é essencial, pois a saúde dos norte-americanos está entrelaçada à saúde do resto do mundo (Jones, 2010; Daulaire, 2012). Segundo Kevany et al. (2012), o sucesso da implementação de adaptações específicas locais (religiosas, dialé- ticas, políticas, ambientais e de infraestrutura) tem implicações para a intervenção tanto do consumo de serviços quanto da perspectiva de DSG. A cooperação Sul-Sul no Brasil é um exemplo de “cooperação estruturante para a saúde”, pois baseia- -se, fundamentalmente, no fortalecimento dos sis- temas locais de saúde a partir da potencialização das capacidades e recursos endógenos em cada país (Buss, 2011). No Brasil várias instituições e atores atuam no âmbito da cooperação internacional em saúde nessa perspectiva. A criação da União de Na- ções Sul-Americanas (Unasul) e o Plano Estratégico de Cooperação em Saúde da Comunidade de Países de Língua Portuguesa (CPLP) são experiências de- correntes do aprimoramento do diálogo diplomático estabelecido em iniciativas de integração regional na América do Sul (Mercosul, Comunidade Andina e Organização do Tratado da Cooperação Amazôni- ca) (Buss; Ferreira, 2010a). Estratégias dos países para fortalecer a DSG e a cooperação internacional Em con- cordância, Seiff (2013) reforçou essa afirmação ao constatar que a liderança da Indonésia no campo da DSG contribuiu para que conseguisse diplomatica- mente a manutenção da Association of Southeast Asian Nations (Asean), além de ocupar cargos na ONU e no Fundo Global contra aids, malária e tu- berculose. A divulgação do National Health System, pela In- glaterra, contribuiu para o aprendizado da implan- tação de sistemas de saúde com cobertura universal em países emergentes, além de gerar oportunidades comerciais (Chalkidou; Vega, 2013). Desde a institucionalização da cooperação in- ternacional na década de 1950 até os dias atuais, países industrializados têm buscado abordar e Saúde Soc. São Paulo, v.26, n.1, p.229-239, 2017 235 235 As estratégias dos países para fortalecer a DSG e a cooperação internacional promovem a paz entre as nações com boa relação custo-benefício, por meio da participação das ONGs internacionais. As ações humanitárias melhoram problemas de curto prazo e auxiliam países em situação de conflito e em casos de desastre. Capacitar os recursos humanos para forta- lecer os sistemas universais de saúde e desenvolver resultados de saúde sustentáveis a médio e longo pra- zo devem ser um dos objetivos dessas estratégias, a fim de colaborar para emancipar países emergentes. resolver questões globais de pobreza e exclusão social (Almeida et al., 2010). O modelo tradicional de cooperação internacional é caracterizado pela ajuda humanitária de países desenvolvidos a países em desenvolvimento, unilateral, verticalizado e com predominância das visões políticas e práticas dos governos mais fortes economicamente ou das organizações não governamentais, filantrópicas e empresariais dessas nações (Buss; Ferreira, 2010b). Entretanto, modelos alternativos de cooperação internacional em saúde estão em desenvolvimento na América do Sul, através das experiências da cooperação Sul-Sul. Referências A DSG contribui para melhorar o acesso uni- versal, a equidade e diminuir os gastos em saúde, podendo também ajudar na redistribuição de renda de acordo com o estágio de desenvolvimento de cada país e a estabilização das sociedades. Ajudar outros países a resolverem esses desafios globais de saúde contribui para o crescimento sustentável da economia global (Abe, 2013) – um exemplo são os pro- gramas de inclusão social desenvolvidos pelos EUA. 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Por fim, Kickbusch e Szabo (2014) reforçaram o conceito de saúde como um bem comum entre as na- ções, que redefine o papel do Estado em um período de globalização, comercialização e individualização. ALMEIDA, C. et al. A concepção brasileira de “cooperação Sul-Sul estruturante em saúde”. Reciis, Rio de Janeiro, v. 4, n. 1, p. 25-35, 2010. Diplomacia da saúde e o fortalecimento da saúde global Os principais objetivos dessas organizações são fortalecer os sistemas e serviços de saúde e suas instituições estruturantes e avançar no desenvolvimento de recursos humanos para a saúde. A DSG, mediante parcerias entre Estado e organizações não governamentais, é um potente instrumento para garantir o sucesso das interven- ções na saúde global e gerar resultados de saúde sustentáveis e redes de vigilância em saúde global entre as nações. O sucesso das intervenções depende da adaptação a normas culturais, religiosas, sociais locais e de segurança da equipe. 236 Saúde Soc. 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A formação e a capacitação de recursos humanos para atuar na DSG, o desenvolvimento de resulta- dos de saúde sustentáveis a médio e longo prazo, a adaptação cultural e a segurança das equipes que desempenham atividades em outros países, a pro- moção da saúde bucal global e a emancipação de países emergentes com base em intervenções feitas são grandes desafios no âmbito da DSG. Considerações finais 1722- 1723, 2011. FEINSILVER, J. M. Fifty years of Cuba’s medical diplomacy: from idealism to pragmatism. Cuban Studies, Pittsburgh, v. 41, p. 85-104, 2010. BUSS, P. M. Global health and health diplomacy. Cadernos de Saúde Pública, Rio de Janeiro, v. 29, n. 1, p. 1-2, 2013. FIDLER, D. P. The globalization of public health: the first 100 years of international health diplomacy. Bulletin of the World Health Organization, Geneva, v. 79, n. 9, p. 842-849, 2001. BUSS, P. M.; FERREIRA, J. R. 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Considerações finais BADEAU, J. S. Diplomacy and medicine. Bulletin of the New York Academy of Medicine, New York, v. 46, n. 5, p. 303-312, 1970. A produção científica sobre o tema é recente e abrange o campo teórico-conceitual. A DSG é uma ferramenta para promover a saúde global e nacional que, mediante parcerias entre governos e organi- zações não governamentais, influencia na política externa, na economia e no desenvolvimento das nações. A DSG melhora a equidade e gera cooperação internacional para resolver e priorizar problemas comuns de saúde, com o intuito de alcançar resul- tados de saúde sustentáveis. Em outra perspectiva, colabora para disseminar o conceito de que a saúde é um bem comum para as nações e redefine o papel do Estado nos cenários político, econômico e social mundiais. Para atingir esses objetivos, precisa promover a cooperação internacional em pesquisa, a fim de divulgar estratégias dos países para forta- BARDIN, L. Análise de conteúdo. 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https://openalex.org/W2592645432	https://www.nature.com/articles/srep43558.pdf	English	null	Serum snoRNAs as biomarkers for joint ageing and post traumatic osteoarthritis	Scientific reports	2,017	cc-by	9,101	Serum snoRNAs as biomarkers for joint ageing and post traumatic osteoarthritis received: 07 October 2016 accepted: 25 January 2017 Published: 02 March 2017 Mandy M. F. Steinbusch1, Yongxiang Fang2, Peter I. Milner3, Peter D. Clegg3, David A. Young4, Tim J. M. Welting1 & Mandy J. Peffers3 received: 07 October 2016 accepted: 25 January 2017 Published: 02 March 2017 The development of effective treatments for the age-related disease osteoarthritis and the ability to predict disease progression has been hampered by the lack of biomarkers able to demonstrate the course of the disease. Profiling the expression patterns of small nucleolar RNAs (snoRNAs) in joint ageing and OA may provide diagnostic biomarkers and therapeutic targets. This study determined expression patterns of snoRNAs in joint ageing and OA and examined them as potential biomarkers. Using SnoRNASeq and real-time quantitative PCR (qRT-PCR) we demonstrate snoRNA expression levels in murine ageing and OA joints and serum for the first time. SnoRNASeq identified differential expression (DE) of 6 snoRNAs in young versus old joints and 5 snoRNAs in old sham versus old experimental osteoarthritic joints. In serum we found differential presence of 27 snoRNAs in young versus old serum and 18 snoRNAs in old sham versus old experimental osteoarthritic serum. Confirmatory qRT-PCR analysis demonstrated good correlation with SnoRNASeq findings. Profiling the expression patterns of snoRNAs is the initial step in determining their functional significance in ageing and osteoarthritis, and provides potential diagnostic biomarkers and therapeutic targets. Our results establish snoRNAs as novel markers of musculoskeletal ageing and osteoarthritis. Osteoarthritis (OA) is an age-related musculoskeletal disease and a common cause of chronic disability world- wide1. In addition it is a significant contributor to both individual and socioeconomic burden and the number of disability adapted life years globally2. If the deterioration in musculoskeletal health and development of OA can be identified and treated early serious life impairment may be abrogated. Ageing is the time-dependent reduction of functional capacity and stress resistance, associated with an increased risk of morbidity and mortality. The joint and its articular cartilage is particularly affected by ageing3. There is evidence that the rate of ageing, that is the ‘biological age’, differs significantly between individuals’ actual age in years (i.e. the ‘chronological age’). Defining markers of joint ageing may enable a prediction of the risk of onset of OA, enabling early intervention. OA is characterised by a non-symptomatic, pre-radiographical phase that if identified would allow earlier diag- nosis. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports SCIeNtIFIC RePOrTS \| 7:43558 \| DOI: 10.1038/srep43558 Materials and Methodsh All reagents were from Thermo-Fisher-Scientific, unless stated. All reagents were from Thermo-Fisher-Scientific, unless stated. Animals. C57BL6/J male mice were used for the study. For SnoRNASeq old mice were 18 months old (n = 6), young 8 months old (n = 6)16 and mice used for destabilisation of the medial meniscus (DMM) 24 months old (sham n = 3; DMM n = 6). Mice were group housed in individually ventilated cages at a 12 hour light/dark cycle, with ad libitum access to food and water. Experimental animal protocols were performed in accordance with the guidelines of the Animals (Scientific Procedures) Act 1986 following ethical review. Animal usage and protocols for this study was approved by the University of Liverpool Animal Welfare Committee. Surgical induction of OA by DMM in mice. DMM surgery was perform as previously reported17. Briefly, under anaesthesia a 3 mm skin incision was made over the medial aspect of the patellar ligament through the joint capsule into the femorotibial joint of the left knee. The medial meniscotibial ligament was transected to destabilise the cranial pole of the medial meniscus from the anterior tibial plateau. In sham operated mice the medial menis- cotibial ligament was visualised but not transected. Mice were sacrificed 8 weeks post-surgery. Joint and serum collection for SnoRNASeq. Following euthanasia, knee joints were collected from young, old, DMM (n = 6 each group) and sham n = 3 for SnoRNASeq. Joints were harvested free of soft tissues at 7 mm from the joint into RNALater. Serum was collected using cardiac puncture. One old serum sample was not processed further due to extensive haemolysis. OARSI scoring of histological sections of mouse knee joints. For histology, as the total knee joint was used in the study for RNA extraction, joints were collected (into 4% paraformaldehyde) from additional equivalent aged and treated young (n = 8), old (n = 4); sham (n = 5); and DMM (n = 6) mice in order to eval- uate the extent of OA. The procedure, surgeon and duration of the studies were identical. Knees were decalci- fied in 0.5 M ethylenediaminetetraacetic acid (pH 7.4) for 4 weeks at 4 °C and coronally embedded in paraffin. Sectioning, Safranin-O Fast-Green staining and histological scoring (defined as the severity and extent of OA) was undertaken on a scale from 1 to 6 by two blinded independent observers using the OARSI histopathology initiative18. www.nature.com/scientificreports/ mRNA, whilst others exhibit miR-like activity5. Aberrant expression of snoRNAs has been associated with disease development5 such as lung tumorigenesis6. p g g Emerging evidence shows that there is an increased level of circulating RNAs in the serum of cancer patients7. Circulating microRNAs (miRs) have been extensively described as biomarkers for diseases like pancreatic/breast cancer8,9, Alzheimer’s disease10 and inflammatory diseases like asthma, inflammatory bowel disease and rheu- matoid arthiritis11, but with the recent discovery of stable12 snoRNAs in serum, interest in their potential as circulating biomarkers of cancers (reviewed5) has been stimulated. We have previously identified dysregulation of a defined set of snoRNAs in cartilage13 and tendon14 ageing and OA15 and in man, snoRNA SNORD38 and SNORD48 were identified as potential non-age-dependant serum biomarkers for OA progression following cru- ciate ligament injury12.ii Expression profiling of snoRNAs in ageing and OA may help in determining their functional significance in the development and progression of disease and provide much needed diagnostic biomarkers for ageing and OA development. This study compared serum and joint snoRNA expression in ageing and OA from knee joint tissues from young and old adult mice and old mice using a traumatic in vivo model of OA. Because OA involves the whole joint as an organ; we undertook our analysis on whole mouse joints, which included cartilage, meniscus, subchondral bone, and joint capsule with synovium. Serum snoRNAs as biomarkers for joint ageing and post traumatic osteoarthritis However radiographic changes are only evident later in disease progression. Magnetic resonance imaging techniques have been developed for early-stage evaluation of cartilage damage in OA but are expensive and con- traindicated in some individuals.hf The development of effective treatments for OA and the ability to predict disease progression has been ham- pered by the lack of substantive biomarkers, able to demonstrate pathological disturbances preceding identifiable tissue alterations. Others have attempted to identify products of tissue turnover in serum and synovial fluid (reviewed4). This has been challenging due to patient and disease heterogeneity and dilution effects either by tissue fluids or with similar products from other joints or diseases. In addition, the variability of antibody assays has been problematic. p SnoRNAs are a class of evolutionary conserved non-coding small guide RNAs of which the majority direct the chemical modification of other RNA substrates, including ribosomal RNAs and spliceosomal RNAs. In addition, some snoRNAs are involved in the regulation of alternative splicing and post-transcriptional modification of 1Department of Orthopedic Surgery, Caphri School for Public Health and Primary Care, Maastricht University Medical Center, Maastricht, the Netherlands. 2Centre for Genomic Research, Institute of Integrative Biology, Biosciences Building, Crown Street, University of Liverpool, Liverpool L69 7ZB, UK. 3Institute of Ageing and Chronic Disease, University of Liverpool, Apex Building, 6 West Derby Street, Liverpool, L7 9TX, UK. 4Musculoskeletal Research Group, Newcastle University, Newcastle upon Tyne NE2 4HH, UK. Correspondence and requests for materials should be addressed to M.M.F.S. (email: m.steinbusch@maastrichtuniversity.nl) SCIeNtIFIC RePOrTS \| 7:43558 \| DOI: 10.1038/srep43558 1 www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ Name Sequence (5′-3′) Tm (°C) RTQ primer poly(A) cDNA synthesis CGAATTCTAGAGCTCGAGGCAGGC GACATGGCTGGCTAGTTAAGCTTG GTACCGAGCTCGGATCCACTAGTCC TTTTTTTTTTTTTTTTTTTTTTTTTVN 75 Snora28 CATGAGACAAGCCGTTATATAGGC 50 Snora30 TGTACCAGTGGCAGCTGTTACTC 50 Snora31 CTTTGTGGCAGTTCAGATTGAATTAG 50 Snora64 GTGGCCTCTCTTGCCTAGAG 65 Snora73 ACAGTGACTGAGGAGGCAAAC 50 Snord46 AATGCAAGGACTTGTCATAGTTACAC 50 Snord85 TTAGACCAGAGGTCGATGATGAG 50 Snord88 ACCTTTGACACCTGGAGATCTGA 50 Snord116 TGTACCGCCACTCTCATCGG 65 U2 TGGTATTGCAGTACCTCCAGGAACG 55 U3 AGTGAGAGGGAGAGAACGCGGTC 55 U6 GATGACACGCAAATTCGTGAAGCGTTC 55 RTQ-UNIr-50 AATTCTAGAGCTCGAGGCAGG 50 RTQ-UNIr-55 CGAATTCTAGAGCTCGAGGCAGG 55 RTQ-UNIr-65 CTAGAGCTCGAGGCAGGCGACATGGCTGGC 65 Table 1. Oligonucleotides sequences used in qRT-PCR. DE analysis was performed in R environment using package edgeR22. The processes and technical details of the analysis include: assessing data variation and detecting outlier samples through comparing variations of within and between sample groups using principle component analysis (PCA; 3-D PCA plots were generated using R function in package plot3D) and correlation analysis; handling library size variation respectively for joint samples and serum samples through data normalisation; formulating data variation using negative binomial distributions; modelling data using a generalised linear model; computing log2 Fold Change (logFC) values for required contrasts based on model fitting results through contrast fitting approach, assigning P-values to logFC values by LR23 testing; dealing with the effects of multiple tests using FDR approach to obtain FDR adjusted P-values; and defining significantly DE snoRNAs as those with FDR-adjusted p-value < 5%. Sequence data have been submitted to National Centre for Biotechnology Information Gene Expression Omnibus (NCBI GEO); E-MTAB-4878. RNA isolation, poly(A) cDNA synthesis and snoRNA qRT-PCR. qRT-PCR of snoRNAs was performed24. Total RNA was isolated using the mirVana kit. Isolated RNA samples were polyadenylated at 37 °C for 60 minutes in a 50 μL reaction volume containing 1 μg RNA and 1.5 U poly(A) polymerase (NEB, Ipswich, USA). 500 μL lysis binding buffer was added. Then, an equal volume of acid-phenol:chloroform was added, vor- texed and samples were centrifuged for 10 minutes and the aqueous phase removed. The poly(A)-tailed total RNA was extracted using the filter cartridge provided by the mirVana kit. To generate poly(A) cDNA, 500 ng poly(A)-tailed RNA and 250 ng RTQ primer (Eurogentec, Seraing, Belgium) (Table 1) were mixed in a 26 μL reac- tion volume, incubated at 65 °C for 10 minutes and annealed at 4 °C for 20 minutes. Materials and Methodsh All four quadrants of the section (medial tibial plateau, lateral tibial plateau, medial femoral condyle, lateral femoral condyle) were scored individually and added for each histological section. For statistical analyses mean summed score values of joints of 3–5 section per knee joint at 4 depths throughout the joint was determined (thus a maximum score of 24 was possible). Inter-observer variability was calculated using Cohen’s Kappa statis- tics using an online software tool: (http://www.statstodo.com/CohenKappa). RNA isolation, RNA-Seq analysis, cDNA library preparation and sequencing. Total RNA was isolated from equal weights of joints and 500 μl serum using miRNeasy or RNeasy Serum kits respectively with DNase treatment (all Qiagen, Crawley, UK) to remove residual gDNA. Total RNA integrity (RIN) was confirmed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, USA). Ribosomal RNA was depleted using the Ribo-Zero™ rRNA Removal Kit (Epicentre, Madison, USA). From 41 samples 100 ng of rRNA-depleted RNA was submitted for library preparation using NEB small RNA library kit (New England Biolabs (NEB), Ipswich, USA). To reduce workflow bias we used tobacco acid pyrophosphatase (Epicentre, Madison, USA) to remove potential 5′ caps found on some snoRNAs. Samples were amplified for 15 cycles, mixed into 3 pools, and size selected. The size-selected material was purified with Ampure beads (Agencourt, Beckman-Coulter, High-Wycombe, UK). SnoRNA sequencing was undertaken on the Illumina HiSeq 2000 platform (Illumina, San Diego, USA) using 100 base paired-end reads. SnoRNASeq data analysis. Sequence data measured from 5 lanes of an Illumina HiSeq2000 were pro- cessed through a number of steps to obtain snoRNA expression values. The processes include basecalling and de-multiplexing of indexed reads using CASAVA version 1.8.219; adapter and quality trimming using Cutadapt version 1.2.120 and Sickle version 1.200 to obtain fastq files of trimmed reads; aligning reads to Ensembl GRCm38.77 mouse genome reference sequences which contains 1,555 annotated snoRNA features using Bowtie221 version 2.0.10 with option “–very-sensitive-local”; counting aligned reads against snoRNA features using THSeq-count. The count values were used as snoRNA expression measurements for the DE analysis. SCIeNtIFIC RePOrTS \| 7:43558 \| DOI: 10.1038/srep43558 2 Results OARSI scoring of joints. OARSI scoring of joints (mean ± 95%CI) for young and old were 0.5 ± 0.3 and 2.8 ± 2.7 (p = 0.01), and old sham and old DMM were 1.25 ± 1.1 and 6.5 ± 0.7 (p < 0.001), respectively. Mice exhibited typical histological features of OA in the DMM knees. Cohen’s Kappa statistic was 0.4 indicating a fair agreement. Representative histological images and OARSI scoring are in Fig. 1. Preliminary analysis SnoRNASeq. To identify DE of snoRNAs in mouse joints and serum in response to age and OA 41 cDNA libraries representing old and young joints (old = HJO; young = HJY) and serum (old = HSO; young = HSY), and old sham and old DMM joints and serum (sham joint = DJC, DMM joint = DJM, sham serum = DSC and DMM serum = DSM) were constructed and subjected to Illumina deep sequencing. Summaries of raw, trimmed reads and sequencing alignment to mouse snoRNAs are in Supplementary files 1 and 2 respectively. Reads mapping percentages for joint libraries were between 12~29%, and for serum libraries 0.03~0.9%. Between 42~53% of the 1555 mouse snoRNA reference sequences were aligned for joints and 16~26% for serum. Identification of DE snoRNAs using SnoRNASeq. The 3-D PCA plot (Fig. 2A) shows that the joint samples and serum samples are clearly separated by the 1st component, which explains 93.02% of the data var- iation. For serum samples, the group sham and the group DMM scatter separately on the 2nd component, fur- thermore, based on the 3rd component a clear separation between serum samples of young-healthy and samples of old-healthy exists. For joint samples, there is also a separation between DMM and sham samples, though the separation is not as clear as shown for serum samples. In addition, 5 DMM joint samples scatter far away from other joint samples on the 3rd component. Therefore, it can be expected that disease response small RNA can be detected from this data set. The heat map of hierarchical clusters of correlations among samples (Fig. 2B) depicts that the joint and serum groups of samples are very different in snoRNA expression. In addition, joint samples correlated to each other much more closely than serum samples confirming the phenomena revealed by the PCA analysis shown in Fig. 2A. This indicates that major disease responses in the data were contributed from serum samples. www.nature.com/scientificreports/ Reverse transcription was per- formed with 200 U M-MLV reverse transcriptase, 20 U RNAsin (both Promega, Southampton, UK), 2 μL dNTP mix (10 mM each; Eurogentec, Seraing, Belgium) and 8 μL 5x M-MLV buffer (Promega, Sothampton, USA) in a total reaction volume of 40 μL at 50 °C for 60 minutes. The reverse transcriptase was inactivated at 70 °C for 15 minutes. Finally, 1.5 U of RNAse H (NEB, Ipswich, USA) was added to remove small RNAs. A snoRNA-specific forward primer and a universal reverse primer (RTQ-UNIr, matched to the Tm of each individual snoRNA) were used for the amplification of each snoRNA target (all Eurogentec, Seraing, Belgium) (Table 1). For each cDNA sample a mix was prepared with Mesagreen qPCR Mastermix Plus for SYBR Green (Eurogentec, Seraing, Belgium) and 300 nM forward and reverse oligonucleotides. An ABI-7300 Detection System was used for ampli- fication using the following protocol: denaturation at 95 °C for 5 minutes, followed by 50 cycles of DNA ampli- fication (15 seconds 95 °C and 45 seconds annealing at 62–68 °C). The annealing temperature was optimized for each snoRNAs target. Serially diluted standard curves were utilized to quantify snoRNA expression and data was normalized to a validated housekeeping snoRNA (joint: U2, young-old serum: SNORD85, old sham-old DMM serum and equine serum: U6). Validation of SNORD116 as a marker of OA in equine serum. We determined the reproducibility of the expression of SNORD116 in OA serum of another species. There are well-published studies on the application of metacarpophalangeal (MCP) joint OA changes in the horse, a joint with similarities to the human knee joint25. We studied equine serum from normal and MCP OA horses collected from eight normal (mean age ± standard deviation 5.3 ± 2.1 years) and four OA (7.5 ± 1.0 years) castrated male thoroughbred horses at post-mortem. Samples were collected under the regulations of the Hong Kong Jockey Club with owner consent and stored at −80 °C. OA diagnosis was based on histological (modified Mankin)26 and synovitis scoring27 of MCP joint tissues. SCIeNtIFIC RePOrTS \| 7:43558 \| DOI: 10.1038/srep43558 3 www.nature.com/scientificreports/ Figure 1. Histological changes of in the mouse knee showing the medial femoral condyle (above) and medial tibial plateau (below). (A) Safranin O with Fast-Green counterstain. Scale bar, 100 μm. Red indicates proteoglycan. (B/C) Assessment of osteoarthritis development was evaluated by OARSI scores; young (n = 8) vs. www.nature.com/scientificreports/ old (n = 4) (B); sham (n = 5) vs. DMM (n = 6) (C). Data represents the mean + 95% confidence interval (CI) for each scorer. For statistical evaluation an independent samples t-test was performed using GraphPad Prism 5 (San Diego); p-values are indicated. Figure 1. Histological changes of in the mouse knee showing the medial femoral condyle (above) and medial tibial plateau (below). (A) Safranin O with Fast-Green counterstain. Scale bar, 100 μm. Red indicates proteoglycan. (B/C) Assessment of osteoarthritis development was evaluated by OARSI scores; young (n = 8) vs. old (n = 4) (B); sham (n = 5) vs. DMM (n = 6) (C). Data represents the mean + 95% confidence interval (CI) for each scorer. For statistical evaluation an independent samples t-test was performed using GraphPad Prism 5 (San Diego); p-values are indicated. Statistical analysis. For statistical evaluation of histological scoring non-parametric Mann-Whitney-U test was used. Inter-observer agreement of histological scoring systems was calculated using Cohen’s kappa coefficient (www.statstodo.com/CohenKappa_Pgm.phpl). qRT-PCR data was log-transformed prior to statistical evaluation with an independent samples t-test. Statistical evaluation was performed between young and old or old sham versus old DMM using GraphPad Prism 5 (San Diego); p-values are indicated. Statistical analysis. For statistical evaluation of histological scoring non-parametric Mann-Whitney-U test was used. Inter-observer agreement of histological scoring systems was calculated using Cohen’s kappa coefficient (www.statstodo.com/CohenKappa_Pgm.phpl). qRT-PCR data was log-transformed prior to statistical evaluation with an independent samples t-test. Statistical evaluation was performed between young and old or old sham versus old DMM using GraphPad Prism 5 (San Diego); p-values are indicated. Results A read length distribution graph was generated to highlight the constitutional difference of non-coding RNAs in joint and serum samples at 100 bp or below (Fig. 2C). For this one serum and one joint sample with approximately the same library size were chosen. The plots of the read length distribution for joint and serum SCIeNtIFIC RePOrTS \| 7:43558 \| DOI: 10.1038/srep43558 4 www.nature.com/scientificreports/ ntificreports/ Figure 2. Variation data between the expressions for 41 samples. (A) A 3-D PCA plot of the first three components from principal component analysis of logarithm-transformed small RNA abundance data. Variance (%) associated with each principle component is depicted on the respective axis. Abbreviations; Joint: young healthy (HJY; black), old healthy (HJO; red), old sham (DJC; magenta), old DMM (DJM; light blue). Serum: young healthy (HSY; green), old healthy (HSO; dark blue), old sham (DSC; grey), old DMM (DSM; yellow). (B) The heat map of hierarchical clusters of correlations among samples. Pearson’s correlation coefficients were computed using logarithm transformed small RNA expression data from all known snoRNAs that were detected. (C) The plots of read length distribution for two representative joint and serum samples. Figure 2. Variation data between the expressions for 41 samples. (A) A 3-D PCA plot of the first three components from principal component analysis of logarithm-transformed small RNA abundance data. Variance (%) associated with each principle component is depicted on the respective axis. Abbreviations; Joint: young healthy (HJY; black), old healthy (HJO; red), old sham (DJC; magenta), old DMM (DJM; light blue). Serum: young healthy (HSY; green), old healthy (HSO; dark blue), old sham (DSC; grey), old DMM (DSM; yellow). (B) The heat map of hierarchical clusters of correlations among samples. Pearson’s correlation coefficients were computed using logarithm transformed small RNA expression data from all known snoRNAs that were detected. (C) The plots of read length distribution for two representative joint and serum samples. samples reveal that a peak value of frequency for read length in joint samples is 22 bp (vertical, dotted black line). Therefore the corresponding reads are generally well annotated miRNAs. Another peak is noted at around 100 bp, which is consistent with non-coding RNAs whose length is a hundred bp and over. In contrast, a peak is presented at 30 bp long for serum samples (vertical, dotted red line). More than 50% of the reads are 30 bp long in the serum library (data not shown). www.nature.com/scientificreports/ Contrast Gene ID Name Log2 FC FDR Host Gene Young vs. Old joint ENSMUSG00000098372 SNORD113 −0.92 0.05 Predicted gene ENSMUSG00000065016 SNORA3 −0.80 0.01 Ribosomal protein L27A ENSMUSG00000080352 SNORD88 −0.55 0.00 RIKEN cDNA 2410002F23 gene ENSMUSG00000064380 SNORA73 0.56 0.03 Ribosomal protein SA ENSMUSG00000087883 SNORA17 1.21 0.05 Contactin associated protein-like 5B ENSMUSG00000064536 SNORD38 1.49 0.05 PDZ domain containing 4 Young vs. Old serum ENSMUSG00000089255 SNORA78 −2.29 0.01 Ribosomal protein S2 ENSMUSG00000080396 SNORD111 −1.38 0.01 Splicing factor 3b, subunit 3 ENSMUSG00000065883 U3 −2.53 0.00 Predicted gene ENSMUSG00000064500 SNORD95 1.16 0.01 Guanine nucleotide binding protein, beta polypeptide 2 like 1 ENSMUSG00000095118 SNORD14D 1.23 0.00 Heat shock protein 8 ENSMUSG00000065219 SNORD32A 1.55 0.04 Ribosomal protein L13A ENSMUSG00000088678 SNORA17 1.90 0.03 Predicted gene ENSMUSG00000064938 miR3068/SNORA58 1.96 0.00 AlkB, alkylation repair homolog 1 ENSMUSG00000065282 SNORA18 1.99 0.00 Predicted gene ENSMUSG00000064387 SNORA73A 2.02 0.01 Regulator of chromosome condensation 1 ENSMUSG00000064602 SNORA41 2.12 0.00 Eukaryotic translation elongation factor 1 beta 2 ENSMUSG00000088670 SNORA31 2.14 0.03 Regulatory associated protein of MTOR, complex 1 ENSMUSG00000064918 SNORD18 2.18 0.02 Ribosomal protein L4 ENSMUSG00000065158 SNORA73 2.26 0.02 ERO1-like beta ENSMUSG00000064493 SNORA28 2.39 0.03 Eukaryotic translation initiation factor 5 ENSMUSG00000077167 SNORA53 2.43 0.00 Solute carrier family 25 ENSMUSG00000064495 SNORA5 2.49 0.00 Transforming growth factor beta regulated gene 4 ENSMUSG00000064569 SNORD71 2.56 0.00 Adaptor-related protein complex 1, gamma 1 subunit ENSMUSG00000064791 SNORD14E 2.63 0.00 Heat shock protein 8 ENSMUSG00000077714 SNORD17 2.76 0.00 Sorting nexin 5 ENSMUSG00000065353 SNORD73B 2.79 0.00 Regulator of chromosome condensation 1 and an lnc ENSMUSG00000064696 SNORD20 3.09 0.00 Nucleolin ENSMUSG00000065778 SNORA55 3.10 0.02 Poly(A) binding protein, cytoplasmic 4 ENSMUSG00000080478 SNORD23 4.42 0.00 Glioma tumor suppressor candidate region gene 2 ENSMUSG00000095176 SNORA44 5.65 0.00 Hypothetical protein PNAS-123 ENSMUSG00000088040 SNORA17 12.14 0.00 Hypothetical protein MGC16037, homology human collagen alpha 2 ENSMUSG00000065852 SNORA2 13.40 0.00 Hypothetical protein, 152aa Sham vs. DMM joint ENSMUSG00000064655 SNORA55 −0.88 0.04 Poly(A) binding protein, cytoplasmic 4 ENSMUSG00000098971 SNORD113 −0.99 0.04 Lnc-NA imprinted and accumulated in nucleus ENSMUSG00000064858 SNORA43 −1.12 0.04 Lnc- small nucleolar RNA host gene 7 ENSMUSG00000065105 SNORA29 −1.22 0.04 T-complex protein 1 ENSMUSG00000077191 SNORA64 −1.62 0.04 Small nuclear ribonucleoprotein N Sham vs. Explanation: positive Log2 FC = increased in old or DMM. Negative Log2 FC = is decreased in old or DMM. Table 2. Differentially expressed snoRNAs between contrasts including host gene identification. ab e .f e e t a y e p essed s oRN s betwee co t asts c ud g ost ge e de ti cat o . Explanation: positive Log2 FC = increased in old or DMM. Negative Log2 FC = is decreased in old or DMM. Results In contrast, the joint library has just 1.14% of reads that are 30 bp long (data not shown). Such reads may come from piwi-interacting RNAs (piRNAs). There were 498–646 snoRNAs expressed in serum samples and 1068–1286 in joint samples. The DE snoRNAs between contrasts are in Table 2. These included 6 snoRNAs in young versus old joints, 5 snoRNAs in old sham versus old DMM joints. In serum we identified DE of 27 snoRNAs in young versus old serum and 18 snoRNAs in old sham versus old DMM serum. Validation of SnoRNASeq data using qRT-PCR. Very little is known about the roles of snoRNAs as biomarker for disease or about the functional roles of snoRNAs in mammalian cell biology. We thus could not define functional criteria to select snoRNAs for validation based on prior knowledge. To stay unbiased we thus selected snoRNAs from the RNAseq data that displayed a moderate fold significant expression difference, higher fold significant expression difference, non-significant expression difference and selected box H/ACA (SNORAs) as well as box C/D (SNORDs) snoRNAs for validation. Levels of candidate snoRNAs for further qRT-qPCR analysis were determined using the original RNA from all donors used to perform the SnoRNASeq experiment. There was good concordance between SnoRNASeq and qRT-PCR platforms (Table 2 and Fig. 3). SNORD88 SCIeNtIFIC RePOrTS \| 7:43558 \| DOI: 10.1038/srep43558 5 www.nature.com/scientificreports/ Table 2. Differentially expressed snoRNAs between contrasts including host gene identification. Explanation: positive Log2 FC = increased in old or DMM. Negative Log2 FC = is decreased in old or DMM. SCIeNtIFIC RePOrTS \| 7:43558 \| DOI: 10.1038/srep43558 Discussion SnoRNAs are emerging as important regulators of cell functions, such as alternative splicing28, metabolic stress29 and development of disease; in cancer6, Prader-Willi Syndrome and autism5. Through expression profiling of snoRNAs using deep-sequencing we reveal novel molecular features relating to joint ageing and OA. g p q g g j g g Whilst gene expression has been evaluated in animal models of OA, including the rat anterior cruciate transection30 and meniscal tear models31, and mouse DMM model32 these were primarily interrogating protein-coding genes. Furthermore, apart from the final study these experiments evaluated a single tissue, primarily articular cartilage. In musculoskeletal ageing single tissues have been investigated13,14. OA is a process that involves the whole joint as an organ; therefore we undertook our analysis on whole mouse joints, which included cartilage, meniscus, subchondral bone, and joint capsule with synovium. j y Mice are considered skeletally mature at around 3-months-old (approximate equivalent of a teenaged human) while a 12-month-old mouse would signify a 40 to 50 year human33. Thus, to investigate the effects of joint age we used 8-month-old equivalent to 25 to 28 year human (referred to as young) and 24-month-old mice (referred to as old). To study the development of post-traumatic OA we measured OA severity histologically and analysed snoRNAs expression in joints and serum from 24-month-old mice following DMM. Limitations of this study were that we did not additionally undertake the DMM model in young mice. Additionally the ‘old’ age in young versus old contrast was not the same age as ‘old’ age in sham versus DMM. OARSI scores that were observed in the sham group (DMM surgery cohort; 24 months old) were lower than the old group (young versus old cohort; 18 months old). At this moment we cannot explain this difference, but different housing and environments of the two cohorts may have influenced this. The DMM model is a post-injury model in which the histological lesions within the affected joint are similar to those observed in human OA17. Mild OA-like pathology was present in the old and sham mice implying that mice at this age are in the early stages of acquiring naturally occurring OA comparable to studies of human knees at the equivalent age of approximately 40 years-old34. However the effect of the DMM model exacerbated these changes. g Detection of snoRNAs in serum has previously been demonstrated12,35,36. Discussion SnoRNAs are serum stable, and although normally resident in the nucleolus it is thought in serum they are present as unidentified protein com- plexes12. It is however not clear whether disease-associated RNAs detected in the circulation result from local tissue disturbances and cell death, or whether they are actively locally secreted via exosomes or microvesicles or are a systemic response upon local tissue damage8,37,38. This may even depend on the specific pathology or spe- cific RNA species. Determining the average read-length distribution of representative joint and serum samples (Fig. 2C) revealed an unexpected finding. When looking at the read-length distribution the serum reads spe- cifically contain a large peak of RNAs with a length of approximately 30 nt. The combined realisation that quite a large portion of the reads could not be mapped back to the used genome database, that a large portion of the piRNA class of non-coding RNAs lacks annotation in the used genome database and that piRNAs are typically 30 nt long, makes it is reasonable to think that this explains the presence of the large 30 nt peak specifically in serum. Only recently the presence of piRNAs in human blood has been described39 and this significant difference in the constitution of small RNAs for joint and serum samples is a phenomenon calling for further investigation.i j p p g g In the young versus old serum (27 snoRNAs) more snoRNAs were significantly up-regulated compared to the old sham versus old DMM serum (18 snoRNAs) indicating ageing per se had a greater effect on differential snoRNA presence in serum than OA. This could be due to different ‘old’ ages in the comparisons, but also maybe expected as in ageing serum snoRNAs will be from many tissues whereas in the DMM model we are most likely highlighting primarily OA joint-related snoRNAs. As we were studying the whole joint this may be due to vary- ing expression of snoRNAs in joint tissues, as it is known that there is tissue-specific snoRNAs expression40. We are unable to determine the amount each tissue type contributed to overall expression of snoRNAs. Each tissue will vary in its cellularity and hence RNA and snoRNAs content. Thus whilst a novel aspect of this study was that snoRNAs were extracted from the multiple tissues that form the joint, our approach may be less sensitive in detecting snoRNAs that change in a single tissue. www.nature.com/scientificreports/ DMM serum ENSMUSG00000096017 SNORD116 10.56 0.01 Predicted gene ENSMUSG00000064938 miR3068 3.31 0.03 AlkB, alkylation repair homolog 1 ENSMUSG00000089014 SNORA36 3.05 0.01 Dyskerin ENSMUSG00000077222 SNORA66 2.78 0.05 Ribosomal protein L5 ENSMUSG00000064918 SNORD18 2.21 0.01 Ribosomal protein L4 ENSMUSG00000064400 U3 1.92 0.01 Predicted gene ENSMUSG00000089417 U90 1.90 0.01 Importin ENSMUSG00000077709 SNORA64 1.85 0.04 Small nuclear ribonucleoprotein N ENSMUSG00000065734 SNORD49A −1.03 0.05 RIKEN cDNA 2410006H16 gene ENSMUSG00000064450 SNORD68 −1.06 0.04 Ribosomal protein L13 ENSMUSG00000064844 SNORD58 −1.18 0.01 Ribosomal protein L17 ENSMUSG00000064453 SNORD21 −1.22 0.01 Ribosomal protein L5 ENSMUSG00000065281 SNORD27 −1.31 0.01 Small nucleolar RNA host gene 1 ENSMUSG00000064871 SNORD58B −1.40 0.05 Ribosomal protein L17 ENSMUSG00000087819 SNORA48 −1.41 0.04 Nucleosome assembly protein 1-like 1 ENSMUSG00000064751 SNORD46 −1.46 0.01 Ribosomal protein S8 ENSMUSG00000077756 SNORD90 −1.48 0.01 Ring finger and CCCH-type zinc finger domains 2 ENSMUSG00000089093 SNORD11 −1.86 0.03 NOP58 ribonucleoprotein Table 2. Differentially expressed snoRNAs between contrasts including host gene identification. Explanation: positive Log2 FC = increased in old or DMM. Negative Log2 FC = is decreased in old or DMM. Table 2. Differentially expressed snoRNAs between contrasts including host gene identification. Explanation: positive Log2 FC = increased in old or DMM. Negative Log2 FC = is decreased in old or DMM. SCIeNtIFIC RePOrTS \| 7:43558 \| DOI: 10.1038/srep43558 6 www.nature.com/scientificreports/ was significantly decreased in young versus old joint (Fig. 3A), while SNORA73 was validated to be increased in young versus old joint (Fig. 3A). In agreement with its absence in the DE group (Table 2), SNORA30 was not differentially expressed in young versus old joint (Fig. 3A) and SNORD88 was not differentially expressed in sham versus DMM joint (Fig. 3A). SNORA31, SNORA28, SNORD23 and SNORA73 were confirmed to be significantly increased in young versus old serum (Fig. 3B) and SNORD116, SNORA64 and U3 were significantly increased in sham versus DMM serum (Fig. 3C). SNORD46 was confirmed to be significantly decreased in DMM serum as compared to sham serum (Fig. 3C). SNORD116 in equine serum in OA. To confirm increased SNORD116 in OA serum levels in a differ- ent species using qRT-PCR, we measured SNORD116 in equine serum samples. Normal equine donors had a Mankin’s score 1.25 ± 0.9 (mean ± 95%CI) and OA donors 7.75 ± 7.6. Synovial membrane from normal donors had a synovitis score27 of 3.25 ± 2.3 and OA donors 3.25 ± 3.2. www.nature.com/scientificreports/ There was a significant increase in serum expres- sion of SNORD116 in OA compared to normal donors (p = 0.0010) (Fig. 4). SCIeNtIFIC RePOrTS \| 7:43558 \| DOI: 10.1038/srep43558 Discussion However it has the advantage of determining snoRNAs that could be more globally implicated in OA. Despite the potential limitations, we identified a number of potentially interesting snoRNAs for future studies. g SNORA73 was increased in old joint and serum (Table 2, Fig. 3) and represents a potential joint ‘biological ageing’ marker. A reliable measurement of the state of ageing and a prediction of the risk of the onset of morbid- ity for chronic age-related diseases such as OA would be beneficial. Such a strategy could serve as a measure of ‘biological’ age and predict an age-related biological response more accurately than chronological age. SNORA64 was increased and SNORD46 was decreased in DMM serum, but both were not DE in young versus old serum, SCIeNtIFIC RePOrTS \| 7:43558 \| DOI: 10.1038/srep43558 7 www.nature.com/scientificreports/ Figure 3. SnoRNA expression from SnoRNASeq was validated with qRT-PCR. (A) Gene expression patterns of SNORD88, SNORA73 and SNORA30 were confirmed in young and old joint. SNORD88 expression was verified in sham and DMM joint. (B) SNORA31, SNORA28, SNORD23 and SNORA73 were confirmed to be increased in old serum. (C) Gene expression patterns of SNORD116, SNORA64, U3 and SNORD46 were validated in sham and DMM serum. Gene expression is depicted as fold induction relative to control (i.e. young or sham). Data represents the mean + 95% CI. For statistical evaluation an independent samples t-test was performed using GraphPad Prism 5 (San Diego) on log-transformed data; p-values are indicated. Figure 3. SnoRNA expression from SnoRNASeq was validated with qRT-PCR. (A) Gene expression patterns of SNORD88, SNORA73 and SNORA30 were confirmed in young and old joint. SNORD88 expression was verified in sham and DMM joint. (B) SNORA31, SNORA28, SNORD23 and SNORA73 were confirmed to be increased in old serum. (C) Gene expression patterns of SNORD116, SNORA64, U3 and SNORD46 were validated in sham and DMM serum. Gene expression is depicted as fold induction relative to control (i.e. young or sham). Data represents the mean + 95% CI. For statistical evaluation an independent samples t-test was performed using GraphPad Prism 5 (San Diego) on log-transformed data; p-values are indicated. Figure 4. Increased gene expression of SNORD116 in equine OA serum as compared to serum from normal donors. Data represents the mean + 95% CI. For statistical evaluation an independent samples t-test was performed using GraphPad Prism 5 (San Diego) on log-transformed data; p = 0.0010. Figure 4. Discussion Increased gene expression of SNORD116 in equine OA serum as compared to serum from normal donors. Data represents the mean + 95% CI. For statistical evaluation an independent samples t-test was performed using GraphPad Prism 5 (San Diego) on log-transformed data; p = 0.0010. indicating these snoRNAs as possible OA markers. SNORD18 was increased in serum both in ageing and follow- ing DMM (Table 2) signifying that they are affected in ageing and also OA. It is difficult to speculate how much this snoRNA changes in age-related OA versus age from this study. Histology identified mild increase in OARSI score with age and the level of OA changes in the DMM model was mild. It would be beneficial to investigate this snoRNAs further in tissues and serum in more severe OA. indicating these snoRNAs as possible OA markers. SNORD18 was increased in serum both in ageing and follow- ing DMM (Table 2) signifying that they are affected in ageing and also OA. It is difficult to speculate how much this snoRNA changes in age-related OA versus age from this study. Histology identified mild increase in OARSI score with age and the level of OA changes in the DMM model was mild. It would be beneficial to investigate this snoRNAs further in tissues and serum in more severe OA. indicating these snoRNAs as possible OA markers. SNORD18 was increased in serum both in ageing and follow- ing DMM (Table 2) signifying that they are affected in ageing and also OA. It is difficult to speculate how much this snoRNA changes in age-related OA versus age from this study. Histology identified mild increase in OARSI score with age and the level of OA changes in the DMM model was mild. It would be beneficial to investigate this snoRNAs further in tissues and serum in more severe OA. An interesting finding was an increase in SNORD38 in ageing joint. Zhang et al.12 demonstrated a strong association between serum levels of SNORD38 and severe cartilage damage, in anterior cruciate ligament (ACL) injury, enabling distinction between ACL injury patients from normal donors. They were unable to determine An interesting finding was an increase in SNORD38 in ageing joint. Zhang et al.12 demonstrated a strong association between serum levels of SNORD38 and severe cartilage damage, in anterior cruciate ligament (ACL) injury, enabling distinction between ACL injury patients from normal donors. Discussion They were unable to determine An interesting finding was an increase in SNORD38 in ageing joint. Zhang et al.12 demonstrated a strong association between serum levels of SNORD38 and severe cartilage damage, in anterior cruciate ligament (ACL) injury, enabling distinction between ACL injury patients from normal donors. They were unable to determine SCIeNtIFIC RePOrTS \| 7:43558 \| DOI: 10.1038/srep43558 8 www.nature.com/scientificreports/ age effect of SNORD38 in serum from normal donors as it was undetectable. One year post-surgery there was no relationship between donor age and serum SNORD38. Our study demonstrated an increase in SNORD38 in mouse joints (but not serum) with age. The human study did not assess tissue snoRNAs and the serum samples were primarily from middle-aged donors. Our old mice group represents an equivalent older age than that in the human study, which could contribute to this disparity. y p y The most DE snoRNA in DMM serum was SNORD116. Additionally we demonstrated an increase in serum SNORD116 in horses with MCP OA. We have previously identified SNORD116 as increased in OA compared to normal human cartilage in an array study15. A loss of SNORD116 is a significant contribution to the aetiology of the neurodegenerative genetic condition Prader-Willi syndrome (PWS)41. This paternally imprinted disor- der results in developmental delay and genetic obesity due to hyperphagia42. Clinical signs include short stature and low bone mineral density43. In a recent mouse transgenic study the loss of PWS critical region (including SNORD116) resulted in reduced bone mineral density (BMD), delayed skeletal development and reduced bone size and osteoblastic suppression44. Humans with OA have an increased BMD in affected joints45. Thus our find- ings not only elucidate a potential marker of OA but a snoRNA with a potential role in the pathogenesis of OA.i g y p p p g Previously we have identified DE snoRNAs in ageing cartilage13 and tendon14 and together with results in this study we propose that in musculoskeletal tissues snoRNAs potentially modulate the ageing process as previously described46. While determining the transcriptomic signature of ageing equine cartilage13 we found the differen- tial expression of a number of snoRNAs associated with ageing. When comparing the differentially expressed snoRNAs between the equine and this mouse study it is important to realize that in our previous equine study we did not analyse serum or the whole joint, but specifically the articular cartilage. Conclusion Our results implicate specific changes in snoRNA abundance in joint ageing (SNORD88 and SNORD38 were respectively decreased and increased) and OA suggesting the potential use of snoRNAs such as SNORA73 and SNORD23 as a novel biomarker for joint ageing, SNORA64, SNORD46 and SNORD116 for OA, SNORD18 for ageing and OA. Discussion Overlapping differential snoRNA expression between studies was identified for SNORD113, SNORA53, SNORA48 and SNORA5. SNORA53 was decreased in old equine cartilage, but increased in old mouse serum. SNORA48 was decreased in old equine cartilage, and decreased in old DMM mouse serum. SNORA5 was increased in old equine cartilage and increased in old mouse serum. Possibly the best consistency was found for SNORD113, which was decreased in old mouse joint (and old DMM mouse joint) and equine cartilage. The apparent cross-species conservations of differentially expressed snoRNAs in ageing and OA strengthen our belief that snoRNAs could indeed be used as biomarkers. Further analysis of snoRNA expression profiles and detailed genetic studies will give new insights into novel molecular networks in musculoskeletal ageing and common mechanisms in ageing and age-related diseases such as OA. In mammalians the majority of snoRNAs are encoded within the introns of protein coding or non-coding genes; host-genes47. There is evidence that genes which host snoRNAs might contribute to the aetiology of cancer through regulation of cell homeostasis and cancer biology (reviewed6). Potentially both the host-gene and the snoRNAs encoded within them may be important in different situations. 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The authors wish to thank the Wellcome Trust for MJP’s Wellcome Trust Clinical Intermediate Fellowship (grant 107471/Z/15/Z) and the Medical Research Council (MRC) and Arthritis Research UK as part of the MRC-Arthritis Research UK Centre for Integrated research into Musculoskeletal Ageing (CIMA) (grant MR/K006312/1) (MJP.PC), NWO-DFG (grant #DN82-304; NL; TJMW) and the Dutch Arthritis Foundation (grant #LLP14; NL; TJMW) for funding; Chris Riggs, Hong Kong Jockey Club for equine serum sample provision. Funding sources are (MJP, PC) Wellcome Trust Clinical Intermediate Fellowship (grant 107471/Z/15/Z) and the Medical Research Council (MRC) and Arthritis Research UK as part of the MRC-Arthritis Research UK Centre for Integrated research into Musculoskeletal Ageing (CIMA) (MR/K006312/1) (MJP.PC), NWO-DFG (grant #DN82-304; NL; TJMW) and the Dutch Arthritis Foundation (grant #LLP14; NL; TJMW). SCIeNtIFIC RePOrTS \| 7:43558 \| DOI: 10.1038/srep43558 10 www.nature.com/scientificreports/ Author Contributions M.J.P., M.M.F.S.; conception and design, collection and assembly of data, data analysis and interpretation, manuscript writing, final approval of the manuscript. Y.F.; assembly of data, data analysis, manuscript writing, final approval of the manuscript. P.M.; provision of study material, assembly of data, manuscript writing and final approval of the manuscript. P.D.C., T.J.M.W., D.Y.; conception and design, manuscript writing, final approval of the manuscript. SCIeNtIFIC RePOrTS \| 7:43558 \| DOI: 10.1038/srep43558 © The Author(s) 2017 Additional Information upplementary information accompanies this paper at http://www.nature.com/srep Supplementary information accompanies this paper at http://www.nature.com/srep Competing Interests: The authors declare no competing financial interests. Competing Interests: The authors declare no competing financial interests. How to cite this article: Steinbusch, M. M. F. et al. Serum snoRNAs as biomarkers for joint ageing and post traumatic osteoarthritis. Sci. Rep. 7, 43558; doi: 10.1038/srep43558 (2017). How to cite this article: Steinbusch, M. M. F. et al. Serum snoRNAs as biomarkers for joint ageing and post traumatic osteoarthritis. Sci. Rep. 7, 43558; doi: 10.1038/srep43558 (2017). Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps an institutional affiliations. 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. 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https://openalex.org/W3132555342	https://www.frontiersin.org/articles/10.3389/fmed.2020.592901/pdf	English	null	Metformin Use Is Associated With a Lower Incidence of Hospitalization for Atrial Fibrillation in Patients With Type 2 Diabetes Mellitus	Frontiers in medicine	2,021	cc-by	9,220	Chin-Hsiao Tseng 1,2,3* Chin-Hsiao Tseng 1,2,3* 1 Department of Internal Medicine, National Taiwan University College of Medicine, Taipei, Taiwan, 2 Division of Endocrinology and Metabolism, Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan, 3 Division of Environmental Health and Occupational Medicine of the National Health Research Institutes, Zhunan, Taiwan Background: The effect of metformin on the risk of atrial ﬁbrillation (AF) requires conﬁrmation. This retrospective cohort study compared the incidence of hospitalization for AF in ever and never users of metformin. Background: The effect of metformin on the risk of atrial ﬁbrillation (AF) requires conﬁrmation. This retrospective cohort study compared the incidence of hospitalization for AF in ever and never users of metformin. Methods: Patients with newly diagnosed type 2 diabetes mellitus during 1999–2005 were enrolled from Taiwan’s National Health Insurance database. Analyses were conducted in both an unmatched cohort of 173,398 ever users and 21,666 never users and in a propensity score-matched cohort of 21,662 pairs of ever and never users. They were free from a diagnosis of AF before January 1, 2006 and were followed up until December 31, 2011. Hazard ratios were estimated by Cox regression incorporated with the inverse probability of treatment weighting using the propensity score. Edited by: Horng-Yih Ou, National Cheng Kung University Hospital, Taiwan Reviewed by: Kai Pi Cheng, National Cheng Kung University Hospital, Taiwan Mei-Yueh Lee, Kaohsiung Medical University Hospital, Taiwan Results: A total of 303 ever users and 86 never users in the unmatched cohort and 56 ever users and 86 never users in the matched cohort developed hospitalization for AF during follow-up. The respective incidence rates were 37.72 and 92.45 per 100,000 person-years in the unmatched cohort and were 56.98 and 92.46 per 100,000 person-years in the matched cohort. The hazard ratio for ever vs. never users was 0.405 (95% conﬁdence interval: 0.319–0.515) in the unmatched cohort and 0.617 (0.441–0.864) in the matched cohort. Hazard ratios for the tertiles of cumulative duration of metformin therapy vs. never users showed a dose-response effect. The ﬁndings were consistent in sensitivity analyses. Correspondence: Chin-Hsiao Tseng ccktsh@ms6.hinet.net Specialty section: This article was submitted to Family Medicine and Primary Care, a section of the journal Frontiers in Medicine Specialty section: This article was submitted to Family Medicine and Primary Care, a section of the journal Frontiers in Medicine Received: 08 August 2020 Accepted: 03 December 2020 Published: 22 February 2021 Specialty section: This article was submitted to Family Medicine and Primary Care, a section of the journal Frontiers in Medicine Conclusion: Metformin use is associated with a lower risk of hospitalization for AF in patients with type 2 diabetes mellitus. Received: 08 August 2020 Accepted: 03 December 2020 Published: 22 February 2021 Keywords: atrial ﬁbrillation, diabetes mellitus, metformin, Taiwan, National Health Insurance ORIGINAL RESEARCH published: 22 February 2021 doi: 10.3389/fmed.2020.592901 Citation: Tseng C-H (2021) Metformin Use Is Associated With a Lower Incidence of Hospitalization for Atrial Fibrillation in Patients With Type 2 Diabetes Mellitus. Front. Med. 7:592901. doi: 10.3389/fmed.2020.592901 Atrial ﬁbrillation (AF) is a common arrhythmia that can lead to increased hospitalization, stroke, or even life-threatening thromboembolic events (1, 2). Risk factors of AF may include aging, male sex, smoking, alcohol consumption, diabetes mellitus, hypertension, obesity, thyroid dysfunction, obstructive sleep apnea, high-level physical training, left ventricular dysfunction, valvular heart February 2021 \| Volume 7 \| Article 592901 Frontiers in Medicine \| www.frontiersin.org Metformin and Atrial Fibrillation Tseng disease, myocardial infarction, and heart failure (2–4). The estimated lifetime risk of AF is 22–26% and diabetes patients suﬀer from a 1.4–1.6-fold higher risk (2, 3). While compared to diabetes patients without AF, diabetes patients with AF have a 61% higher risk of total mortality, 77% higher risk of cardiovascular death, and 68% higher risk of heart failure (5). of follow-up. Finally, this study did not consider the requirement of a suﬃcient induction time and did not address the potential bias due to immortal time. Therefore, selection bias, confounding by indication, and immortal time bias could not be excluded in this study. In the study by Liou et al., a nested case-control design was used by including 11,528 diabetes patients without AF and 2,882 diabetes patients with AF (12). The investigators cross-sectionally estimated the odds ratios of AF for metformin users vs. metformin non-users (12). The cross-sectional design did not assure a temporal correctness of a cause (metformin use) that should happen before an eﬀect (AF incidence). It is interesting that although a matching algorithm was used, the enrolled cases (AF group) and controls (non-AF group) were not well-matched in most of the important confounders. For example, the AF group was characterized by signiﬁcantly higher prevalent rates of potential risk factors of AF including hypertension, congestive heart failure, chronic kidney disease, acute myocardial infarction, and ischemic stroke than the non-AF group. Furthermore, the enrolled AF group was characterized by signiﬁcantly higher prevalence rates of the use of insulin, sulfonylurea, glinide, alpha-glucosidase inhibitors, and dipeptidyl-peptidase 4 inhibitors (12). Because insulin and insulin-secreting drugs may induce hypoglycemia which would lead to AF, residual confounding from other antidiabetic drugs could not be excluded. Abbreviations: AF, atrial ﬁbrillation; HAF, hospitalization for atrial ﬁbrillation; ICD-9-CM, International Classiﬁcation of Diseases, Ninth Revision, Clinical Modiﬁcation; IPTW, inverse probability of treatment weighting; NHI, National Health Insurance; PS, propensity score. Citation: Therefore, this study could not clarify the temporal correctness of a cause-eﬀect relationship and might have suﬀered from selection bias, prevalent user bias, immortal time bias, and confounding by indication. Metformin, now a ﬁrst-line oral antidiabetic drug recommended for the treatment of type 2 diabetes mellitus, exerts an insulin sensitizing eﬀect (6), and may have anti- inﬂammatory, anti-aging, anti-cancer, and even anti-microbial eﬀects (7–10). Two previous pharmacoepidemiological studies conducted in Taiwan that used the nationwide reimbursement database of the National Health Insurance (NHI) showed that patients with type 2 diabetes mellitus treated with metformin might have a 20% lower risk of AF (11, 12). However, these earlier studies suﬀered from some methodological limitations. g There are several methodological limitations in the study by Chang et al. (11). First, the metformin users and non- users enrolled were highly imbalanced in the use of statin with a signiﬁcantly higher rate of statin use among metformin users. Because statin may reduce the incidence of AF by 40– 50% (13, 14), the lower risk of AF among incident users of metformin could be potentially ascribed to the eﬀect of statin which had been in use for a certain period before metformin was initiated. Second, the signiﬁcantly higher prevalence rates of important risk factors of AF among non-users of metformin, such as male patients, older age, congestive heart failure, chronic kidney disease, asthma, hyperthyroidism, myocardial infarction, ischemic stroke and peripheral arterial disease, might have exerted residual confounding eﬀects even though some of them were considered in the adjusted model. Third, imbalance in the use of other antidiabetic drugs between metformin users and non-users was highly probable and this might have led to a biased result. The investigators excluded, at the beginning of the study, users of other antidiabetic drugs, and compared the incident cases of AF between metformin users and non- users over time by using a time-dependent approach. In this sense metformin non-users represented a highly selective group of patients who had a diagnosis of diabetes mellitus but had not been treated with any other antidiabetic drugs at the beginning of the study. During subsequent years of follow-up, non-users of metformin might have been prescribed metformin or other antidiabetic drugs. Once metformin was prescribed in the subsequent years, the patients would be reclassiﬁed as metformin users. MATERIALS AND METHODS Non-random observational studies tend to be biased by confounding by indication because treatment allocation may be associated with baseline characteristics. Methodological approaches by using PS have been recommended to reduce such a potential risk. PS is the probability of treatment assignment estimated by a set of baseline characteristics and it is always calculated by logistic regression. PS can be used for matching in selecting an exposed group and a non-exposed group for study or can be used for stratiﬁcation or adjustment in analyses (17). Additionally, Cox proportional hazards model can be incorporated with the inverse probability of treatment weighting (IPTW) using the PS (17). Austin recommended the use of matching on PS for cohort selection and the IPTW method for analyses (17). These two approaches were applied in the present study. The NHI has been implemented in Taiwan since March 1995. It is a unique and universal health care system with a high coverage rate of >99% of the Taiwan’s population. The Bureau of the NHI has contracts with all hospitals and with 93% of all medical settings. The reimbursement database of the NHI kept computerized records of disease diagnoses, medication prescriptions, and clinical procedures and can be used for academic research if approved after ethics review. The present study was approved number 99274 by the Ethics Committee of the National Health Research Institutes. According to local regulations, the National Health Research Institutes de-identiﬁed the individuals in the database for the protection of privacy and the Ethics Committee approved the analyses of the database without the requirement of obtaining informed consent from the participants. The unmatched cohort and the PS-matched cohort were enrolled according to the procedures shown in Figure 1. At ﬁrst, 423,949 patients who were newly diagnosed of diabetes mellitus during 1999–2005 in the outpatient clinics and had been prescribed antidiabetic drugs for two or more times were identiﬁed. The following ineligible patients were then excluded: (1) metformin ever users who had been treated with other Detailed description of the database has been reported in a previously published paper (16). During the study period, the International Classiﬁcation of Diseases, Ninth Revision, Clinical Modiﬁcation (ICD-9-CM) was used for disease diagnoses; and diabetes mellitus was coded as 250.XX and AF as 427.31. Citation: It was expected that insulin or insulin- secreting drugs, which could cause hypoglycemia and thus AF, would have been prescribed to metformin non-users for the purpose of glycemic control in the subsequent years of follow- up. Therefore, the proportions of the use of other antidiabetic drugs that could cause hypoglycemia and AF would be much higher among metformin non-users during any subsequent year Some other common limitations found in these two earlier studies (11, 12) included a lack of investigating a dose-response eﬀect and an ignorance of the potential impact of the regularity of metformin use. Additionally, the diagnosis of AF was made mainly at outpatient settings in these two studies, which was prone to be misclassiﬁed than a diagnosis made at the discharge of a hospitalization that is always supported by laboratory tests performed during admission. A recent study conducted in the USA showed an increasing trend of emergency department visits and hospitalization for AF (HAF) from 2007 to 2014 in a nationwide level and called for a “need for widespread implementation of eﬀective strategies aimed at improving the management of patients with AF to reduce hospital admissions and the economic burden of AF” (15). Because metformin is considered the ﬁrst-line antidiabetic drug for patients with type 2 diabetes mellitus, whether the use of metformin can prevent the potentially fatal disease of AF in these high-risk diabetes patients is of immense clinical importance. The purpose of the present study was to clarify whether metformin could reduce the incidence of AF made at the discharge of a hospitalization by using the Taiwan’s NHI reimbursement database. The potential methodological limitations seen in the previous studies (11, 12) were especially considered. February 2021 \| Volume 7 \| Article 592901 Frontiers in Medicine \| www.frontiersin.org 2 Metformin and Atrial Fibrillation Tseng MATERIALS AND METHODS FIGURE 1 \| Flowchart showing the procedures in creating an unmatched cohort and a cohort of 1:1 propensity score-matched-pairs of metformin ever users and never users derived from the reimbursement database of the Taiwan’s National Health Insurance (PS, propensity score). FIGURE 1 \| Flowchart showing the procedures in creating an unmatched cohort and a cohort of 1:1 propensity score-matched-pairs of metformin ever users and never users derived from the reimbursement database of the Taiwan’s National Health Insurance (PS, propensity score). FIGURE 1 \| Flowchart showing the procedures in creating an unmatched cohort and a cohort of 1:1 propensity score-matched-pairs of metformin ever users and never users derived from the reimbursement database of the Taiwan’s National Health Insurance (PS, propensity score). February 2021 \| Volume 7 \| Article 592901 Frontiers in Medicine \| www.frontiersin.org 3 Tseng Metformin and Atrial Fibrillation antidiabetic drugs before metformin was initiated (n = 183,837); (2) type 1 diabetes mellitus (n = 2,062), (3) patients who died before the start of follow-up (n = 65); (4) missing data (n = 358), (5) diagnosis of AF before the start of follow-up or within 6 months of diabetes diagnosis (n = 812), (6) diagnosis of any cancer before the start of follow-up or within 6 months of diabetes diagnosis (n = 26,675), and (7) follow-up duration <180 days (n = 15,076). As a result, 173,398 ever users and 21,666 never users of metformin were identiﬁed as the unmatched cohort. PS was created from all characteristics listed in Table 1 plus the date of enrollment by logistic regression. A cohort of PS- matched pairs of 21,662 ever users and 21,662 never users (the matched cohort) was then created from the unmatched cohort by matching the PS using the Greedy 8 –>1 digit match algorithm proposed by Parsons (18). Standardized diﬀerence was calculated according to the methods proposed by Austin and Stuart for each covariate as a test of balance diagnostic (21). A value of >10% was used as a cutoﬀfor potential confounding from the variable. Cumulative duration of metformin therapy in months was calculated and its tertiles were used for dose-response analyses. Incidence density of HAF was calculated for never users, ever users, and users categorized according to the tertiles of cumulative duration of metformin therapy. MATERIALS AND METHODS The numerator of the incidence was the case number of newly diagnosed HAF as a primary diagnosis at the discharge of a hospitalization observed during follow-up. The denominator expressed in person-years was the follow-up time since January 1, 2006 until December 31, 2011, when a new diagnosis of HAF was made, or on the date of death or the last reimbursement record, whichever occurred ﬁrst. The start of follow up was set on January 1, 2006 and all comorbidities and covariates were determined as a status/diagnosis at any time before the start of follow-up. Potential confounders included: (1) demographic data: age, sex, occupation, and living region; (2) major comorbidities: hypertension (401-405), dyslipidemia (272.0-272.4), and obesity (278); (3) diabetes-related complications: nephropathy (580-589), eye diseases (250.5: diabetes with ophthalmic manifestations, 362.0: diabetic retinopathy, 369: blindness and low vision, 366.41: diabetic cataract, and 365.44: glaucoma associated with systemic syndromes), hemorrhagic stroke (430-432), non-hemorrhagic stroke (433-435), other types of stroke (436-438), ischemic heart disease (410-414), and peripheral arterial disease (250.7, 785.4, 443.81, and 440-448); (4) antidiabetic drugs: insulin, sulfonylurea, meglitinide, acarbose, rosiglitazone, and pioglitazone (rosiglitazone and pioglitazone are both thiazolidinediones but they may have diﬀerent cardiovascular eﬀects and thus were included separately); (5) commonly encountered comorbidities and potential risk factors of AF: chronic obstructive pulmonary disease (a surrogate for smoking, 490-496), tobacco abuse (305.1, 649.0 and 989.84), alcohol-related diagnoses (291, 303, 535.3, 571.0-571.3 and 980.0), cancer (140-208), heart failure (398.91, 402.11, 402.91, 404.11, 404.13, 404.91, 404.93, and 428), gout (274), hyperthyroidism (242), sleep apnea syndrome (327.2, 780.51, 780.53, and 780.57) and valvular heart disease (394-396, 424, and 746); and (6) commonly used medications in diabetes patients: angiotensin-converting enzyme inhibitors/angiotensin receptor blockers, calcium channel blockers, beta-blockers, statins, ﬁbrates, and aspirin. The accuracy of disease diagnoses in the NHI database has been studied previously. Agreements between claim data and medical records are moderate to substantial, with Kappa values range from 0.55 to 0.86 (19). Kaplan-Meier curves for HAF-free probability were plotted for never users and ever users of metformin and for never users and users categorized according to the tertiles of cumulative duration of metformin therapy in the unmatched cohort and the matched cohort, respectively. Logrank test was used to test the signiﬁcance in diﬀerent subgroups of metformin exposure. In main analyses, hazard ratios and their 95% conﬁdence intervals were estimated by the IPTW method (17) in the unmatched cohort and the matched cohort, respectively. Frontiers in Medicine \| www.frontiersin.org MATERIALS AND METHODS Models were created for ever users vs. never users and for users in each tertile of cumulative duration of metformin therapy in comparison to never users. For sensitivity analyses, the following models were created in the unmatched cohort by using the IPTW method. At ﬁrst, we excluded patients with irregular reﬁll of metformin, based on two consecutive prescriptions of metformin spanning a period of >4 months (Model I, the Bureau of NHI allows a maximum of 3 months at each time of drug prescription for chronic diseases and these patients with delayed reﬁll of metformin for more than 1 month after a previous 3- month prescription might represent those with poor adherence). We then excluded patients who happened to be treated with incretin-based therapies, i.e., dipeptidyl peptidase 4 inhibitors and glucagon-like peptide 1 receptor agonists, during follow- up (Model II, incretin-based therapies were not reimbursed by the Bureau of NHI in Taiwan until after 2009 and this analysis was aimed at excluding their potential impact). Because metformin may be contraindicated in patients with nephropathy or chronic kidney disease and an early study suggested that chronic kidney disease is associated with the incidence of AF in a dose-response pattern (22), additional analyses were conducted in patients with nephropathy (Model III), in patients without nephropathy (Model IV), after excluding patients with a diagnosis of chronic kidney disease (ICD-9-CM 585, Model V), after excluding patients in a renal dialysis status (ICD- 9-CM V45.1, Model VI) and after excluding patients with a diagnosis of chronic kidney disease and/or in a renal dialysis status (Model VII). The classiﬁcations of living region and occupation were detailed elsewhere (20). In brief, the living region was classiﬁed as Taipei, Northern, Central, Southern, and Kao- Ping/Eastern. Occupation was classiﬁed as class I (civil servants, teachers, employees of governmental or private businesses, professionals, and technicians), class II (people without a speciﬁc employer, self-employed people or seamen), class III (farmers or ﬁshermen), and class IV (low-income families supported by social welfare, or veterans). Analyses were conducted using SAS statistical software, version 9.4 (SAS Institute, Cary, NC). P < 0.05 was considered statistically signiﬁcant. February 2021 \| Volume 7 \| Article 592901 4 Metformin and Atrial Fibrillation Tseng TABLE 1 \| Characteristics in never users and ever users of metformin in the unmatched and matched cohorts. MATERIALS AND METHODS Unmatched cohort Matched cohort Variable Never users Ever users Never users Ever users (n = 21666) (n = 173398) Standardized difference (n = 21662) (n = 21662) Standardized difference n % n % n % n % Demographic data Age (years) 68.77 13.25 64.23 11.98 −41.47 68.76 13.25 68.70 12.09 0.18 Sex (men) 11,793 54.43 91,570 52.81 −3.47 11,790 54.43 11,731 54.15 −0.68 Occupation I 7,825 36.12 64,789 37.36 7,824 36.12 7,930 36.61 II 3,646 16.83 36,710 21.17 12.61 3,646 16.83 3,634 16.78 −0.29 III 5,216 24.07 39,817 22.96 −2.67 5,215 24.07 5,187 23.95 −0.10 IV 4,979 22.98 32,082 18.50 −12.97 4,977 22.98 4,911 22.67 −0.69 Living region Taipei 7,328 33.82 54,416 31.38 7,325 33.81 7,279 33.60 Northern 2,310 10.66 20,016 11.54 2.87 2,309 10.66 2,280 10.53 −0.48 Central 3,769 17.40 31,678 18.27 2.12 3,769 17.40 3,770 17.40 0.01 Southern 3,729 17.21 29,840 17.21 0.37 3,729 17.21 3,797 17.53 1.02 Kao–Ping and Eastern 4,530 20.91 37,448 21.60 2.59 4,530 20.91 4,536 20.94 0.22 Major comorbidities Hypertension 18,476 85.28 144,909 83.57 −5.99 18,473 85.28 18,528 85.53 0.98 Dyslipidemia 15,127 69.82 141,918 81.85 32.61 15,127 69.83 15,181 70.08 0.67 Obesity 518 2.39 7,364 4.25 11.02 518 2.39 485 2.24 −1.04 Diabetes–related complications Nephropathy 8,376 38.66 50,614 29.19 −24.85 8,373 38.65 8,280 38.22 −1.36 Eye diseases 3,741 17.27 55,119 31.79 35.93 3,741 17.27 3,646 16.83 −1.65 Hemorrhagic stroke 1,670 7.71 8,229 4.75 −15.04 1,670 7.71 1,604 7.40 −1.17 Non–hemorrhagic stroke 6,249 28.84 41,367 23.86 −14.07 6,248 28.84 6,286 29.02 0.47 Other types of stroke 6,512 30.06 40,938 23.61 −17.92 6,510 30.05 6,483 29.93 −0.08 Ischemic heart disease 11,689 53.95 84,499 48.73 −12.66 11,688 53.96 11,703 54.03 0.29 Peripheral arterial disease 5,678 26.21 47,465 27.37 2.04 5,676 26.20 5,648 26.07 −0.38 Antidiabetic drugs Insulin 1,891 8.73 3,953 2.28 −35.13 1,890 8.72 1,641 7.58 −6.31 Sulfonylurea 15,450 71.31 123,550 71.25 9.13 15,449 71.32 16,096 74.31 6.22 Meglitinide 1,958 9.04 6,930 4.00 −22.90 1,956 9.03 1,902 8.78 −1.17 Acarbose 2,443 11.28 9,223 5.32 −20.88 2,441 11.27 2,606 12.03 0.56 Rosiglitazone 618 2.85 8,158 4.70 10.82 618 2.85 615 2.84 −0.99 Pioglitazone 510 2.35 4,356 2.51 2.45 510 2.35 516 2.38 −0.67 Commonly encountered comorbidities and potential risk factors of atrial ﬁbrillation Chronic obstructive pulmonary disease 12,054 55.64 89,210 51.45 −11.07 12,052 55.64 11,977 55.29 −0.56 Tobacco abuse 507 2.34 6,439 3.71 8.85 507 2.34 490 2.26 −0.54 Alcohol–related diagnoses 1,406 6.49 11,443 6.60 −0.12 1,406 6.49 1,300 6.00 −2.27 Cancer 2,254 10.40 13,711 7.91 −9.65 2,253 10.40 2,207 10.19 −0.68 Heart failure 6,237 28.79 35,109 20.25 −24.33 6,235 28.78 6,033 27.85 −2.13 Gout 8,921 41.18 63,552 36.65 −10.69 8,919 41.17 8,908 41.12 0.08 Hyperthyroidism 1,030 4.75 8,637 4.98 1.28 1,030 4.75 1,055 4.87 0.58 Sleep apnea syndrome 413 1.91 3,378 1.95 0.42 413 1.91 401 1.85 −0.41 Valvular heart disease 3,233 14.92 18,333 10.57 −16.23 3,233 14.92 3,111 14.36 −1.60 Commonly used medications in diabetes patients Angiotensin-converting enzyme inhibitors/angiotensin receptor blockers 15,959 73.66 130,207 75.09 2.71 15,957 73.66 15,880 73.31 −0.76 Calcium channel blockers 14,776 68.20 109,003 62.86 −12.95 14,773 68.20 14,794 68.29 0.47 Beta–blockers 15,951 73.62 119,761 69.07 −11.19 15,948 73.62 15,983 73.78 0.59 Statin 11,326 52.28 112,600 64.94 29.08 11,326 52.29 11,274 52.05 −0.56 Fibrate 7,090 32.72 73,563 42.42 22.48 7,090 32.73 7,013 32.37 −0.69 Aspirin 13,582 62.69 111,115 64.08 1.90 13,581 62.70 13,619 62.87 0.54 *Age is expressed as mean and standard deviation. RESULTS the ﬁndings were similar to those observed in the unmatched cohort shown in Figure 2. Table 1 shows the characteristics in never users and ever users of metformin in the unmatched cohort and the matched cohort, respectively. Before matching, ever users and never users showed imbalanced distribution of many covariates with standardized diﬀerence >10%. However, after matching, the two groups were well-balanced in all covariates and none of the variables had a value of standardized diﬀerence >10%. Table 2 shows the incidence of HAF and the hazard ratios by metformin exposure in the unmatched cohort and the matched cohort in the main analyses, respectively. A signiﬁcantly lower risk of HAF in metformin ever users could be demonstrated by the overall hazard ratios and the tertile analyses supported a dose-response relationship. Table 3 shows the ﬁndings in the sensitivity analyses. All analyses supported a lower risk of HAF associated with metformin use. The beneﬁcial eﬀect of metformin was independent of the regularity of metformin reﬁll (Model I), the prescription of incretin-based therapies during follow-up (Model II), the diagnosis of diabetic nephropathy (Models III and IV), and the diagnosis of chronic kidney disease and/or in a renal dialysis status (Models V, VI, and VII). Figure 2 shows the Kaplan-Meier curves comparing HAF- free probability in never users and ever users of metformin (Figure 2A) and in never users and users in each of the tertiles of cumulative duration of metformin therapy (Figure 2B) in the unmatched cohort. The logrank test supported signiﬁcant diﬀerences among the various subgroups of metformin exposure. Figure 3 shows the respective curves in the matched cohort and FIGURE 2 \| Kaplan-Meier curves comparing hospitalization for atrial ﬁbrillation (HAF)-free probability in never users and ever users of metformin (A) and in never users and users in each tertile of cumulative duration of metformin therapy (B) in the unmatched cohort. The 95% conﬁdence intervals are shown in shaded areas. HR, hazard ratio; CI, conﬁdence interval. FIGURE 2 \| Kaplan-Meier curves comparing hospitalization for atrial ﬁbrillation (HAF)-free probability in never users and ever users of metformin (A) and in never users and users in each tertile of cumulative duration of metformin therapy (B) in the unmatched cohort. The 95% conﬁdence intervals are shown in shaded areas. HR, hazard ratio; CI, conﬁdence interval. MATERIALS AND METHODS Refer to “Materials and Methods” for the classiﬁcation of occupation TABLE 1 \| Characteristics in never users and ever users of metformin in the unmatched and matched cohorts. February 2021 \| Volume 7 \| Article 592901 5 Frontiers in Medicine \| www.frontiersin.org Metformin and Atrial Fibrillation Tseng Tseng RESULTS FIGURE 3 \| Kaplan-Meier curves comparing hospitalization for atrial ﬁbrillation (HAF)-free probability in never users and ever users of metformin (A) and in never users and users in each tertile of cumulative duration of metformin therapy (B) in the matched cohort. The 95% conﬁdence intervals are shown in shaded areas. HR, hazard ratio; CI, conﬁdence interval. FIGURE 3 \| Kaplan-Meier curves comparing hospitalization for atrial ﬁbrillation (HAF)-free probability in never users and ever users of metformin (A) and in never users and users in each tertile of cumulative duration of metformin therapy (B) in the matched cohort. The 95% conﬁdence intervals are shown in shaded areas. HR, hazard ratio; CI, conﬁdence interval. FIGURE 2 \| Kaplan-Meier curves comparing hospitalization for atrial ﬁbrillation (HAF)-free probability in never users and ever users of metformin (A) and in never users and users in each tertile of cumulative duration of metformin therapy (B) in the unmatched cohort. The 95% conﬁdence intervals are shown in shaded areas. HR, hazard ratio; CI, conﬁdence interval. FIGURE 3 \| Kaplan-Meier curves comparing hospitalization for atrial ﬁbrillation (HAF)-free probability in never users and ever users of metformin (A) and in never users and users in each tertile of cumulative duration of metformin therapy (B) in the matched cohort. The 95% conﬁdence intervals are shown in shaded areas. HR, hazard ratio; CI, conﬁdence interval. FIGURE 2 \| Kaplan-Meier curves comparing hospitalization for atrial ﬁbrillation (HAF)-free probability in never users and ever users of metformin (A) and in never users and users in each tertile of cumulative duration of metformin therapy (B) in the unmatched cohort. The 95% conﬁdence intervals are shown in shaded areas. HR, hazard ratio; CI, conﬁdence interval. February 2021 \| Volume 7 \| Article 592901 Frontiers in Medicine \| www.frontiersin.org 6 Metformin and Atrial Fibrillation Tseng TABLE 2 \| Incidence of hospitalization for atrial ﬁbrillation and hazard ratios by metformin exposure in the main analyses. DISCUSSION received regular reﬁll of metformin (Model I of Table 3). The present study deﬁned AF as a primary diagnosis made at hospital discharge which should have been supported by laboratory information such as electrocardiograms or electrophysiological studies for the reimbursement purpose. This study conﬁrmed a lower risk of HAF in patients with type 2 diabetes mellitus who had been treated with metformin (Tables 2, 3, Figures 2, 3). A dose-response pattern was seen and the beneﬁcial eﬀect was especially remarkable when metformin had been used for more than 2 years in either the main analyses (Table 2) or the sensitivity analyses (Table 3). p p It is worthy to note that, by using the diagnostic code mainly identiﬁed at the outpatient clinics, the hazard ratio of 0.81 (95% conﬁdence interval 0.76–0.86) estimated by Chang et al. (11) and the odds ratio of 0.81 (95% conﬁdence interval 0.71– 0.93) estimated by Liou et al. (12) showed a less magnitude of protection than what we have seen in the present study (Tables 2, 3). This could be because that the diagnostic code of AF labeled at the outpatient clinics was not as accurate as the diagnosis made during hospitalization. The misclassiﬁcation made at outpatient clinics was probably non-diﬀerential and thus biased the hazard ratios toward the null (23). It is true that when we used a less stringent deﬁnition of AF by including the diagnostic code identiﬁed either at the outpatient clinics or during hospitalization in secondary analyses, the estimated hazard ratios did move toward the null (overall hazard ratio for the unmatched cohort: 0.426, 95% conﬁdence interval: 0.395– 0.459; and for the matched cohort: 0.667, 95% conﬁdence interval: 0.601–0.740), indicating a prone to non-diﬀerential misclassiﬁcation by using the diagnosis made at the outpatient clinics. However, despite the variation in the estimated risk ratios, all studies consistently supported a protection of metformin against AF by using diﬀerent study designs and diﬀerent statistical analyses and by deﬁning AF either as a diagnosis made at the outpatient clinics or made at the discharge of a hospitalization. While compared with the previous two studies conducted in Taiwan that investigated the eﬀect of metformin on AF (11, 12), the present study has several merits and methodological improvements. The study by Chang et al. Frontiers in Medicine \| www.frontiersin.org RESULTS Cohort/Metformin use n N Person–year Incidence rate (per 100,000 person-years) Hazard ratio 95% Conﬁdence interval P value Unmatched cohort Never users 86 21,666 93021.27 92.45 1.000 Ever users 303 173,398 803329.81 37.72 0.405 (0.319–0.515) <0.0001 Tertiles of cumulative duration of metformin therapy (months) Never users 86 21,666 93021.27 92.45 1.000 <25.9 121 57,224 194246.52 62.29 0.664 (0.502–0.878) 0.0041 25.9–57.0 107 57,143 274026.03 39.05 0.419 (0.316–0.557) <0.0001 >57.0 75 59,031 335057.26 22.38 0.230 (0.168–0.314) <0.0001 Matched cohort Never users 86 21,662 93008.43 92.46 1.000 Ever users 56 21,662 98284.01 56.98 0.617 (0.441–0.864) 0.0049 Tertiles of cumulative duration of metformin therapy (months) Never users 86 21,662 93008.43 92.46 1.000 <25.3 23 7,152 23486.22 97.93 1.022 (0.643–1.625) 0.9260 25.3–56.5 20 7,142 33396.19 59.89 0.654 (0.402–1.064) 0.0873 >56.5 13 7,368 41401.59 31.40 0.339 (0.189–0.608) 0.0003 n, incident case number of hospitalization for atrial ﬁbrillation; N, case number followed. n, incident case number of hospitalization for atrial ﬁbrillation; N, case number followed. DISCUSSION Patients with nephropathy Never users 46 8,376 1.000 Ever users 111 50,614 0.347 (0.246–0.490) <0.0001 Tertiles of cumulative duration of metformin therapy (months) Never users 46 8,376 1.000 <25.2 38 17,930 0.454 (0.294–0.700) 0.0004 25.2–56.4 38 16,903 0.343 (0.223–0.527) <0.0001 >56.4 35 15,781 0.275 (0.177–0.428) <0.0001 IV. Patients without nephropathy Never users 40 13,290 1.000 Ever users 192 122,784 0.494 (0.351–0.694) <0.0001 Tertiles of cumulative duration of metformin therapy (months) Never users 40 13,290 1.000 <25.2 83 39,294 0.895 (0.611–1.311) 0.5704 25.2–56.4 69 40,240 0.525 (0.355–0.775) 0.0012 >56.4 40 43,250 0.227 (0.146–0.353) <0.0001 V. Excluding patients with a diagnosis of chronic kidney disease Never users 49 17,254 1.000 Ever users 246 155,114 0.524 (0.386–0.712) <0.0001 Tertiles of cumulative duration of metformin therapy (months) Never users 49 17,254 1.000 <25.2 101 50,213 0.911 (0.645–1.288) 0.5989 25.2–56.4 86 51,063 0.540 (0.380–0.768) 0.0006 >56.4 59 53,838 0.280 (0.191–0.410) <0.0001 VI. Excluding patients in a renal dialysis status Never users 81 21,082 1.000 Ever users 301 172,642 0.418 (0.327–0.535) <0.0001 Tertiles of cumulative duration of metformin therapy (months) Never users 81 21,082 1.000 <25.2 120 56,839 0.684 (0.515–0.910) 0.0091 25.2–56.4 107 56,865 0.435 (0.326–0.581) <0.0001 >56.4 74 58,938 0.235 (0.171–0.322) <0.0001 VII. Excluding patients with chronic kidney disease and/or in a renal dialysis status Never users 49 17,252 1.000 Ever users 245 155,099 0.522 (0.384–0.709) <0.0001 Tertiles of cumulative duration of metformin therapy (months) Never users 49 17,252 1.000 <25.2 100 50,206 0.901 (0.638–1.274) 0.5566 25.2–56.4 86 51,057 0.540 (0.380–0.768) 0.0006 >56.4 59 53,836 0.280 (0.191–0.410) <0.0001 n, incident case number of hospitalization for atrial ﬁbrillation, N, case number followed. endothelial dysfunction, inﬂammation, and left atrial dilatation and ﬁbrosis (3). These can lead to electrical and structural remodeling and AF (3). Therefore, even though the mechanisms of a reduced risk of AF associated with metformin use are not yet elucidated, the biological actions of metformin targeting the pathophysiology of the development of AF might have contributed to such a clinical beneﬁt. Metformin improves insulin resistance by increasing the expression of insulin receptor and activation of tyrosine kinase (24). In the absence of co-administration of insulin or insulin- stimulating drugs, metformin per se rarely induce hypoglycemia. On the other hand, microvascular dysfunction and relative ischemia with reduced oxygen/nutrient delivery and/or increased energy demand in the heart may lead to metabolic stress and induce AF (1). DISCUSSION showed a high imbalance between metformin users and non-users in the use of statin, the distribution of major risk factors and the use of other antidiabetic drugs (11). All of these have been considered in the present study and the distributions of these potential confounders were well-balanced in our analyses in the matched cohort (Table 1). Furthermore, Chang et al. did not address the potential bias resulting from immortal time (11) and this has been considered in the present study (discussed later). The study by Liou et al. used a cross-sectional design and also had imbalanced distributions of potential confounders and the use of other antidiabetic drugs (12). The present study considered the balance of potential confounders and the use of other antidiabetic drugs in the matched cohort (Table 1) and used a retrospective cohort design that addressed the correct temporality between exposure (metformin use) and eﬀect (HAF). Additionally, both previous studies did not consider a dose-response eﬀect and the regularity in the prescription of metformin and the investigators included the diagnosis of AF made at the outpatient clinics (11, 12). The present study reinforced the robustness of the preventive eﬀect of metformin on AF by showing a dose-response eﬀect (Tables 2, 3) and a consistency of the ﬁndings after excluding patients who had not The higher risk of AF in patients with type 2 diabetes mellitus is assumed to be related to metabolic syndrome and the pathogenesis may involve insulin resistance, hypertension, greater glycemic excursion, hypoglycemia, myocardial steatosis, February 2021 \| Volume 7 \| Article 592901 7 Metformin and Atrial Fibrillation Tseng Tseng TABLE 3 \| Sensitivity analyses. Model/metformin use n N Hazard ratio 95% Conﬁdence interval P-value I. Excluding two consecutive prescriptions of metformin spanning more than 4 months Never users 86 21,666 1.000 Ever users 93 59,513 0.393 (0.293–0.527) <0.0001 Tertiles of cumulative duration of metformin therapy (months) Never users 86 21,666 1.000 <25.2 35 20,075 0.708 (0.474–1.058) 0.0922 25.2–56.4 27 16,197 0.415 (0.269–0.640) <0.0001 >56.4 31 23,241 0.245 (0.162–0.370) <0.0001 II. Excluding patients treated with incretin–based therapies during follow–up Never users 84 20,402 1.000 Ever users 269 134,549 0.458 (0.359–0.585) <0.0001 Tertiles of cumulative duration of metformin therapy (months) Never users 84 20,402 1.000 <25.2 115 48,775 0.713 (0.536–0.947) 0.0196 25.2–56.4 94 44,083 0.465 (0.347–0.625) <0.0001 >56.4 60 41,691 0.258 (0.185–0.361) <0.0001 III. DISCUSSION Through the activation of 5′-adenosine monophosphate-activated protein kinase, metformin promotes fatty acid oxidation, increases ketone body metabolism, reduces lipid accumulation, and induces the expression of glucose transporter in cardiomyocytes, thus facilitates more eﬃcient energy use with reduction of metabolic stress (1, 25). Studies also suggested that metformin may reduce AF by alleviating the dysfunction of epicardial adipose tissue (26). In an in vitro study, metformin reduces the production of reactive oxygen species and myolysis during tachypacing cell culture of atrial myocytes (11). Pro-ﬁbrotic biomarkers such as interleukin-6, transforming growth factor-beta one, matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 are important biomarkers of atrial remodeling in AF (1, 27). Interestingly, metformin inhibits the signaling pathways of transforming growth factor-beta one (28). Taken together, the mechanisms of a reduced risk of AF associated with metformin use may be multifactorial and are related to a reduction of insulin resistance and metabolic stress, inhibition of inﬂammation, and alleviation of cardiac ﬁbrosis. The ﬁndings of the present study have some clinical implications. First, because diabetes patients have a higher risk of AF (2–4) which may contribute to the signiﬁcantly higher risk of thromboembolic events and mortality (5), the reduced risk of AF associated with metformin use provided a good rationale for the recommendation of metformin as the ﬁrst- line treatment for glucose lowering in patients with type 2 diabetes mellitus. Second, it would be a good strategy to continue the use of metformin when the addition of other antidiabetic drugs is required for better control of hyperglycemia because its protection against AF may or may not be directly related to glucose lowering and such a protection was observed mainly after 2 years of its use (Tables 2, 3). Furthermore, the dose- response pattern (Tables 2, 3) not only implicates a cause-eﬀect relationship but also provides a good rationale for its continuous use when other antidiabetic drugs are added for the improvement of hyperglycemia. Third, the consistent ﬁndings of a protective role of metformin on AF in the diabetes patients might provide rationale for more vigorous investigation of its usefulness in non-diabetes people who are at a high risk of developing AF. Pharmacoepidemiological studies using administrative databases to evaluate long-term safety or beneﬁcial/adverse eﬀects of medications have become popular in recent years. DISCUSSION As the incidence of HAF was low, the absolute risk reduction calculated from the matched cohort was too small (86/21,662–56/21,662 = 0.14%) and the calculated number needed to treat was too large (the reciprocal of absolute risk reduction = 722). Therefore, the cost eﬀectiveness of using metformin to prevent HAF remains to be investigated. Seventh, because this is a retrospective cohort study, the ﬁndings should better be conﬁrmed by prospective cohort study designs or by clinical trials. Finally, we did not have measurement data of some other confounders like biochemical and hormonal data, anthropometric factors, lifestyle, physical activity, dietary pattern, cigarette smoking, alcohol drinking, family history, and genetic parameters. Immortal time refers to the follow-up period during which the outcome cannot happen. Immortal time bias can be introduced when treatment status and/or follow-up time are inappropriately assigned (29). To prevent misclassiﬁcation of non-diabetes people as diabetes patients, those with uncertain diabetes diagnosis have been excluded by enrolling only patients who had been prescribed antidiabetic drugs for two or more times (Figure 1). Misclassiﬁcation of the treatment status of metformin was not likely because the NHI is a universal health care system and the information of all prescriptions is available during the long period of follow-up. The immortal time during the period between diabetes diagnosis and the initiation of antidiabetic drugs and during the initial short follow-up period of <180 days were not included in the calculation of the follow-up person-years. Lastly, the immortal time during the waiting period between drug prescription and drug dispense when a patient is discharged from the hospital [as pointed out by Lévesque et al. (29)] is not a problem in Taiwan because the patient can get all discharge medications immediately from the hospital at the time of his/her discharge. There are some additional strengths. First, recall bias resulting from self-reporting would not happen in the study because medical records were used. Second, although the detection rate of a disease might be aﬀected by socioeconomic status of the patients in studies conducted in other countries, this was less likely in Taiwan because the drug cost-sharing in the NHI health care system is low and much expenses can be waived in veterans, in patients with low-income or when the patients receive prescription reﬁlls for chronic disease. DISCUSSION February 2021 \| Volume 7 \| Article 592901 Frontiers in Medicine \| www.frontiersin.org 8 Tseng Metformin and Atrial Fibrillation These studies are most useful for outcomes with low incidence or when randomized controlled trials are not feasible. However, some methodology limitations should be carefully addressed for not getting a biased result. Methodological limitations such as selection bias, prevalent user bias, immortal time bias, and confounding by indication as seen in the two previous studies (11, 12) have been carefully addressed in the present study during study design, patient enrollment and statistical analyses. Selection bias was avoided by using the nationwide database covering > 99% of the population and prevalent user bias was prevented by enrolling patients with new-onset diabetes and new users of metformin. The impacts or carryover eﬀects of other antidiabetic drugs used before metformin was initiated were also excluded by enrolling only ever users of metformin who received metformin as the ﬁrst antidiabetic drug (Figure 1). There are some limitations in the present study. First, we did not have electrocardiograms for conﬁrming the diagnosis of AF. Therefore, misclassiﬁcation of AF could not be completely excluded. However, if the misclassiﬁcations were not diﬀerential in ever and never users of metformin, the hazard ratios would only have been underestimated (23). Second, blood glucose levels were not available in the database. Because a cause should happen before an eﬀect, the lack of blood glucose levels prevents us from more aﬃrmative assurance of the correctness of temporality between diabetes as a cause and AF as an eﬀect in some patients. Third, in the matched cohort, the additional treatment with metformin in ever users who had balanced use of other antidiabetic drugs while compared to never users of metformin suggested that metformin users might have a higher intensity of medications. This either implied that ever users might have a more severe disease condition or that blood glucose control was not balanced between the two groups. Because ever users and never users of metformin were balanced in all covariates in the matched cohort (Table 1), a discrepancy in disease severity in terms of diabetes complications or comorbidities was less likely. However, a discrepancy in blood glucose control between the two groups could not be ruled out. Frontiers in Medicine \| www.frontiersin.org DISCUSSION Because blood glucose ﬂuctuations and insulin resistance may be related to the pathogenesis of AF and the information of these parameters was not available in the database, future studies are required to clarify the role of glycemic control and insulin resistance in the discrepant eﬀects observed in ever users and never users of metformin. Fourth, in the sensitivity analyses, although we have separately conducted subgroup analyses in patients with nephropathy (Model III, Table 3) and without nephropathy (Model IV, Table 3); and after excluding patients with a diagnosis of chronic kidney disease and/or in a renal dialysis status (Models V, VI, and VII, Table 3), we did not have data of urinary albumin excretion rate or estimated glomerular ﬁltration rate for more accurate diagnosis. Fifth, obesity and tobacco use might have been underestimated by using the diagnostic codes because these diagnoses were mostly not directly related to reimbursement purpose. However, because clinical diseases related to obesity and tobacco use such as hypertension, dyslipidemia, chronic obstructive pulmonary disease, cancer, and cardiovascular diseases have also been considered as potential confounders in the analyses (Table 1), it is believed that their eﬀects on the estimation of hazard ratios might also have been adjusted for. Sixth, knowledge of absolute risk reduction and number needed to treat may be of clinical importance (30). As the incidence of HAF was low, the absolute risk reduction calculated from the matched cohort was too small (86/21,662–56/21,662 = 0.14%) and the calculated number needed to treat was too large (the reciprocal of absolute risk reduction = 722). Therefore, the cost eﬀectiveness of using metformin to prevent HAF remains to be investigated. Seventh, because this is a retrospective cohort study, the ﬁndings should better be conﬁrmed by prospective cohort study designs or by clinical trials. Finally, we did not have measurement data of some other confounders like biochemical and hormonal There are some limitations in the present study. First, we did not have electrocardiograms for conﬁrming the diagnosis of AF. Therefore, misclassiﬁcation of AF could not be completely excluded. However, if the misclassiﬁcations were not diﬀerential in ever and never users of metformin, the hazard ratios would only have been underestimated (23). Second, blood glucose levels were not available in the database. DISCUSSION Because a cause should happen before an eﬀect, the lack of blood glucose levels prevents us from more aﬃrmative assurance of the correctness of temporality between diabetes as a cause and AF as an eﬀect in some patients. Third, in the matched cohort, the additional treatment with metformin in ever users who had balanced use of other antidiabetic drugs while compared to never users of metformin suggested that metformin users might have a higher intensity of medications. This either implied that ever users might have a more severe disease condition or that blood glucose control was not balanced between the two groups. Because ever users and never users of metformin were balanced in all covariates in the matched cohort (Table 1), a discrepancy in disease severity in terms of diabetes complications or comorbidities was less likely. However, a discrepancy in blood glucose control between the two groups could not be ruled out. Because blood glucose ﬂuctuations and insulin resistance may be related to the pathogenesis of AF and the information of these parameters was not available in the database, future studies are required to clarify the role of glycemic control and insulin resistance in the discrepant eﬀects observed in ever users and never users of metformin. Fourth, in the sensitivity analyses, although we have separately conducted subgroup analyses in patients with nephropathy (Model III, Table 3) and without nephropathy (Model IV, Table 3); and after excluding patients with a diagnosis of chronic kidney disease and/or in a renal dialysis status (Models V, VI, and VII, Table 3), we did not have data of urinary albumin excretion rate or estimated glomerular ﬁltration rate for more accurate diagnosis. Fifth, obesity and tobacco use might have been underestimated by using the diagnostic codes because these diagnoses were mostly not directly related to reimbursement purpose. However, because clinical diseases related to obesity and tobacco use such as hypertension, dyslipidemia, chronic obstructive pulmonary disease, cancer, and cardiovascular diseases have also been considered as potential confounders in the analyses (Table 1), it is believed that their eﬀects on the estimation of hazard ratios might also have been adjusted for. Sixth, knowledge of absolute risk reduction and number needed to treat may be of clinical importance (30). FUNDING This study was supported by the Ministry of Science and Technology (MOST 107-2221-E-002-129-MY3) of Taiwan and the Yee Fong Charity Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. DISCUSSION The consistency of the ﬁndings in both the unmatched cohort and the matched cohort (Table 2) and in the sensitivity analyses (Table 3) suggested that the results were reproducible in diﬀerent cohorts. The use of a PS-matched cohort (Table 1) and the estimation of hazard ratios by using the Cox proportional hazards model incorporated with IPTW (Tables 2, 3) was aimed at reducing potential confounding by indication. The possibility of residual confounding from the covariates should be small, especially in the matched cohort for whom the values of standardized diﬀerence between ever and never users of metformin were < 10% for all covariates (Table 1). February 2021 \| Volume 7 \| Article 592901 9 Tseng Tseng Metformin and Atrial Fibrillation CONCLUSION kin was not required to participate in this study in accordance with the national legislation and the institutional requirements. kin was not required to participate in this study in accordance with the national legislation and the institutional requirements. This study supports a lower risk of HAF in patients with type 2 diabetes mellitus who have been treated with metformin. However, additional prospective observational studies and/or clinical trials are necessary to conﬁrm a cause-eﬀect relationship. Because metformin is inexpensive and safe and does not cause hypoglycemia when it is used as a monotherapy, the usefulness of metformin as a protection against AF in high risk patients is worthy of more intensive investigation in both the diabetes patients and the non-diabetes people. ACKNOWLEDGMENTS The study is based in part on data from the National Health Insurance Research Database provided by the Bureau of National Health Insurance, Department of Health and managed by National Health Research Institutes. The interpretation and conclusions contained herein do not represent those of Bureau of National Health Insurance, Department of Health or National Health Research Institutes. ETHICS STATEMENT The studies involving human participants were reviewed and approved by National Health Research Institutes. Written informed consent from the participants’ legal guardian/next of DATA AVAILABILITY STATEMENT The datasets presented in this article are not readily available because public availability of the dataset is restricted by local regulations to protect privacy. Requests to access the datasets should be directed to Chin-Hsiao Tseng, ccktsh@ms6.hinet.net. C-HT researched data and wrote manuscript. C-HT researched data and wrote manuscript. REFERENCES 12. Liou YS, Yang FY, Chen HY, Jong GP. Antihyperglycemic drugs use and new- onset atrial ﬁbrillation: a population-based nested case control study. PLoS ONE. (2018) 13:e0197245. doi: 10.1371/journal.pone.0197245 1. Harada M, Melka J, Sobue Y, Nattel S. Metabolic considerations in atrial ﬁbrillation - mechanistic insights and therapeutic opportunities. Circ J. (2017) 81:1749–57. doi: 10.1253/circj.CJ-17-1058 1. Harada M, Melka J, Sobue Y, Nattel S. 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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. Copyright © 2021 Tseng. 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. 27. Stanciu AE, Vatasescu RG, Stanciu MM, Serdarevic N, Dorobantu M. The role of pro-ﬁbrotic biomarkers in paroxysmal and persistent atrial ﬁbrillation. Cytokine. (2018) 103:63–8. doi: 10.1016/j.cyto.2017.12.026 February 2021 \| Volume 7 \| Article 592901 Frontiers in Medicine \| www.frontiersin.org 11
https://openalex.org/W2604467988	https://research-information.bris.ac.uk/ws/files/109170056/e012674.full.pdf	English	null	What are the risks and benefits of temporarily discontinuing medications to prevent acute kidney injury? A systematic review and meta-analysis	BMJ open	2,017	cc-by	8,227	Whiting, P., Savović, J., Morden, A., Caskey, F., Blakeman, T., Tomson , C., Stone, T., Richards, A., Horwood, J., & Tomlinson, L. (2017). What are the risks and benefits of temporarily discontinuing medications to prevent acute kidney injury? A systematic review and meta-analysis. BMJ Open, 7(4), Article e012674. https://doi.org/10.1136/bmjopen-2016-012674 Whiting, P., Savović, J., Morden, A., Caskey, F., Blakeman, T., Tomson , C., Stone, T., Richards, A., Horwood, J., & Tomlinson, L. (2017). What are the risks and benefits of temporarily discontinuing medications to prevent acute kidney injury? A systematic review and meta-analysis. BMJ Open, 7(4), Article e012674. https://doi.org/10.1136/bmjopen-2016-012674 Whiting, P., Savović, J., Morden, A., Caskey, F., Blakeman, T., Tomson , C., Stone, T., Richards, A., Horwood, J., & Tomlinson, L. (2017). What are the risks and benefits of temporarily discontinuing medications to prevent acute kidney injury? A systematic review and meta-analysis. BMJ Open, 7(4), Article e012674. https://doi.org/10.1136/bmjopen-2016-012674 Link to publication record on the Bristol Research Portal PDF-document This is the final published version of the article (version of record). It first appeared online via BMJ at http://bmjopen.bmj.com/content/7/4/e012674. Please refer to any applicable terms of use of the publisher. Strengths and limitations of this study Objectives: To summarise evidence on temporary discontinuation of medications to prevent acute kidney injury (AKI). Design: Systematic review and meta-analysis of randomised and non-randomised studies. Participants: Adults taking diuretics, ACE inhibitors (ACEI), angiotensin receptor blockers (ARB), direct renin inhibitors, non-steroidal anti-inflammatories, metformin or sulfonylureas, experiencing intercurrent illnesses, radiological or surgical procedures. ▪We have conducted a thorough systematic review of the evidence from studies that have examined interventions involving temporary discontinuation of medications to prevent or minimise the sever- ity, or consequences, of acute kidney injury (AKI). Design: Systematic review and meta-analysis of randomised and non-randomised studies. Participants: Adults taking diuretics, ACE inhibitors (ACEI), angiotensin receptor blockers (ARB), direct renin inhibitors, non-steroidal anti-inflammatories, metformin or sulfonylureas, experiencing intercurrent illnesses, radiological or surgical procedures. Participants: Adults taking diuretics, ACE inhibitors (ACEI), angiotensin receptor blockers (ARB), direct renin inhibitors, non-steroidal anti-inflammatories, ▪This is a topic of major importance due to inter- ventions currently being implemented to reduce the risk of AKI throughout the UK and internationally. metformin or sulfonylureas, experiencing intercurrent illnesses, radiological or surgical procedures. Interventions: Temporary discontinuation of any of the medications of interest. ▪Broad eligibility criteria included randomised and non-randomised studies; primary and secondary care; intercurrent illness or a radiological/surgical procedure; planned and unplanned settings. Primary and secondary outcome measures: Risk of AKI. Secondary outcome measures were estimated glomerular filtration rate and creatinine post-AKI, urea, systolic and diastolic blood pressure, death, clinical outcomes and biomarkers. ▪The strength of the conclusion is limited by the quality and number of studies, and absence of evidence for important settings and classes of medications. ▪The strength of the conclusion is limited by the quality and number of studies, and absence of evidence for important settings and classes of medications. Results: 6 studies were included (1663 participants), 3 randomised controlled trials (RCTs) and 3 prospective cohort studies. The mean age ranged from 65 to 73 years, and the proportion of women ranged from 31% to 52%. All studies were in hospital settings; 5 evaluated discontinuation of medication prior to coronary angiography and 1 prior to cardiac surgery. 5 studies evaluated discontinuation of ACEI and ARBs and 1 small cohort study looked at discontinuation of non- steroidal anti-inflammatory drugs. No studies evaluated discontinuation of medication in the community following an acute intercurrent illness. University of Bristol – Bristol Research Portal General rights This document is made available in accordance with publisher policies. Please cite only the published version using the reference above. Full terms of use are available: http://www.bristol.ac.uk/red/research-policy/pure/user-guides/brp-terms/ What are the risks and beneﬁts of temporarily discontinuing medications to prevent acute kidney injury? A systematic review and meta-analysis Penny Whiting,1,2 Andrew Morden,1,2 Laurie A Tomlinson,3,4 Fergus Caskey,2,3 Thomas Blakeman,5,6 Charles Tomson,7 Tracey Stone,1,2 Alison Richards,1,2 Jelena Savović,1,2 Jeremy Horwood1,2 Open Access Research group.bmj.com on April 11, 2017 - Published by http://bmjopen.bmj.com/ Downloaded from R h group.bmj.com on April 11, 2017 - Published by http://bmjopen.bmj.com/ Downloaded from group.bmj.com on April 11, 2017 - Published by http://bmjopen.bmj.com/ Downloaded from group.bmj.com on April 11, 2017 - Published by http://bmjopen.bmj.com/ Downloaded from Open Access Research What are the risks and beneﬁts of temporarily discontinuing medications to prevent acute kidney injury? A systematic review and meta-analysis Penny Whiting,1,2 Andrew Morden,1,2 Laurie A Tomlinson,3,4 Fergus Caskey,2,3 Thomas Blakeman,5,6 Charles Tomson,7 Tracey Stone,1,2 Alison Richards,1,2 Jelena Savović,1,2 Jeremy Horwood1,2 ABSTRACT Objectives: To summarise evidence on temporary discontinuation of medications to prevent acute kidney injury (AKI). ABSTRACT Objectives: To summarise evidence on temporary discontinuation of medications to prevent acute kidney injury (AKI). To cite: Whiting P, Morden A, Tomlinson LA, et al. What are the risks and benefits of temporarily discontinuing medications to prevent acute kidney injury? A systematic review and meta-analysis. BMJ Open 2017;7:e012674. doi:10.1136/bmjopen-2016- 012674 Strengths and limitations of this study There was an increased risk of AKI of around 15% in those in whom medication was continued compared with those in whom it was discontinued (relative risk (RR) 1.17, 95% CI 0.99 to 1.38; 5 studies). When only results from RCTs were pooled, the increase in risk was almost 50% (RR 1.48, 95% CI 0.84 to 2.60; 3 RCTs), but the CI was wider. There was no difference between groups for any secondary outcomes. Received 16 May 2016 Revised 21 September 2016 Accepted 25 October 2016 Penny Whiting,1,2 Andrew Morden,1,2 Laurie A Tomlinson,3,4 Fergus Caskey,2,3 Thomas Blakeman,5,6 Charles Tomson,7 Tracey Stone,1,2 Alison Richards,1,2 Jelena Savović,1,2 Jeremy Horwood1,2 Received 16 May 2016 Revised 21 September 2016 Accepted 25 October 2016 METHODS Randomised controlled trials (RCTs) were assessed for methodological quality using a draft version of the new Cochrane risk of bias tool13 that includes items covering allocation bias (random sequence generation, allocation concealment and baseline imbalance), departures from interventions (participant and study personnel blinding, deviations from intended interventions and analysis in groups to which they were randomised), attrition bias (incomplete outcome data and robustness of results to missing data), detection bias (blinding of outcome asses- sors and likelihood of blinding to have inﬂuenced results) and reporting bias (selective reporting of outcome domain being assessed). The ROBINS-I tool was used to assess the risk of bias in non-randomised studies.14 It includes domains covering bias due to con- founding, bias in the selection of participants into the study, bias due to departures from intended interven- tions, bias due to missing data, bias in taking measure- ments and bias in the selection of the reported result. Systematic review methods followed guidance from the Centre for Reviews and Dissemination (CRD)9 and the Cochrane Collaboration;10 this review is reported accord- ing to the PRISMA guidelines.11 The review followed a predeﬁned published protocol.12 Identification and selection of studies The following databases were searched from inception to January 2016: Embase, MEDLINE, PsycINFO, BIOSIS Citation Index (Web of Science), CINAHL (Cumulative Index to Nursing and Allied Health Literature), Science Citation Index (SCI) (Web of Science) and the Cochrane Central Register of Controlled Trials (CENTRAL). Supplementary searches were undertaken to identify grey literature, completed and ongoing trials, in the following Data extraction and assessment of risk of bias Data extraction and assessment of risk of bias We extracted data on baseline characteristics (number of participants, participant characteristics, study settings, study design, country, inclusion and exclusion criteria), intervention/exposure related to stopping medication and outcomes. The primary outcome was incidence of AKI. Secondary outcomes included urinary biomarkers, clinical outcomes, creatinine, eGFR, urea and blood pressure. For dichotomous data (eg, incidence of AKI), we extracted the number of events and participants in each treatment group and calculated the relative risk (RR) and 95% CI. For continuous data, we extracted the mean and SD in each treatment group and calcu- lated mean differences (MD) and 95% CIs. We conducted a systematic review and meta-analysis of the randomised and non-randomised studies that have examined temporary discontinuation of all or any of these medications in patients in primary or secondary care at risk of AKI or with newly diagnosed AKI as a result of an intercurrent illness or a radiological/surgical procedure (planned or unplanned). BACKGROUND Acute kidney injury (AKI) is a sudden decline in renal function, affecting up to 20% of people admitted to hospital, and is strongly associated with increased mortality and longer duration of hospital stay.1 Historically, recognition and treatment of AKI has been poor.2 Recent comprehensive initiatives in the UK have focused on impro- ving awareness and treatment of people with or at risk of AKI.3 It is thought that a substan- tial proportion of AKI is triggered or exacer- bated by prescribed medications, particularly during times of physiological stress such as For numbered affiliations see end of article. For numbered affiliations see end of article. Conclusions: There is low-quality evidence that withdrawal of ACEI/ARBs prior to coronary angiography and cardiac surgery may reduce the incidence of AKI. There is no evidence of the impact of drug cessation interventions on AKI incidence during intercurrent illness in primary or secondary care. Correspondence to Dr Penny Whiting; penny.whiting@bristol.ac.uk 1 Whiting P, et al. BMJ Open 2017;7:e012674. doi:10.1136/bmjopen-2016-012674 group.bmj.com on April 11, 2017 - Published by http://bmjopen.bmj.com/ Downloaded from group.bmj.com on April 11, 2017 - Published by http://bmjopen.bmj.com/ Downloaded from Open Access intercurrent illness, surgery or radiocontrast imaging.4 These medications include ACE inhibitors (ACEI), angiotensin receptor blockers (ARB), diuretics, non- steroidal anti-inﬂammatory drugs (NSAIDs). Under the same circumstances, reduced excretion of metformin is associated with an increased risk of lactic acidosis, while sulfonylureas can lead to a greater incidence of hypogly- caemia. Therefore, many clinicians, expert consensus statements and guidelines recommend that some or all of these medications are stopped prior to elective or emergency procedures, or when patients become unwell with symptoms of severe infection.5 6 Initiatives advising patients prescribed these medications to temporarily stop taking them when they become unwell (so-called ‘sick-day rules’) have been implemented throughout Scotland and in local initiatives across the UK.7 However, the evidence base to support these recommendations is unclear, and the overall beneﬁt remains controversial.8 resources: NIH ClinicalTrials.gov (http://www.clinicaltrials. gov), metaRegister of Controlled Trials (http://www. controlled-trials.com), WHO International Clinical Trials Registry Platform (ICTRP) (http://www.who.int/ictrp/ en), relevant guidelines (eg, NICE in the UK) regarding management of AKI. Reference lists of included studies were screened. Details of the MEDLINE search strategy are available as an online supplementary appendix. Search results and full-text articles were independently assessed for inclusion by two reviewers; disagreements were resolved through consensus or referral to a third reviewer where necessary. Study eligibility criteria Studies, randomised and non-randomised, that evalu- ated adults (age ≥18 years) who were taking a speciﬁed medication and experiencing an intercurrent illness or undergoing a radiological/surgical procedure (planned or unplanned) in whom the medication was temporarily discontinued for any reason were eligible for inclusion. Medications of interest were diuretics, ACEIs, ARBs, direct renin inhibitors, NSAIDs, metformin or sulfony- lureas. Studies had to report a measure of kidney func- tion (eg, incidence of AKI, estimated glomerular ﬁltration rate (eGFR) or serum creatinine) and include a comparator group consisting of placebo, no treatment or usual care. Data were extracted by one reviewer using a standard data extraction form designed for this review, and checked by a second reviewer. The risk of bias assess- ment was performed independently by two reviewers. Any disagreements were resolved by consensus or refer- ral to a third reviewer. Data synthesis We grouped studies by design (randomised vs non- randomised), population (coronary angiography vs surgery) and outcome. If there were two or more studies assessing the same outcome, data were plotted on a 2 Whiting P, et al. BMJ Open 2017;7:e012674. doi:10.1136/bmjopen-2016-012674 group.bmj.com on April 11, 2017 - Published by http://bmjopen.bmj.com/ Downloaded from group.bmj.com on April 11, 2017 - Published by http://bmjopen.bmj.com/ Downloaded from Open Access (522 participants)16–18 and three prospective cohort studies (1141 participants).19–21 One study was available only as a conference abstract and so limited details were available for this study.21 forest plot. If data were considered statistically and clinic- ally sufﬁciently homogeneous, then summary estimates were produced using random effects meta-analysis. When the same outcomes were assessed in randomised and non-randomised studies that were considered similar in terms of population and intervention, we ﬁrst stratiﬁed the analysis based on study design. If summary estimates from stratiﬁed analyses were consid- ered sufﬁciently similar, we then produced an overall summary estimate combining data from randomised and non-randomised studies. For dichotomous out- comes, we estimated summary RRs and 95% CIs; for continuous data, we estimated summary MDs and 95% CIs. Heterogeneity was investigated using forest plots and the I2 statistic. Where data were considered too heterogeneous to pool, a narrative synthesis was pro- vided. We used GRADE to rate the overall quality of the evidence for risk of bias, publication bias, impreci- sion, inconsistency, indirectness and magnitude of effect.15 All studies were conducted in hospital settings: ﬁve evaluated discontinuation of medication prior to coron- ary angiography and one prior to cardiac surgery.19 All but one study16 restricted inclusion to patients deemed at higher risk of AKI such as those with chronic kidney disease (3 studies),17 18 20 diabetes (1 study)21 or a set of criteria that deﬁned patients at high risk (1 study).19 The most commonly reported comorbidities included diabetes, hypertension and congestive heart failure. No studies of discontinuation of medication in the commu- nity following an acute intercurrent illness were found. Studies were conducted in North America, Turkey and Israel. The mean age, where reported, ranged from 65 to 73 years and the proportion of women in the studies ranged from 31% to 52%. Five studies evaluated discon- tinuation of ACEI and ARBs, one small cohort study looked at discontinuation of NSAIDs.20 The time point at which the medication was stopped varied between studies. Data synthesis Three studies reported that medication was stopped 24 hours prior to the procedure,16 18 21 two (including the one study of surgery) that it was stopped on the morning of the procedure17 19 and one (the study of NSAIDs)20 did not provide details on when Risk of bias medication was stopped. The time point at which medi- cation was started again also varied. One study stated that medication was started up to 96 hours postproce- dure,18 one RCT16 included two intervention arms that compared restarting immediately postprocedure with restarting 24 hours after the procedure and four studies did not report on this. No studies were found that assessed discontinuation of diuretics, metformin or sul- fonylureas. Table 1 provides an overview of included studies. The risk of bias assessment was performed for the primary outcome of incidence of AKI. Two RCTs were judged to have ‘some concerns’ regarding risk of bias16 17 and one was rated ‘low risk of bias’.18 The two judged at ‘some concern’ had issues with the randomisa- tion process; all other bias domains were rated ‘low risk’. Search results The searches identiﬁed 4316 hits (records) of which 42 were considered potentially relevant and obtained for full-text review (ﬁgure 1). A total of six studies (1663 participants) were included in the review: three RCTs Figure 1 Flow of studies through the review process. Whiti P t l BMJ O 2017 7 012674 d i 10 1136/b j 2016 012674 Figure 1 Flow of studies through the review process. Whiting P, et al. BMJ Open 2017;7:e012674. doi:10.1136/bmjopen-2016-012674 3 group.bmj.com on April 11, 2017 - Published by http://bmjopen.bmj.com/ Downloaded from group.bmj.com on April 11, 2017 - Published by http://bmjopen.bmj.com/ Downloaded from Risk of bias One provided no information on the methods used to allocate participants to the two treatment groups, the other randomised patients by means of a coin toss but Table 1 Details of studies included in the review Bainey et al18 Rosenstock et al17 Wolak et al16 Coca et al19 Goksuluk et al21* Weisbord et al20 Study design RCT RCT RCT Prospective cohort Prospective cohort Prospective cohort Sample size 208 220 94 1017 80 44 Country Canada USA Israel North America Turkey USA Population Coronary angiography Coronary angiography Coronary angiography Cardiac surgery Coronary angiography Coronary angiography Risk group CKD CKD None High risk of AKI Diabetes CKD Mean age (SD) Intervention: 73 (9) Control: 72 (8) Intervention: 72 (10) Control: 72 (10) 65 (12) Intervention: 71 (11) Control: 70 (12) NR NR Female (%) 26 52 33 31 NR NR AKI definition Increase in SCr ≥25% or ≥0.5 mg from baseline Increase in SCr >25% or 0.5 mg from baseline Increase in SCr ≥25% from baseline Increase in SCr ≥50% or ≥0.3 mg from baseline Increase in SCr ≥25% or ≥0.5 mg from baseline Increase in SCr ≥25% from baseline or ≥0.5 mg from baseline Comorbidities Diabetes (54%), hypertension (47%), congestive heart failure (14%), liver cirrhosis (1%) Hypotension (97%), diabetes (55%) Diabetes (50%), unstable angina (62%) Diabetes (47%), Hypertension (88%), congestive heart failure (23%) Diabetes (100%) NR Study drug ACE/ARB ACE/ARB ACE/ARB ACE/ARB ACE/ARB NSAIDs Intervention: timing of hold 24 hours prior to procedure Day of procedure 24 hours prior to procedure Morning of surgery 24 hours before procedure No details Intervention: timing of restart Up to 96 hours postprocedure 24 hours postprocedure (1) Immediately afterwards; (2) 24 hours after No details No details No details Control Continued throughout study Continued throughout study Continued throughout study Continued throughout study Continued throughout study Continued throughout study Risk of bias Low Some: randomised by coin toss, no information on allocation concealment. Baseline difference compatible with chance Some; no information on treatment allocation, baseline difference compatible with chance Moderate; controlled for confounding but possibility of residual confounding Critical; no control for confounding Not assessed Available only as conference abstract. ACE, angiotensin-converting enzyme inhibitors; AKI, acute kidney infection; ARB, angiotensin receptor blockers; CKD, chronic kidney Available only as conference abstract. Risk of bias ACE, angiotensin-converting enzyme inhibitors; AKI, acute kidney infection; ARB, angiotensin receptor blockers; CKD, chronic kidney disease; NSAIDs, non-steroidal anti-inflammatory drugs; RCT, randomised controlled trial; SCr, serum creatinine. 4 Whiting P, et al. BMJ Open 2017;7:e012674. doi:10.1136/bmjopen-2016-012674 group.bmj.com on April 11, 2017 - Published by http://bmjopen.bmj.com/ Downloaded from group.bmj.com on April 11, 2017 - Published by http://bmjopen.bmj.com/ Downloaded from group.bmj.com on April 11, 2017 - Published by http://bmjopen.bmj.com/ Downloaded from Open Access Open Access did not provide any information on whether allocation was concealed. Both studies provided a reasonable over- view of baseline characteristics, including similarities in timings of baseline kidney function, which suggested that any differences between groups were compatible with chance. The risk of bias assessment highlighted that none of the studies provided information on blind- ing of participants, study personnel or outcome asses- sors. However, there do not appear to have been any departures from the intended interventions, thus knowl- edge of the assigned intervention appears unlikely to have inﬂuenced the study result. The outcome measure was considered relatively objective and therefore also unlikely to have been inﬂuenced by knowledge of treat- ment assignment. of NSAIDs, only found 3 patients out of 44 NSAID users who were advised to discontinue their medication prior to coronary angiography. It reported that discontinuation of NSAID was not associated with a lower rate of AKI, but this was limited by the small number of patients in whom medication was discontinued. All other studies assessed the incidence of AKI (or contrast-induced nephropathy) in those in whom medi- cation was stopped prior to the procedure compared with those in whom medication was continued (ﬁgure 2). Three studies deﬁned AKI as an increase in creatinine of 25% or 0.5 mg/dL above baseline,17 18 21 one as an increase in creatinine of 25% above baseline16 and one used a slightly different deﬁnition of an increase in creatinine of 50% or 0.3 mg/dL above base- line.19 All but one suggested an increased risk of AKI in those in whom medication was continued, but CIs were generally wide. There was an increased risk of AKI of around 15% in those in whom medication was contin- ued compared with those in whom it was discontinued (RR 1.17, 95% CI 0.99 to 1.38). Omitting the study judged at critical risk of bias had very little effect on the summary estimate (RR 1.16, 95% CI 0.98 to 1.37). Risk of bias When only results from RCTs were pooled, the increase in risk was almost 50% (RR 1.48, 95% CI 0.84 to 2.60; 3 RCTs), but the CI was much wider. There was no evidence of heterogeneity for any of these analyses (I2=0%). Based on GRADE, the quality of the evidence was judged as low for the analysis restricted to RCTs and very low when non-randomised studies were included (table 2). The evidence was downgraded due to imprecision and the likelihood of publication bias for the analysis that included RCTs and for study quality and publication bias for the analysis that included non-randomised trials. It was not possible to conduct a risk of bias assessment for one of the non-randomised studies20 as this study did not provide any numerical data and the risk of bias assessment is performed at the result level. One of the non-randomised studies was judged at moderate risk of bias,19 and the other at critical risk of bias.21 The study judged at critical risk of bias only presented crude outcome data with no adjustment for potential con- founding factors. It was judged at low risk of bias for all other domains with the exception of measurement of interventions which was judged at moderate risk of bias as it was not clear exactly how exposure to ACEI and ARBs was measured. The study judged at moderate risk of bias was judged to have appropriately controlled for confounding factors, but the guidance for the ROBINS-I tool states that this domain can only be rated as low risk of bias if the study is considered comparable to a well- performed randomised trial. DISCUSSION The results of our meta-analysis demonstrate a ∼15% increased risk of AKI in those in whom medication was continued compared with those in whom it was discon- tinued (RR 1.17, 95% CI 0.99 to 1.38). When only results from RCTs were pooled, the increase in risk was almost 50% (RR 1.48, 95% CI 0.84 to 2.60), but the CI was much wider. Based on the GRADE approach, the quality of the evidence was low when restricted to RCTs and very low when non-randomised studies were included. There was no difference between groups in which medication was stopped and groups in which it was continued for any secondary outcomes, but these were mainly assessed in single studies. The results of our meta-analysis demonstrate a ∼15% increased risk of AKI in those in whom medication was continued compared with those in whom it was discon- tinued (RR 1.17, 95% CI 0.99 to 1.38). When only results from RCTs were pooled, the increase in risk was almost 50% (RR 1.48, 95% CI 0.84 to 2.60), but the CI was much wider. Based on the GRADE approach, the quality of the evidence was low when restricted to RCTs and very low when non-randomised studies were included. There was no difference between groups in which medication was stopped and groups in which it was continued for any secondary outcomes, but these were mainly assessed in single studies. y g This is the ﬁrst systematic review into a topic of major importance, as interventions of this type are currently being implemented throughout the UK and internation- ally, with the aim of reducing the incidence and/or severity of AKI. We have used broad inclusion criteria in many databases to capture randomised and non- randomised studies, in primary and secondary care, and for a range of AKI precipitants including intercurrent illness and planned or emergency radiological and surgical procedure. However, we have found that the published evidence was sparse and has important limita- tions. It is focused in hospital settings, mainly in patients undergoing coronary angiography, restricted to patients who were considered high risk for AKI and predomin- antly evaluates discontinuation of ACEI and ARBs. The primary deﬁnition of AKI in all studies was based on short-term changes in serum creatinine, although deﬁni- tions varied across studies. Incidence of AKI Secondary outcomes Two studies16 17 assessed GFR and creatinine at 24 hours postintervention (ﬁgures 3 and 4). Both suggested no One study did not provide any numerical data on the effect of discontinuation of medication on patient out- comes.20 This cohort study, which assessed discontinuation Figure 2 Forest plot showing the risk of acute kidney injury in those who stopped medication prior to procedure compared with those who continued medication. Figure 2 Forest plot showing the risk of acute kidney injury in those who stopped medication prior to procedure compared with those who continued medication. Figure 2 Forest plot showing the risk of acute kidney injury in those who stopped medication prior to procedure compared with those who continued medication. Figure 2 Forest plot showing the risk of acute kidney injury in those who stopped medication prior to procedure compared with those who continued medication. Whiting P, et al. BMJ Open 2017;7:e012674. doi:10.1136/bmjopen-2016-012674 5 group.bmj.com on April 11, 2017 - Published by http://bmjopen.bmj.com/ Downloaded from group.bmj.com on April 11, 2017 - Published by http://bmjopen.bmj.com/ Downloaded from Open Access Table 2 GRADE evidence profile: risks and benefits of temporarily discontinuing medications to prevent acute kidney injury Quality assessment Number of patients Effect Studies Risk of bias Inconsistency Indirectness Imprecision Other considerations Continuation Discontinuation Relative (95% CI) Absolute (95% CI) Quality Incidence of acute kidney injury 3 RCTs Not serious Not serious Not serious Serious Publication bias strongly suspected† 27/248 (10.9%) 21/274 (7.7%) RR 1.48 (0.84 to 2.60) 52 more per 1000 (from 17 fewer to 174 more) ⊕⊕○○ Low 3 RCTs3 Cohorts Very serious‡ Not serious Not serious Not serious Publication bias strongly suspected† 134/520 (25.8%) 323/1099 (29.4%) RR 1.14 (0.96 to 1.36) 36 more per 1000 (from 10 fewer to 93 more) ⊕○○○ Very low Wide CI and few events. †Non-randomised studies appear would have been unlikely to have been written up for publication if findings had been negative; therefore, similar studies with negative findings considered likely. ‡RCTS, no serious concerns regarding risk of bias. Two cohort studies, one judged moderate risk of bias due to possibility of residual confound, one judged critical risk of bias as did not control difference in these measures between intervention groups, although CIs were wide. Other outcomes reported in single studies included urinary biomarkers (structural AKI), clinical end points (mortality, myocar- dial infarction, stroke, congestive heart failure, rehospita- lisation, hypertensive treatment) and blood pressure. Incidence of AKI Generally, there was no difference between groups in which medication was stopped and groups in which it was continued for any of these outcomes. Table 3 pro- vides an overview of key outcomes; other outcomes reported in included studies were different ways of meas- uring these outcomes (eg, continuous rather than dichotomous data, or change from baseline rather than absolute value). Table 2 GRADE evidence profile: risks and benefits of temporarily discontinuing medications to prevent acute kidney injury Quality assessment Number of patients Effect Studies Risk of bias Inconsistency Indirectness Imprecision Other considerations Continuation Discontinuation Relative (95% CI) Absolute (95% CI) Quality Incidence of acute kidney injury 3 RCTs Not serious Not serious Not serious Serious Publication bias strongly suspected† 27/248 (10.9%) 21/274 (7.7%) RR 1.48 (0.84 to 2.60) 52 more per 1000 (from 17 fewer to 174 more) ⊕⊕○○ Low 3 RCTs3 Cohorts Very serious‡ Not serious Not serious Not serious Publication bias strongly suspected† 134/520 (25.8%) 323/1099 (29.4%) RR 1.14 (0.96 to 1.36) 36 more per 1000 (from 10 fewer to 93 more) ⊕○○○ Very low Wide CI and few events. †Non-randomised studies appear would have been unlikely to have been written up for publication if findings had been negative; therefore, similar studies with negative findings considered likely. ‡RCTS, no serious concerns regarding risk of bias. Two cohort studies, one judged moderate risk of bias due to possibility of residual confound, one judged critical risk of bias as did not control DISCUSSION The deﬁnitions of AKI used in four of the studies may have overestimated the in- cidence of AKI compared with the currently accepted deﬁnition of AKI, which was used in only one study.19 Since ACEIs/ARBs reduce glomerular ﬁltration rate but preserve tubular blood ﬂow, a more marked short- term reduction in eGFR may be associated with lower rates of established AKI due to ongoing tubular injury.22 Indeed, the only study19 that examined alternate biomarker-based deﬁnitions of AKI found no effect related to drug cessation. In addition, the longer term 6 Whiting P, et al. BMJ Open 2017;7:e012674. doi:10.1136/bmjopen-2016-012674 group.bmj.com on April 11, 2017 - Published by http://bmjopen.bmj.com/ Downloaded from Figure 3 Forest plot showing the mean difference in glomerular filtration rate at 24 hours in those who stopped medication prior to procedure compared with those who continued medication. Open Access group.bmj.com on April 11, 2017 - Published by http://bmjopen.bmj.com/ Downloaded from Figure 3 Forest plot showing the mean difference in glomerular filtration rate at 24 hours in those who stopped medication prior to procedure compared with those who continued medication. Open Access group.bmj.com on April 11, 2017 - Published by http://bmjopen.bmj.com/ Downloaded from Figure 3 Forest plot showing the mean difference in glomerular filtration rate at 24 hours in those who stopped medication prior to procedure compared with those who continued medication. Figure 3 Forest plot showing the mean difference in glomerular filtration rate at 24 hours in those who stopped medication prior to procedure compared with those who continued medication. Figure 4 Forest plot showing the mean difference in creatinine at 24 hours in those who stopped medication prior to procedure compared with those who continued medication. Figure 4 Forest plot showing the mean difference in creatinine at 24 hours in those who stopped medication prior to procedure compared with those who continued medication. impact of AKI in terms of the development of CKD or reductions in baseline GFR was not reported. The reduc- tion in glomerular ﬁltration rate caused by ACEI and ARB treatment is reversible on stopping the drug.23 This temporary rise in GFR among patients who discontinued the drugs might have masked AKI in the studies included here, given that AKI was deﬁned as a change in serum creatinine from a baseline measurement that was taken prior to drug withdrawal. DISCUSSION Recognising the potential for physiological rather than pathological changes in kidney function,22 future studies will beneﬁt from examining later clinical outcomes including incom- plete recovery from AKI (ie, failure of serum creatinine concentration to return to baseline), chronic kidney disease and all-cause mortality. have used the ROBINS-I tool14 and the new Cochrane tool for randomised trials.13 The majority of studies were small and there were some concerns regarding risk of bias in some studies, especially one of the non- randomised studies which was judged at critical risk of bias. Publication bias was not formally assessed in this review because the number of studies was too small for such an assessment to be meaningful. However, our search strategy included a variety of routes to identify unpublished studies and resulted in the inclusion of one conference abstract. Despite this, we consider the likeli- hood of publication bias in this area to be high. Importantly, there are no studies which evaluate the beneﬁts of stopping medication in the community fol- lowing acute infection, and no studies that assessed dis- continuation of diuretics that could exacerbate AKI, or metformin and/or sulfonylureas, which may accumulate during an episode of AKI. Only one study assessed To further quantify the limitations of the studies, we conducted a formal risk of bias assessment using the most recently developed tools. DISCUSSION This is the ﬁrst review to Table 3 Summary of outcomes evaluated in single studies Outcome Study Effect size (95% CI) Urea (24 hours) Wolak et al16 MD=2.17 (−5.22 to 9.56) Diastolic blood pressure (48 hours) Wolak et al16 MD=0.30 (−5.01 to 5.61) Systolic blood pressure (48 hours) Wolak et al16 MD=−2.10 (−12.98 to 8.78) Hypertensive treatment Wolak et al16 RR=0.17 (0.01 to 3.69) Death Bainey et al18 RR=3.15 (0.13 to 78.17) Myocardial infarction Bainey et al18 No events Stroke Bainey et al18 RR=3.15 (0.13 to 78.17) Congestive heart failure Bainey et al18 No events Rehospitalisation Bainey et al18 RR=7.49 (0.38 to 146.89) Interleukin 18 (IL 18) (≥120 ng/mL) Coca et al19 0.89 (0.65 to 1.23) Kidney injury molecule 1 (KIM 1) (≥1.15 ng/mL) Coca et al19 1.09 (0.82 to 1.44)* Liver fatty acid binding protein (L-FABP) (≥170 ng/mL) Coca et al19 0.97 (0.73 to 1.3)* Neutrophilgelatinase-associated lipocalin (NGAL) (≥120 ng/mL) Coca et al19 0.84 (0.60 to 1.16)* Adjusted for sex, age, white, CKD-EPI eGFR, diabetes, hypertension, congestive heart failure, myocardial infarction, cardiac cauterisation in past 48 hours, electic surgery and type of surgery (CABG, valve, both). MD, mean difference; RR, relative risk. Table 3 Summary of outcomes evaluated in single studies Outcome Table 3 Summary of outcomes evaluated in single studies Outcome Study Effect size (95% CI) Urea (24 hours) Wolak et al16 MD=2.17 (−5.22 to 9.56) Diastolic blood pressure (48 hours) Wolak et al16 MD=0.30 (−5.01 to 5.61) Systolic blood pressure (48 hours) Wolak et al16 MD=−2.10 (−12.98 to 8.78) Hypertensive treatment Wolak et al16 RR=0.17 (0.01 to 3.69) Death Bainey et al18 RR=3.15 (0.13 to 78.17) Myocardial infarction Bainey et al18 No events Stroke Bainey et al18 RR=3.15 (0.13 to 78.17) Congestive heart failure Bainey et al18 No events Rehospitalisation Bainey et al18 RR=7.49 (0.38 to 146.89) Interleukin 18 (IL 18) (≥120 ng/mL) Coca et al19 0.89 (0.65 to 1.23) Kidney injury molecule 1 (KIM 1) (≥1.15 ng/mL) Coca et al19 1.09 (0.82 to 1.44)* Liver fatty acid binding protein (L-FABP) (≥170 ng/mL) Coca et al19 0.97 (0.73 to 1.3)* Neutrophilgelatinase-associated lipocalin (NGAL) (≥120 ng/mL) Coca et al19 0.84 (0.60 to 1.16)* *Adjusted for sex, age, white, CKD-EPI eGFR, diabetes, hypertension, congestive heart failure, myocardial infarction, cardiac cauterisation in past 48 hours, electic surgery and type of surgery (CABG, valve, both). MD, mean difference; RR, relative risk. 7 Whiting P, et al. BMJ Open 2017;7:e012674. Open Access Subsequent evidence regarding the safety of community medication cessation interventions has come from an ongoing evaluation of hospital admissions following introduction of the NHS Scotland scheme, which has shown a stabilisation or fall in hospital admissions with AKI.24 However, a concurrent fall in heart failure admis- sions (which might have been expected to increase as a consequence of discontinuation of ACEIs or ARBs among patients previously stabilised on these drugs for treatment of heart failure) suggest a secular trend in hospital admissions unrelated to the introduction of the intervention, and interpretation is also limited by the absence of a control population. There remains ongoing disagreement about how the general evidence base should be interpreted to consider the balance of risks and beneﬁts of drug-cessation interventions, particularly during acute illness.8 discontinuation of NSAIDs and only a very small number of patients discontinued these drugs in this study and so it was not possible to draw conclusions regarding the effects of discontinuing NSAIDs. At present, a number of national organisations provide guidance about medication cessation, as well as many regional schemes and guidelines. The UK NICE guidance published in 2013 recommends consideration of temporarily stopping ACEI and ARBs in adults having iodinated contrast agents if they have chronic kidney disease with an eGFR <40 mL/min/1.73 m2, and in adults, children and young people with diarrhoea, vomit- ing or sepsis.6 In 2015, NHS Scotland and the Scottish Patient Safety Programme initiated a more wide ranging medication cessation intervention. Predominantly via community pharmacists, patients are issued with Sick-Day Rules cards, advising them to stop taking ACEIs/ARBs, NSAIDS, diuretics and metformin when they become unwell with vomiting or diarrhoea, and/or fevers sweats and shaking.24 Under similar circum- stances, guidance from the Canadian Diabetes Association Clinical Practice Guidelines for Chronic Kidney Disease in Diabetes recommends physicians and patients to withhold ACEIs, ARBs, NSAIDs, diuretics, metformin, direct renin inhibitors and sulfonylureas.25 This guidance is based on the commonly held belief that there is an association between the use of ACEI/ ARBs, diuretics and NSAIDs and the development of AKI, particularly during illness or other physiological insult. Open Access The potentially strongest source of evidence, the incidence of AKI in RCTs of ACEIs and ARBs compared with placebo is poorly described due to variable deﬁni- tions or absent reporting of kidney-related adverse events.26 A number of observational studies have demon- strated a higher risk of AKI among patients among ACEI/ARB users also taking diuretics and/or NSAIDs compared with those taking ACEIs/ARBs alone,27–29 or with ACEI/ARB users compared with non-users during acute illness or after surgery.30 31 As with all observa- tional evidence, these studies carry an inherent risk of associations being due to bias and confounding, particu- larly confounding by indication, in which patients at higher risk of AKI are more likely to be treated with the drugs of interest, making a direct causal effect uncertain g This systematic review includes ﬁve additional studies published since the NICE guidance on AKI. Our results show low-quality evidence that withdrawal of ACEI/ARBs and NSAIDs prior to coronary angiography and cardiac surgery may reduce the incidence of AKI. However, the quality, power and limited scope of these studies reduce the emphasis that can be placed on this ﬁnding and have not substantially clariﬁed the evidence base. There is no published evidence of the impact of drug cessation interventions on AKI incidence during intercurrent illness in primary or secondary care, of other included medications (NSAIDs, diuretics, sulfonylureas, metfor- min) or of combinations of medications. We also found no evidence of ongoing studies of interventions on any of these topics. The current widespread promotion of ‘sick-day guid- ance’ incurs ﬁnancial and opportunity costs. While the public health impact of sick-day guidance can be evalu- ated through the novel data ﬂows recently established by NHS England and the UK Renal Registry,3more formal controlled evaluation in the form of stepped wedge or cluster randomised trials could be applied to ensure we achieve maximal overall public health beneﬁt. DISCUSSION doi:10.1136/bmjopen-2016-012674 Whiting P, et al. BMJ Open 2017;7:e012674. doi:10.1136/bmjopen-2016-012674 group.bmj.com on April 11, 2017 - Published by http://bmjopen.bmj.com/ Downloaded from group.bmj.com on April 11, 2017 - Published by http://bmjopen.bmj.com/ Downloaded from Open Access Open Access 1The National Institute for Health Research Collaboration for Leadership in Applied Health Research and Care West (NIHR CLAHRC West) at University Hospitals Bristol NHS Foundation Trust, Bristol, UK 2School of Social and Community Medicine, University of Bristol, Bristol, UK 3UK Renal Registry, Bristol, UK 4Department of Non-communicable Disease Epidemiology, London School of Hygiene and Tropical Medicine, London, UK 5Centre for Primary Care, Institute of Population Health, The University of Manchester, Manchester, UK 6National Institute for Health Research Collaboration for Leadership in Applied Health Research and Care (NIHR CLAHRC) Greater Manchester, Centre for Primary Care, Institute of Population Health, University of Manchester, Manchester, UK 7D t t f R l M di i F H it l N tl U T Author affiliations 1 All authors contributed to the interpretation of results, commented on draft manuscripts and have given their approval for publication. 11. Moher D, Liberati A, Tetzlaff J, et al. Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement. Ann Intern Med 2009;151:264–9. w64. ; 12. Morden A, Horwood J, Whiting P, et al. The risks and benefits of patients temporarily discontinuing medications in the event of an intercurrent illness: a systematic review protocol. Syst Rev 2015;4:139. y p y 13. Savovic J, Higgins J, Sterne J, et al. Introducing a revised risk of bias tool for randomized trials. Cochrane Colloquium, 2015. http:// www.cochranelibrary.com/dotAsset/5bc8d6fd-0604-4bed-beb2- 8bc7a74aa4b9.pdf p 14. Sterne Jonathan AC, Hernán Miguel A, Reeves Barnaby C, et al. ROBINS-I: a tool for assessing risk of bias in non-randomised studies of interventions. BMJ 2016;355:4919. Funding This research is supported by the National Institute for Health Research (NIHR) Collaboration for Leadership in Applied Health Research and Care (CLAHRC) West at University Hospitals Bristol NHS Foundation Trust. TB was partly funded by the NIHR CLAHRC Greater Manchester. LAT is funded by a Wellcome Trust intermediate clinical fellowship (101143/Z/13/Z). 15. Guyatt GH, Oxman AD, Vist GE, et al. GRADE: an emerging consensus on rating quality of evidence and strength of recommendations. BMJ 2008;336:924–6. 16. Wolak T, Aliev E, Rogachev B, et al. Renal safety and angiotensin II blockade medications in patients undergoing non-emergent coronary angiography: a randomized controlled study. Isr Med Assoc J 2013;15:682–7. Disclaimer The funders had no role in the design of the study, data collection and analysis, decision to publish or preparation of the manuscript. However, the project outlined in this article may be considered to be affiliated to the work of the NIHR CLAHRC Greater Manchester and NIHR CLAHR West. The views expressed in this article are those of the authors and not necessarily those of the NHS, NIHR or the Department of Health. 17. Rosenstock JL, Bruno R, Kim JK, et al. The effect of withdrawal of ACE inhibitors or angiotensin receptor blockers prior to coronary angiography on the incidence of contrast-induced nephropathy. Int Urol Nephrol 2008;40:749–55. p ; 18. Bainey KR, Rahim S, Etherington K, et al. Effects of withdrawing vs continuing renin-angiotensin blockers on incidence of acute kidney injury in patients with renal insufficiency undergoing cardiac catheterization: results from the Angiotensin Converting Enzyme Inhibitor/Angiotensin Receptor Blocker and Contrast Induced Nephropathy in Patients Receiving Cardiac Catheterization (CAPTAIN) trial. Author affiliations 1 1The National Institute for Health Research Collaboration for Leadership in Applied Health Research and Care West (NIHR CLAHRC West) at University Hospitals Bristol NHS Foundation Trust, Bristol, UK 2School of Social and Community Medicine, University of Bristol, Bristol, UK 3UK Renal Registry, Bristol, UK 4Department of Non-communicable Disease Epidemiology, London School of Hygiene and Tropical Medicine, London, UK 5Centre for Primary Care, Institute of Population Health, The University of Manchester, Manchester, UK 6National Institute for Health Research Collaboration for Leadership in Applied Health Research and Care (NIHR CLAHRC) Greater Manchester, Centre for Primary Care, Institute of Population Health, University of Manchester, Manchester, UK 7Department of Renal Medicine Freeman Hospital Newcastle Upon Tyne Only one of the studies17 considered in this review was available at the time of development of the NICE guid- ance for AKI.6 The guideline development group discuss explicitly the difﬁculty of issuing guidance regarding medication cessation (for ACEIs/ARBs only), despite limited evidence.6 They felt that the available evidence for discontinuation was weak but that the ‘con- tinuing use of ACEIs/ARBs [during acute illness or exposure to iodinated contrast agents] is clearly asso- ciated with AKI. In contrast, the temporary suspension of ACEIs/ARBs for a short period seems unlikely to greatly increase the risk of cardiovascular events’. 7Department of Renal Medicine, Freeman Hospital, Newcastle Upon Tyne Hospitals Foundation Trust, Tyne and Wear, UK 7Department of Renal Medicine, Freeman Hospital, Newcastle Upon Tyne Hospitals Foundation Trust, Tyne and Wear, UK Acknowledgements The authors would like to thank Dr Tim Jones, NIHR CLAHRC West, for help with assessing the risk of bias in the included studies. Acknowledgements The authors would like to thank Dr Tim Jones, NIHR CLAHRC West, for help with assessing the risk of bias in the included studies. 8 Whiting P, et al. BMJ Open 2017;7:e012674. doi:10.1136/bmjopen-2016-012674 group.bmj.com on April 11, 2017 - Published by http://bmjopen.bmj.com/ Downloaded from Open Access Open Access Contributors CT, TB and FC conceived the idea for the review. PW, AM, JH and LAT drafted the article with the support of FC. AR developed the search strategy. FC, LAT, TB and CT served as content experts in the field of AKI. JH served as the overall supervisor and provided input on study methodology. JS provided methodological support. AM, PW and TS undertook screening and data extraction. PW and FC performed the risk of bias assessment. PW and JS performed the GRADE assessment. REFERENCES 1. Chertow GM, Burdick E, Honour M, et al. Acute kidney injury, mortality, length of stay, and costs in hospitalized patients. J Am Soc Nephrol 2005;16:3365–70. y g g p y ; 22. Perazella MA, Coca SG. Three feasible strategies to minimize kidney injury in ‘incipient AKI’. Nat Rev Nephrol 2013;9:484–90. y j y p p 23. Bakris GL, Weir MR. Angiotensin-converting enzyme inhibitor-associated elevations in serum creatinine: is this a cause for concern? Arch Intern Med 2000;160:685–93. p 2. National Confidential Enquiry into Patient Outcome and Death. Acute Kidney Injury: Adding Insult to Injury. 2009. http://www. ncepod.org.uk/2009aki.html 24. Morrison C, Wilson M. Medicine sick day rules cards: a safe and effective tool to improve medicines safety in NHS Highland. Int J Pharm Pract 2015;23:92–3. 3. Think Kidneys. Acute Kidney Injury: the NHS campaign to improve the care of people at risk of, or with, acute kidney injury. 2016. (11 April 2016). https://www.thinkkidneys.nhs.uk/aki/ 25. Canadian Diabetes Association. Sick Day Medication List. 2015. http://guidelines.diabetes.ca/browse/appendices/appendix7_2015 4. Pannu N, Nadim MK. An overview of drug-induced acute kidney injury. Crit Care Med 2008;36(Suppl 4):S216–23. p g pp pp 26. Palmer SC, Mavridis D, Navarese E, et al. Comparative efficacy and safety of blood pressure-lowering agents in adults with diabetes and kidney disease: a network meta-analysis. Lancet 2015;385:2047–56. 5. Feehally J, Gilmore I, Barasi S, et al. RCPE UK consensus conference statement: management of acute kidney injury: the role of fluids, e-alerts and biomarkers. J R Coll Physicians Edinb 2013;43:37–8. y y 27. Dreischulte T, Morales DR, Bell S, et al. Combined use of nonsteroidal anti-inflammatory drugs with diuretics and/or renin-angiotensin system inhibitors in the community increases the risk of acute kidney injury. Kidney Int 2015;88:396–403. 6. National Institute for Health and Clinical Excellence. Acute kidney injury: prevention, detection and management. NICE guideline (CG169), 2013. 6. National Institute for Health and Clinical Excellence. Acute kidney injury: prevention, detection and management. NICE guideline (CG169), 2013. y j y y 28. Lapi F, Azoulay L, Yin H, et al. Concurrent use of diuretics, angiotensin converting enzyme inhibitors, and angiotensin receptor blockers with non-steroidal anti-inflammatory drugs and risk of acute kidney injury: nested case-control study. BMJ 2013;346:e8525. 7. Scottish Patient Safety Programme. Medicine Sick Day Rules Card. 2015. http://www.scottishpatientsafetyprogramme.scot.nhs.uk/ programmes/primary-care/medicine-sick-day-rules-card 8. Griffith K Ashley C, Blakeman T, et al. Author affiliations 1 Am Heart J 2015;170:110–16. Competing interests None declared. Competing interests None declared. Provenance and peer review Not commissioned; externally peer reviewed. Provenance and peer review Not commissioned; externally peer reviewed. Data sharing statement No additional data are available. Data sharing statement No additional data are available. Open Access This is an Open Access article distributed in accordance with the terms of the Creative Commons Attribution (CC BY 4.0) license, which permits others to distribute, remix, adapt and build upon this work, for commercial use, provided the original work is properly cited. See: http:// creativecommons.org/licenses/by/4.0/ ( ) 19. Coca SG, Garg AX, Swaminathan M, et al. Preoperative angiotensin-converting enzyme inhibitors and angiotensin receptor blocker use and acute kidney injury in patients undergoing cardiac surgery. Nephrol Dial Transplant 2013;28:2787–99. g y p p ; 20. Weisbord SD, Mor MK, Resnick AL, et al. Prevention, incidence, and outcomes of contrast-induced acute kidney injury. Arch Intern Med 2008;168:1325–32. 21. Goksuluk H, Kerimli N, Atmaca Y, et al. Effects of renin-angiotensin- aldosterone system blockers on contrast-induced nephropathy and its association with NGAL levels in diabetic patients undergoing coronary angiography. Eur Heart J 2015;36:642. REFERENCES ‘Sick day rules’ in patients at risk of Acute Kidney Injury: an Interim Position Statement from the Think Kidneys Board. 2015. http://www.thinkkidneys.nhs.uk/ wp-content/uploads/2015/07/Think-Kidneys-Sick-Day-Rules- 160715.pdf y j y y 29. Loboz KK, Shenfield GM. Drug combinations and impaired renal function—the ‘triple whammy’. Br J Clin Pharmacol 2005;59:239–43. p y 30. Arora P, Rajagopalam S, Ranjan R, et al. Preoperative use of angiotensin-converting enzyme inhibitors/angiotensin receptor blockers is associated with increased risk for acute kidney injury after cardiovascular surgery. Clin J Am Soc Nephrol 2008;3:1266–73. 9. Centre for Reviews and Dissemination. Systematic reviews: CRD’s guidance for undertaking reviews in health care [Internet]. 2009. http://www.york.ac.uk/inst/crd/SysRev/!SSL!/WebHelp/SysRev3.htm g y p 31. Plataki M, Kashani K, Cabello-Garza J, et al. Predictors of acute kidney injury in septic shock patients: an observational cohort study. Clin J Am Soc Nephrol 2011;6:1744–51. 10. Higgins JPT, Green S. Cochrane handbook for systematic reviews of interventions [Internet]. 2011. http://www.cochrane-handbook.org/ Whiting P, et al. BMJ Open 2017;7:e012674. doi:10.1136/bmjopen-2016-012674 9 group.bmj.com on April 11, 2017 - Published by http://bmjopen.bmj.com/ Downloaded from meta-analysis kidney injury? A systematic review and discontinuing medications to prevent acute What are the risks and benefits of temporarily Jelena Savovic and Jeremy Horwood Thomas Blakeman, Charles Tomson, Tracey Stone, Alison Richards, Penny Whiting, Andrew Morden, Laurie A Tomlinson, Fergus Caskey, doi: 10.1136/bmjopen-2016-012674 2017 7: BMJ Open doi: 10.1136/bmjopen-2016-012674 2017 7: BMJ Open doi: 10.1136/bmjopen-2016-012674 2017 7: BMJ Open http://bmjopen.bmj.com/content/7/4/e012674 Updated information and services can be found at: These include: These include: #BIBL http://bmjopen.bmj.com/content/7/4/e012674 This article cites 22 articles, 6 of which you can access for free at: http://creativecommons.org/licenses/by/4.0/ use, provided the original work is properly cited. See: others to distribute, remix, adapt and build upon this work, for commercial the Creative Commons Attribution (CC BY 4.0) license, which permits This is an Open Access article distributed in accordance with the terms of box at the top right corner of the online article. Receive free email alerts when new articles cite this article. Sign up in the Collections Topic Articles on similar topics can be found in the following collections (131) Renal medicine (690) Evidence based practice http://group.bmj.com/group/rights-licensing/permissions To request permissions go to: http://group.bmj.com/group/rights-licensing/permissions To request permissions go to: http://journals.bmj.com/cgi/reprintform To order reprints go to: http://group.bmj.com/subscribe/ To subscribe to BMJ go to:
https://openalex.org/W4390467186	https://journal.lontaradigitech.com/VOICE/article/download/110/119	Indonesian	null	Analisis Tingkat Berpikir Mahasiswa Terkait Literasi Komputer dalam Konteks Pendidikan	Journal of Vocational, Informatics and Computer Education	2,023	cc-by-sa	5,303	ABSTRACT Tujuan penelitian ini ialah pemanfaatan literasi yang dapat dijadikan menjadi kurikulum sekolah,menganalisis tingkat berpikir siswa terkait literasi komputer bagaimana dampak pada kemapuan berpikir siswa dan mengukur dampak literasi komputer..Pada penelitian sebelumnya dengan pembagian kuesioner,Didapati berbagai macam data tingkat kemampuan literasi komputer, literasi digital dan literasi finansial. Hasil penelitian ini menggunakan hasil pernyataan responden (kuesioner) yang dimasukkan aplikasi microsoft Excel.Data ini diambil dari mahasiswa Universita Negeri Makassar dengan 72 responden sebagai sampel dalam observasi menggunakan angket kuesioner. Hasil Data yang telah di hitung menyatakan bahwa penggunaan media umum dapat menaikkan literasi digital siswa.Media umum bisa sebagai asal belajar yang efektif.Peserta didik merasa mempunyai pemahaman yang relatif perihal etika dan integrasi komputer dalam pendidikan.Upaya pendidikan berhasil dalam memberikan pencerahan perihal nilai etika.Internet sudah memudahkan peserta didik dalam mengakses informasi.Literasi komputer memiliki dampak positif terhadap pemahaman Mahasiswa terhadap materi pembelajaran.Literasi personal komputer memainkan peran krusial dalam menaikkan kemampuan Mahasiswa dalam memahami serta mengaplikasikan materi pembelajaran. This is an open access article under the CC BY-SA license Diterima 17 November 2023; Disetujui 28 Desember 2023 Tersedia secara daring 31 Desember 2023 Dipublikasikan oleh Lontara Digitech Indonesia Analisis Tingkat Berpikir Mahasiswa Terkait Literasi Komputer dalam Konteks Pendidikan 1IPOSS Jakarta, 2Universitas Binawan, 3,4,5Universitas Negeri Makassar 1IPOSS Jakarta, 2Universitas Binawan, 3,4,5Universitas Negeri Makassar ARTICLE INFO Kata kunci: Literasi Komputer, Teknologi Pendidikan j Tersedia secara daring 31 Desember 2023 Dipublikasikan oleh Lontara Digitech Indonesia Journal of Vocational, Informatics and Computer Education Vol.1 No. 2 December (2023) Journal of Vocational, Informatics and Computer Education Vol.1 No. 2 December (2023) Fundamental and Applied Management Journal E-ISSN: XXXX-YYYY; P-ISSN: XXXX-YYYY Journal Homepage: http://journal.lontaradigitech.com/FAMJ Journal of Vocational, Informatics and Computer Education E-ISSN: 2988-6325; P-ISSN: 2988-4918 DOI : https://doi.org/10.61220/voice Journal Homepage: http://journal.lontaradigitech.com/VOICE 1. PENDAHULUAN Kemajuan pengetahuan dan teknologi digital sudah mempunyai akibat luas pada global pendidikan. pada zaman teknologi saat ini, teknologi digital telah merubah cara kita menuntut ilmu serta pengajaran [12].Pada satu studi yang dilakukan oleh Geertshuis serta Liu menganalisis tantangan yang dihadapi sang staf akademik ketika mengadopsi sistem manajemen pembelajaran (LMS).[17] Mereka menemukan bahwa tantangan-tantangan tersebut terkait menggunakan identitas profesional, proses pengambilan keputusan, serta beragamnya ciri-ciri profesional guru. Dukungan kelembagaan buat adopsi teknologi tidak sepenuhnya menjawab tantangan-tantangan ini. 102 Journal of Vocational, Informatics and Computer Education E-ISSN: 2988-6325 P-ISSN: 2988-4918 Banyak cara untuk mengatasi tantangan-tantangan yang dihadapi oleh propesional guru .Membahas model konseptual buat integrasi teknologi pada Pendidikan merupakan salah satu model solusi untuk mengatasi tantangan yang ada.Model-contoh ini dapat menjembatani teori dan software mudah, memberikan panduan untuk menggunakan teknologi secara efektif pada pendidikan[18].Dalam konteks pendidikan suku Indian Amerika, Adcock menyoroti upaya waktu ini untuk memakai teknologi pendidikan dengan siswa K-12[7]. Studi ini berpendapat bahwa literatur yang ada tentang kesetaraan digital memarjinalkan siswa Pribumi dan menyerukan lebih poly penelitian perihal bagaimana teknologi bisa melayani serta mendukung peserta didik Pribumi[7].Dalam beberapa dekade terakhir, ada sejumlah tren primer yang sudah mensugesti pendidikan.keliru satu perkembangan terbesar adalah penggunaan internet. penting bagi kita semua buat selalu mengikuti perkembangan teknologi. menggunakan adanya perkembangan tadi akan sangat mensugesti dunia pendidikan yaitu, akan meningkatkan lulusan berkualitas secara terus menerus. Selain itu, teknologi sudah mengganti cara pelajar dan mengajar. Penggunaan software pembelajaran, visualisasi, serta alat-alat interaktif telah membentuk pembelajaran lebih menarik dan efektif. Hal ini dapat memakai teknologi buat mempersonalisasi pengalaman belajar bagi setiap peserta didik. Literasi digital diartikan sebagai usaha belajar dengan teknologi,bisa dikatakan dua hal ini di gabungkan antara belajar dengan teknologi. Nyatanya dikatakan literasi digital sebelumnya susah dilakukan sebab minesnya keinginan pendidik untuk menggunakan digital dijakur mengajarnya dan kurangnya pemahaman dengan peenggunaan teknologi digital, literasi digital zaman ini dipergunakan dengan syarat pengajaran pada tempat menuntut ilmu mulai dipindahkan sebagai pembelajaran dengan tempat yang berbeda-beda [20]. Kemristekdikti (2018) mengatakan zaman 4.0 akan mendisrupsi macam-macam pekerjaan,begitu pula dengan bidang iptek (ilmu pengetahuan dan teknologi) dan pembelajaran nanti[27]. Diera revolusi keempat atau 4.0 yang dikenal menggunakan revolusi digital, seluruh informasi bisa diperoleh menggunakan real-time serta cepat dimana saja dan kapan saja.[23] Berdasarkan [6] Pengembangan bidang teknologi juga akan membuat ancaman baru pada era industry 4.0, keliru diantaranya merupakan pembelajaran. Berdasarkan[5]. 1. PENDAHULUAN Pada artikel ini, tersaji beberapa temuan utama terkait literasi digital yang memegang peranan krusial pada pendidikan,khususnya dalam pemanfaatan teknologi pada pembelajaran. Penggunaan kecerdasan sintesis serta teknologi lainnya juga bisa menaikkan pengalaman belajar[28].Guru bisa mengoptimalkan potensi literasi digital siswa dengan menggunakan software atau media yg dapat mengotomatisasi tugas-tugas seperti memberikan feedback, menentukan materi pembelajaran yang sinkron, dan menyelaraskan kurikulum menggunakan kebutuhan peserta didik.Selain itu, peserta didik perlu mempunyai keterampilan literasi digital yang krusial seperti memahami konteks dunia digital, mengevaluasi konten, bertanggung jawab, kreatif, kritis, dan bertanggung jawab secara sosial. Pada artikel ini, tersaji beberapa temuan utama terkait literasi digital yang memegang peranan krusial pada pendidikan,khususnya dalam pemanfaatan teknologi pada pembelajaran. Penggunaan kecerdasan sintesis serta teknologi lainnya juga bisa menaikkan pengalaman belajar[28].Guru bisa mengoptimalkan potensi literasi digital siswa dengan menggunakan software atau media yg dapat mengotomatisasi tugas-tugas seperti memberikan feedback, menentukan materi pembelajaran yang sinkron, dan menyelaraskan kurikulum menggunakan kebutuhan peserta didik.Selain itu, peserta didik perlu mempunyai keterampilan literasi digital yang krusial seperti memahami konteks dunia digital, mengevaluasi konten, bertanggung jawab, kreatif, kritis, dan bertanggung jawab secara sosial. Meskipun penelitian sebelumnya sudah memberikan pemahaman yg baik perihal literasi digital, masih terdapat beberapa pertanyaan yang belum terjawab. misalnya, apa hubungan literasi komputer dengan berpikir siswa pada konteks pendidikan? Apa praktik terbaik buat mengintegrasikan literasi personal komputer ke dalam kurikulum buat menaikkan keterampilan berpikir peserta didik? Pertanyaan pertanyaan ini akan fokus penelitian ini serta bisa memberikan wawasan baru dalam pengembangan literasi digital. Tujuan penelitian ini ialah pemanfaatan literasi yang dapat dijadikan menjadi kurikulum sekolah,menganalisis tingkat berpikir siswa terkait literasi komputer bagaimana dampak pada kemapuan berpikir siswa dan mengukur dampak literasi komputer. 1. PENDAHULUAN Kesimpulan penelitian menyatakan terampil berpikir keras dan daya literasi sains siswa masih rendah.[24].Keterampilan tersebut adalah keliru satu hal yang berujuk pada berkembangnya teknologi komunikasi. di era 4.0 pula era yang saat ini kita rasakan.Dalam kehidupan insan akan selalu berkaitan dengan dunia teknologi serta berita.[28] Semua pendidikan wajib manfaatkan rintangan dan kesempatan yang terdapat dizaman 4.0, menggunakan memulai dari adaptasi asal segala perubahan yang ada serta bersiap buat menghadapi murid zaman sekarang pada dasarnya pengajaran, terampilan teknologi, literasi awal, literasi perasaan, pendidikann menjadi lebih bagus serta hayati. Selama nyayris 20 tahun sebelumnya semenjak diterbitkan dari PISA, literasi sains Indonesia tidak ada kelebihan yang tinggi. Nilai literasi sains siswa berada rata-rata 393 tahun 2000 hingga 396 tahun 2018. Nilai tersebut sangat jauh pada bawah nilai rata-rata Negara anggota OECD yakni 489. Terdapat banyak faktor sebab rendahnya kemampuan literasi sains siswa Indonesia yang dijelaskan sang peneliti terkait pada hasil PISA Indonesia.antara lain a). Memilih buku pengajaran, b). Miskonsepsi, c). Pengajaran tidak tertata, d). Banyak yang tidak mampu membaca, serta e). Wilayah yang tidak mememungkinkan.[18] Penelitian [2] sebelumnya menunjukkan bahwa telah terlaksana namun belum sepenuhnya. Hal ini didebabkan hambatan yang dihadapi yaitu kompetensi digital pengajar yang 103 Journal of Vocational, Informatics and Computer Education E-ISSN: 2988-6325 P-ISSN: 2988-4918 terbatas,pasilitas yang kurang, dan peserta didik yang belum paham betul tentang literasi digital. Perkembangan literasi akan sebagai krusial karena literasi artinya kemampuan awal yang harus dimiliki bagi setiap individu buat menjalankan kehidupan dimasa depan. Pembelajaran literasi jua akan menerima akibat yg optimal Bila diberikan sejak usia dini sebagai akibatnya dianggap literasi awal. Di usia tersebut akan terjadi masa golden age, dimana masa tadi artinya masa keemasan seorang anak yang akan mengalami suatu perkembangan yg sangat pesat. waktu ini, kemampuan masyarakat dalam tahu isu di ranah digital yg berkembang dalam jaringan internet sudah semakin maju. Mereka telah mulai bisa menyaring mana saja yang layak buat dikonsumsi serta apa saja yang kemudian dikategorikan menjadi informasi negatif. Memikirkan hal serius dimulai ketika orang mengeluarkan keputusan ilmu yang lebih bisa digunakan,banyak sekali pengetahuan yang terdapat pada kepala buat memfiniskan persoalan [11]. Pada keadaan ini,pasti setiap orang mampu menyelesaikan dengan jalan yang berbeda. Ada perbedaan kekuatan di pemaparan konsep setiap orang akan terpengaruh pada menuntaskan dilema [13]. Pada artikel ini, tersaji beberapa temuan utama terkait literasi digital yang memegang peranan krusial pada pendidikan,khususnya dalam pemanfaatan teknologi pada pembelajaran. 1. PENDAHULUAN Penggunaan kecerdasan sintesis serta teknologi lainnya juga bisa menaikkan pengalaman belajar[28].Guru bisa mengoptimalkan potensi literasi digital siswa dengan menggunakan software atau media yg dapat mengotomatisasi tugas-tugas seperti memberikan feedback, menentukan materi pembelajaran yang sinkron, dan menyelaraskan kurikulum menggunakan kebutuhan peserta didik.Selain itu, peserta didik perlu mempunyai keterampilan literasi digital yang krusial seperti memahami konteks dunia digital, mengevaluasi konten, bertanggung jawab, kreatif, kritis, dan bertanggung jawab secara sosial. terbatas,pasilitas yang kurang, dan peserta didik yang belum paham betul tentang literasi digital. Perkembangan literasi akan sebagai krusial karena literasi artinya kemampuan awal yang harus dimiliki bagi setiap individu buat menjalankan kehidupan dimasa depan. Pembelajaran literasi jua akan menerima akibat yg optimal Bila diberikan sejak usia dini sebagai akibatnya dianggap literasi awal. Di usia tersebut akan terjadi masa golden age, dimana masa tadi artinya masa keemasan seorang anak yang akan mengalami suatu perkembangan yg sangat pesat. waktu ini, kemampuan masyarakat dalam tahu isu di ranah digital yg berkembang dalam jaringan internet sudah semakin maju. Mereka telah mulai bisa menyaring mana saja yang layak buat dikonsumsi serta apa saja yang kemudian dikategorikan menjadi informasi negatif. Memikirkan hal serius dimulai ketika orang mengeluarkan keputusan ilmu yang lebih bisa digunakan,banyak sekali pengetahuan yang terdapat pada kepala buat memfiniskan persoalan [11]. Pada keadaan ini,pasti setiap orang mampu menyelesaikan dengan jalan yang berbeda. Ada perbedaan kekuatan di pemaparan konsep setiap orang akan terpengaruh pada menuntaskan dilema [13]. terbatas,pasilitas yang kurang, dan peserta didik yang belum paham betul tentang literasi digital. Perkembangan literasi akan sebagai krusial karena literasi artinya kemampuan awal yang harus dimiliki bagi setiap individu buat menjalankan kehidupan dimasa depan. Pembelajaran literasi jua akan menerima akibat yg optimal Bila diberikan sejak usia dini sebagai akibatnya dianggap literasi awal. Di usia tersebut akan terjadi masa golden age, dimana masa tadi artinya masa keemasan seorang anak yang akan mengalami suatu perkembangan yg sangat pesat. waktu ini, kemampuan masyarakat dalam tahu isu di ranah digital yg berkembang dalam jaringan internet sudah semakin maju. Mereka telah mulai bisa menyaring mana saja yang layak buat dikonsumsi serta apa saja yang kemudian dikategorikan menjadi informasi negatif. Memikirkan hal serius dimulai ketika orang mengeluarkan keputusan ilmu yang lebih bisa digunakan,banyak sekali pengetahuan yang terdapat pada kepala buat memfiniskan persoalan [11]. Pada keadaan ini,pasti setiap orang mampu menyelesaikan dengan jalan yang berbeda. Ada perbedaan kekuatan di pemaparan konsep setiap orang akan terpengaruh pada menuntaskan dilema [13]. METODE PENELITIAN Penemuan ini memakai observasi dengan deskriptif kuantitatif yang dijalankan pada Universitas Negeri Makassar[12]. Saat mengumpulkan data dengan memakai teknik mengumpulkan data kuesioner. kuesioner ialah cara mengumpulkan data yang menggabungkan pengajuan berbagai macam pertanyaan atau pernyataan yang ditulisa pada responden. Teknik ini memungkinkan peneliti buat menerima pemahaman mendalam perihal pandangan, pengalaman, serta persepsi responden sedangkan Penelitian kuantitatif menggunakan teknik pengumpulan data mirip angket atau kuesioner,observasi terstruktur, eksperimen, dan studi survei. Teknik ini memungkinkan peneliti buat mengumpulkan data dalam bentuk angka atau statistik yang dapat dianalisis secara kuantitatif[19].Observasi ini memakai skala likert pada proses mengumpulkan datanya. Skala Likert ialah skala yang bisa dipergunakan buat mendeteksi sikap, pendapat, serta dugaan seorang atau banyak orang terhadap suatu kejadian atau fenomena,terkhusus pada bidang peguruan.Bagi[4]skala yang dipakai buat menghitung persepsi,perilaku atau saran 104 Journal of Vocational, Informatics and Computer Education E-ISSN: 2988-6325 P-ISSN: 2988-4918 Journal of Vocational, Informatics and Computer Education E-ISSN: 2988-6325 P-ISSN: 2988-4918 seorang atau orang-orang mengenai sebuah insiden dan kenyataan social[22].Skala Likert dalam temuan ini mencakup 5 alternatif pilihan respon yang wajib dipilih oleh siswa dengan pilihan jawaban Sangat tidak setuju (1), tidak setuju (2), Netral (3), setuju (4), Sangat Setuju (5).[21] Observasi kualitatif bertujuan buat tahu fenomena secara mendalam melalui interpretasi serta analisis deskriptif, sedangkan penelitian kuantitatif bertujuan buat mengukur serta menganalisis data secara statistik[11]. Cara Observasi dari Sugiyono[25] “Cara penelitian adalah penggunaan ilmiah demi dapat data yang di inginkan dan untuk hal yang khusus” ada juga cara penelitian yang dipergunakan di observasi ini dengan cara survei, menurut [26].“Observasi survei adalah peraturan di observasi dimana peneliti mengadministrasikan survei di suatu hal atau semua orang buat menggambarkan sikap,saran, prilaku atau karakteristik khusus populasi” Observasi dengan survey ini memakai cara mengumpulkan data dengan memakai kuesioner serta untuk menganalisis data secara stastistik buat menguji pertanyaan atau hipotesis yang diajukan pada satu observasi. METODE PENELITIAN seorang atau orang-orang mengenai sebuah insiden dan kenyataan social[22].Skala Likert dalam temuan ini mencakup 5 alternatif pilihan respon yang wajib dipilih oleh siswa dengan pilihan jawaban Sangat tidak setuju (1), tidak setuju (2), Netral (3), setuju (4), Sangat Setuju (5).[21] seorang atau orang-orang mengenai sebuah insiden dan kenyataan social[22].Skala Likert dalam temuan ini mencakup 5 alternatif pilihan respon yang wajib dipilih oleh siswa dengan pilihan jawaban Sangat tidak setuju (1), tidak setuju (2), Netral (3), setuju (4), Sangat Setuju (5).[21] Observasi kualitatif bertujuan buat tahu fenomena secara mendalam melalui interpretasi serta analisis deskriptif, sedangkan penelitian kuantitatif bertujuan buat mengukur serta menganalisis data secara statistik[11]. Cara Observasi dari Sugiyono[25] “Cara penelitian adalah penggunaan ilmiah demi dapat data yang di inginkan dan untuk hal yang khusus” ada juga cara penelitian yang dipergunakan di observasi ini dengan cara survei, menurut [26].“Observasi survei adalah peraturan di observasi dimana peneliti mengadministrasikan survei di suatu hal atau semua orang buat menggambarkan sikap,saran, prilaku atau karakteristik khusus populasi” Observasi dengan survey ini memakai cara mengumpulkan data dengan memakai kuesioner serta untuk menganalisis data secara stastistik buat menguji pertanyaan atau hipotesis yang diajukan pada satu observasi. Tabel.1 Rincian spesifik dari kuesioner dan strukturnya Tabel.1 Rincian spesifik dari kuesioner dan strukturnya No Pernyataan Nomor Pernyataan Referensi 1. Saya meresa media sosial membantu saya dalam dalam meningkatkan kemampuan literasi digital saya. 1. [3] saya merasa lebih mudah mendapat informasi setelah menggunakan internet. 2. saya merasa mudah menggunakan program dalam mengelola kat 3. Saya merasa mudah menggunakan program pengelolaan presentasi. 4. Saya Tahu cara mengelola dan menyimpan file di komputer dengan baik. 5. Saya dapat mengidentifikasi dan mengatasi masalah umum yang terjadi pada komputer saya. 6. Saya sangat paham mengenai konsep jaringan komputer,Saya sangat sering menggunakan internet untuk mencari informasi. 7. Saya sangat memiliki kemampuan literasi digital yang cukup. 8. Saya sangat merasa nyaman menggunakan teknologi 9. Saya merasa mudah menemukan informasi yang saya butuhkan di internet 10. saya sangat paham mengenai pengetahuan dasar sistem operasi komputer 11. 2. Saya merasa aplikasi pembelajaran membantu dalam meningkatkan kemampuan literasi digital Saya. 12. Saya sangat sering menggunakan perangkat mobile 13. Saya sering menggunakan program pengolah presentasi. 14. 105 P-ISSN: 2988-4918 Sejauh ini saya merasa literasi komputer mempengaruhi kemampuan saya dalam memahami materi pelajaran. 15. Saya merasa literasi komputer bermanfaat dalam menyelesaikan tugastugas pendidikan Saya. 16. saya merasa literasi komputer memberi keunggulan dalam mencari pekerjaan atau karier saya di masa depan 17. Metode pengumpulan data yang digunakan yaitu metode angket dan metode tes. kuesioner dipergunakan buat mengukur kemampuan literasi sains. kuesioner pengetahuan ilmiah disusun METODE PENELITIAN merasa media sosial membantu dalam meningkatkan kemampuan literasi digital saya. 18. saya sering menggunakan komputer atau perangkat lainnya untuk kuliah, tugas dan keperluan pribadi 19. saya merasa percaya diri dalam menggunakan perangkat lunak komputer 20. [1] 3. Saya merasa literasi komputer saya telah membantu saya dalam menyelesaikan tugas akademik dengan lebih efisien. 21. Saya menggunakan perangkat komputer dalam proyek-proyek kelompok atau kolaboratif dengan teman-teman saya. 22. Saya menggunakan perangkat komputer untuk mengikuti kuliah online atau program e-learning. 23. Saya merasa nyaman dalam menggunakan aplikasi dan perangkat lunak pendidikan seperti Microsoft Word, Excel, atau Google Drive. 24. Saya merasa penggunan perangkat komputer memudah untuk mengakses sumber-sumber pembelajaran terbuka. 25. Saya merasa literasi komputer membantu saya dalam menghadapi perubahan cepat dalam dunia teknologi. 26. Saya sering menggunakan perangkat komputer untuk mengakses kursus atau pelatihan online tambahan. 27. Saya merasa memiliki pemahaman yang cukup tentang nilai etika dan integritas dalam konteks pendidikan. 28. saya merasa memiliki kemampuan berpikir kritis dalam memecahkan masalah yang dihadapi dalam Pendidikan 29. Saya memiliki akses ke perangkat komputer di luar jam sekolah atau kampus untuk keperluan Pendidikan 30. M t d l d t di k it t d k t d t d t k i Metode pengumpulan data yang digunakan yaitu metode angket dan metode tes. kuesioner dipergunakan buat mengukur kemampuan literasi sains. kuesioner pengetahuan ilmiah disusun 106 Journal of Vocational, Informatics and Computer Education E-ISSN: 2988-6325 P-ISSN: 2988-4918 dari indikator-indikator yang kemudian dikembangkan dengan 30 pertanyaan yang meliputi aspek harapan, aspek pengaruh sosial, serta aspek sikap pengguna perangkat lunak disajikan di Tabel 1.[2] dari indikator-indikator yang kemudian dikembangkan dengan 30 pertanyaan yang meliputi aspek harapan, aspek pengaruh sosial, serta aspek sikap pengguna perangkat lunak disajikan di Tabel 1.[2] Pengukuran ini menyampaikan informasi tentang nilai tengah serta variasi data. dalam artikel "Analisis tingkat Berpikir siswa Terkait Literasi komputer dalam Konteks Pendidikan," pendekatan penelitian yang dipakai adalah kuantitatif dengan desain cross-sectional. Responden atau sampel penelitian dipilih melalui penggunaan kuesioner menjadi metode pengumpulan data. Meskipun referensi tidak memberikan detail lengkap tentang isi serta struktur berita umum yang dipakai. Teknik analisis pada penelitian ini ialah analisis deskriptif, yang mencakup penyajian ukuran- berukuran seperti mean (rata-homogen), median (nilai tengah), modus (nilai yang paling seringkali muncul), jumlah total, nilai maksimum, dan nilai minimum pada data yang dikumpulkan. HASIL DAN DISKUSI Observasi dilakukan di bulan oktober – november 2023.Latar belakang dalam penelitian ini ialah tingkat literasi mahasiswa Universitas Negeri Makassar dikota Makassar.Subjek penelitian adalah mahasiswa Universitas Negeri Makassar di kota Makassaer. Dengan tujuan untuk mengetahui tingkat kemampuan literasi komputer dalam konteks pendidikan pada mahasiswa universitas negeri makassar. Dari Observasi yang sudah dibuat dengan cara penyebaran kuesioner, didapat macam-macam data tentang tingkat kemampuan literasi komputer, literasi digital, dan literasi finansial. Hasil penelitian ini menggunakan hasil pernyataan responden (kuesioner) yang dimasukkan aplikasi microsoft Excel. diambil dari mahasiswa Universita Negeri Makassar dengan 72 responden sebagai sampel bservasi menggunakan angket kuesioner. Data ini diambil dari mahasiswa Universita Negeri Makassar dengan 72 responden sebagai sampel dalam observasi menggunakan angket kuesioner. Data ini diambil dari mahasiswa Universita Negeri Makassar dengan 72 responden sebagai sampel dalam observasi menggunakan angket kuesioner. Tabel.Demografi Responden Gender N Percentage (%) Mean age (years) Male 37 51,4% 20 Female 35 48,6% 19.17143 Total 72 Data dibawah merupakan hasil dari observasi dengan mengunakan kuesioner, nilai Mean,Median,Modus,Minimum,Maksimum,Sum didapatkan dengan Skala Likert menggunakan xl. Tabel.Data Deskriptif Aspek/Faktor No Item/Pernyataan/ Pertanyaan Mean Median Modus Minimum Maksimum Sum 1. Saya meresa media sosial membantu saya dalam dalam meningkatkan kemampuan literasi 3.9722 22222 4 4 1 5 286 107 E-ISSN: 2988-6325 P-ISSN: 2988-4918 digital saya. 2. saya merasa lebih mudah mendapat informasi setelah menggunakan internet. 4.1944 44444 4 4 1 5 302 3. saya merasa mudah menggunakan program dalam mengelola kat 3.625 4 4 1 5 261 4. Saya merasa mudah menggunakan program pengelolaan presentasi. 3.7361 11111 11111 4 4 1 5 269 5. Saya Tahu cara mengelola dan menyimpan file di komputer dengan baik. 3.8055 55556 4 4 1 5 274 6. Saya dapat mengidentifikasi dan mengatasi masalah umum yang terjadi pada komputer saya. 3.5694 44444 3 3 2 5 257 7. Saya sangat paham mengenai konsep jaringan komputer,Saya sangat sering menggunakan internet untuk mencari informasi. 3.7222 22222 4 3 1 5 268 8. Saya sangat memiliki kemampuan literasi digital yang cukup. 3.5972 22222 4 3 2 5 259 9. Saya sangat merasa nyaman menggunakan teknologi 3.9027 77778 4 4 1 5 281 10. Saya merasa mudah menemukan informasi yang saya butuhkan di internet 3.9444 44444 4 4 1 5 284 11. saya sangat paham mengenai pengetahuan dasar sistem operasi komputer 3.8472 22222 4 3 2 5 277 12. Saya merasa aplikasi pembelajaran membantu dalam meningkatkan kemampuan literasi digital Saya. HASIL DAN DISKUSI 3.7222 22222 4 4 2 5 268 108 E ISSN: 2988 6325 P-ISSN: 2988-4918 13. Saya sangat sering menggunakan perangkat mobile 3.9305 55556 4 4 1 5 283 14. Saya sering menggunakan program pengolah presentasi. 3.7361 11111 4 4 2 5 269 15. Sejauh ini saya merasa literasi komputer mempengaruhi kemampuan saya dalam memahami materi pelajaran. 3.7361 11111 4 4 2 5 269 16. Saya merasa literasi komputer bermanfaat dalam menyelesaikan tugastugas pendidikan Saya. 3.9027 77778 4 4 2 5 281 17. saya merasa literasi komputer memberi keunggulan dalam mencari pekerjaan atau karier saya di masa depan 3.8888 88889 4 4 2 5 280 18. merasa media sosial membantu dalam meningkatkan kemampuan literasi digital saya. 3.9444 44444 4 4 2 5 184 19. saya sering menggunakan komputer atau perangkat lainnya untuk kuliah, tugas dan keperluan pribadi 4 4 4 1 5 288 20. saya merasa percaya diri dalam menggunakan perangkat lunak komputer 3.875 4 4 1 6 279 21. Saya merasa literasi komputer saya telah membantu saya dalam menyelesaikan tugas akademik dengan lebih efisien. 3.8611 11111 4 4 2 5 278 22. Saya menggunakan perangkat komputer 3.8611 11111 4 4 2 5 278 109 E-ISSN: 2988-6325 P-ISSN: 2988-4918 dalam proyek-proyek kelompok atau kolaboratif dengan teman-teman saya. 23. Saya menggunakan perangkat komputer untuk mengikuti kuliah online atau program e- learning. 3.8472 22222 4 4 2 5 277 24. Saya merasa nyaman dalam menggunakan aplikasi dan perangkat lunak pendidikan seperti Microsoft Word, Excel, atau Google Drive. 3.8472 22222 4 4 1 5 277 25. Saya merasa penggunan perangkat komputer memudah untuk mengakses sumber- sumber pembelajaran terbuka. 3.9722 22222 4 4 1 5 286 26. Saya merasa literasi komputer membantu saya dalam menghadapi perubahan cepat dalam dunia teknologi. 3.9722 22222 4 4 2 5 286 27. Saya sering menggunakan perangkat komputer untuk mengakses kursus atau pelatihan online tambahan. 4 4 4 2 5 288 28. Saya merasa memiliki pemahaman yang cukup tentang nilai etika dan integritas dalam konteks pendidikan. 3.75 4 4 1 5 270 29. saya merasa memiliki kemampuan berpikir kritis dalam memecahkan masalah yang dihadapi dalam Pendidikan 3.8611 11111 4 4 2 5 278 30. Saya memiliki akses ke perangkat komputer di luar jam sekolah atau 3.8333 33333 4 4 1 5 276 110 Journal of Vocational, Informatics and Computer Education E-ISSN: 2988-6325 P-ISSN: 2988-4918 kampus untuk keperluan Pendidikan Gambar 1. HASIL DAN DISKUSI Diagram Analisis yang menunjukkan bahwa literasi berpengaruh terhadap cara menyelesaikan tugas akademik kampus untuk keperluan Pendidikan kampus untuk keperluan Pendidikan Gambar 1. Diagram Analisis yang menunjukkan bahwa literasi berpengaruh terhadap cara menyelesaikan tugas akademik Gambar 1. Diagram Analisis yang menunjukkan bahwa literasi berpengaruh terhadap cara menyelesaikan tugas akademik Analisis data memberikan bahwa literasi komputer berkontribusi positif terhadap efisiensi dalam menuntaskan tugas akademik. Hal ini menandakan bahwa pemahaman serta keterampilan literasi komputer memainkan peran penting dalam pembelajaran serta penyelesaian tugas-tugas akademik. Gambar 2. Diagram Analisis yang menyatakan bahwa banyak siswa sudah terbantu oleh media social Gambar 2. Diagram Analisis yang menyatakan bahwa banyak siswa sudah terbantu oleh media social Data membagikan bahwa penggunaan media umum dapat menaikkan literasi digital siswa. Hal ini memberikan bahwa media umum bisa sebagai asal belajar yang efektif dalam mengembangkan keterampilan literasi digital. Data membagikan bahwa penggunaan media umum dapat menaikkan literasi digital siswa. Hal ini memberikan bahwa media umum bisa sebagai asal belajar yang efektif dalam mengembangkan keterampilan literasi digital. 111 Journal of Vocational, Informatics and Computer Education E-ISSN: 2988-6325 P-ISSN: 2988-4918 Journal of Vocational, Informatics and Computer Education E-ISSN: 2988-6325 P-ISSN: 2988-4918 Journal of Vocational, Informatics and Computer Education E-ISSN: 2988-6325 P-ISSN: 2988-4918 Gambar 3. Diagram Analisis menyatakan bahwa banyak siswa yang memiliki pemahaman etika dalam integritasi pendidikan Gambar 3. Diagram Analisis menyatakan bahwa banyak siswa yang memiliki pemahaman etika dalam integritasi pendidikan Temuan ini menunjukkan bahwa peserta didik merasa mempunyai pemahaman yang relatif perihal etika dan integrasi komputer dalam pendidikan. Ini dapat diartikan bahwa upaya pendidikan pada hal ini telah berhasil dalam memberikan pencerahan perihal nilai etika serta pentingnya integrasi teknologi pada pembelajaran. Gambar 4. Diagram Analisis yang menyatakan bahwa internet membantu mendapatkan informasi dengan mudah Gambar 4. Diagram Analisis yang menyatakan bahwa internet membantu mendapatkan informasi dengan mudah Data menunjukkan bahwa internet sudah memudahkan peserta didik dalam mengakses informasi. Ini artinya yang akan terjadi yang diperlukan asal literasi personal komputer , yang memungkinkan akses lebih cepat serta luas ke sumber informasi. Data menunjukkan bahwa internet sudah memudahkan peserta didik dalam mengakses informasi. Ini artinya yang akan terjadi yang diperlukan asal literasi personal komputer , yang memungkinkan akses lebih cepat serta luas ke sumber informasi. 112 Journal of Vocational, Informatics and Computer Education E-ISSN: 2988-6325 P-ISSN: 2988-4918 Gambar 5. Diagram Analisis yang menyatakan bahwa literasi berpengaruh terhadap memahami suatu materi Journal of Vocational, Informatics and Computer Education E-ISSN: 2988-6325 P-ISSN: 2988-4918 Gambar 5. HASIL DAN DISKUSI Diagram Analisis yang menyatakan bahwa literasi berpengaruh terhadap memahami suatu materi Temuan ini membagikan bahwa literasi personal komputer memiliki dampak positif terhadap pemahaman Mahasiswa terhadap materi pembelajaran. Ini menggambarkan bahwa literasi personal komputer memainkan peran krusial dalam menaikkan kemampuan Mahasiswa dalam memahami serta mengaplikasikan materi pembelajaran. Temuan ini membagikan bahwa literasi personal komputer memiliki dampak positif terhadap pemahaman Mahasiswa terhadap materi pembelajaran. Ini menggambarkan bahwa literasi personal komputer memainkan peran krusial dalam menaikkan kemampuan Mahasiswa dalam memahami serta mengaplikasikan materi pembelajaran. Literasi digital merupakan kekuatan atau kebijaksanaan pada memakai media digital contohnya alat percakapan atau jaringan dalam mendapat, memilah, memakai, mencetak info, serta pemanfaatan media digital dengan pertanggung jawaban[9].Menurut Ferguson literasi teknologi adalah kemampuan tahu kelengkapan yang mengikuti teknologi mirip peranti keras hardware dan aplikasi, serta etika dan etiket dalam memanfaatkan teknologi[16].Menurut Maryland Technology Education State Curriculum literasi teknologi artinya kemampuan buat menggunakan, memahami, mengatur, serta menilai suatu inovasi yang melibatkan proses serta ilmu pengetahuan buat memecahkan duduk perkara serta memperluas kemampuan seseorang. National Academy of Engineering and National Research Council of The National Academies mendefisinikan literasi teknologi sebagai sebuah pemahaman ihwal teknologi di sebuah strata yang memungkinkan pemanfaatan secara efektif dalam warga teknologi[16].Penelitian yang dilakukan oleh Wandani tentang penggunaan multimedia interaktif pada pembelajaran di materi kedudukan bundar membagikan bahwa penggunaan multimedia interaktif efektif serta efisien pada memahamkan peserta didik di materi kedudukan lingkaran[8]. Mengenai hasil diskusi berdasarkan Data kuesioner diatas menunjukkan bahwa literasi komputer berpengaruh terhadap cara berpikir siswa, tak hanya itu ada beberapa hal yang sangat mempengaruhi tentang literasi komputer cara berpikir siswa, berpengaruh terhadap keektifian mengembangkan keterampilan,menaikkan kemampuan Mahasiswa dalam memahami serta mengaplikasikan materi pembelajaran. Pertama, Observasi ini menunjukkan tentang literasi komputer dapat dampak yang berpengaruh bagi pola berpikir siswa yang menyiratkan pemecahan masalah dan konsep pemahaman konsep yang mendasar dalam dunia teknologi. Terkadang siswa yang betul betul paham dengan literasi komputer akan cenderung lebih mampu untuk mengatasi masalahnya dengan teknis dan berpikir kreatif dalam menemukan solusi. 113 Journal of Vocational, Informatics and Computer Education E-ISSN: 2988-6325 P-ISSN: 2988-4918 Teknologi info serta Kocakapan sudah membangun kesempatan lain demi pembaca dan penulis pada tempat kebanyakan orang yang sudah membarui teknik anak menimba ilmu.Keberhasilan yang didapatkan diObservasi ini ialah dua ruang tempat untuk literasi: satu diumumkan ditempat sekolah yang dipakai pada teks cetak; yang lain berkembang di luar sekolah dan menyukai literasi digital menjadi isu. KESIMPULAN DAN SARAN literasi komputer memiliki dampak positif terhadap tingkat berpikir siswa dalam konteks pendidikan. Penggunaan teknologi dalam pembelajaran, seperti aplikasi pembelajaran dan program pengolah presentasi, dapat membantu meningkatkan kemampuan literasi digital siswa. Selain itu, akses yang mudah terhadap perangkat komputer dan internet juga mempengaruhi kemampuan siswa dalam memahami materi pelajaran dan menyelesaikan tugas-tugas pendidikan. Pemahaman dan keterampilan literasi komputer juga berperan penting dalam efisiensi dalam menuntaskan tugas akademik. Untuk meningkatkan literasi komputer siswa melalui integrasi teknologi dalam pembelajaran. Sekolah dapat menyediakan akses yang memadai terhadap perangkat komputer dan internet di luar jam sekolah atau kampus untuk keperluan pendidikan. Selain itu, penggunaan aplikasi pembelajaran dan program pengolah presentasi dapat ditingkatkan untuk membantu siswa meningkatkan kemampuan literasi digital mereka. Pendidikan tentang etika dan nilai-nilai penting dalam penggunaan teknologi juga perlu diberikan kepada siswa. HASIL DAN DISKUSI Karya ini diakhiri menggunakan kebutuhan buat membentuk jembatan yang menghubungkan kompetensi digital di dalam dan pada luar sekolah melalui penciptaan ruang literasi ketiga[14]. REFERENSI [1] S. Hafsah, “Pengaruh Media Sosial Terhadap Motivasi Belajar Siswa Muhammadiyah Jakarta,” Univ. Muhammadiyah Jakarta, p. 140, 2018. [2] L. G. N. Erayani and I Nyoman Jampel, “Meningkatkan Kemampuan Literasi Sains dan Kemampuan Metakognitif Siswa melalui Model Problem Based Learning Berbantuan Media Interaktif,” J. Penelit. dan Pengemb. Pendidik., vol. 6, no. 2, pp. 248–258, 2022, doi: 10.23887/jppp.v6i2.48525. [3] M. J. Tsai, J. C. Liang, and C. Y. Hsu, “The Computational Thinking Scale for Computer Literacy Education,” J. Educ. Comput. Res., vol. 59, no. 4, pp. 579–602, 2021, doi: 10.1177/0735633120972356. [4] S. Bahrun, S. Alifah, and S. Mulyono, “Rancang Bangun Sistem Informasi Survey Pemasaran dan Penjualan Berbasis Web,” J. 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https://openalex.org/W4286433136	https://zenodo.org/record/6865620/files/50.pdf	English	null	The Horrors of Colonialism: A Special Reference to Amitav Ghosh’s The Hungry Tide	International journal of social science and human research	2,022	cc-by	3,791	The Horrors of Colonialism: A Special Reference to Amitav Ghosh’s The Hungry Tide The delineation of this identity constituent element in their character and collective psyche is not easily susceptible to succinct expression, but the attempt is as fascinating as it is untiring.” 1 Many Indians overseas have revisited and researched the history and culture of their ancient land and produced works of great historical as well as literary merit that help us understand India with its baffling contradictions; its discords and divisions; its tolerance of religious diversity side by side with the painful apparition of bigotry and intolerance even evinced in barbarous violence; its message of one reality and no other, as well as its distinctions of caste and class despite their categorical repudiation in the scriptures and by the saints and sages of so many reform movements. “When the people of a country living under an autocratic ore oppressive rule want to throw off that rule and establish a new political set up of their own choice, the people of that country are said to have been imbued with the spirit of nationalism” 2 “When the people of a country living under an autocratic ore oppressive rule want to throw off that rule and establish a new political set up of their own choice, the people of that country are said to have been imbued with the spirit of nationalism” 2 In their zeal to show that they are by no means inferior to their rulers, they make a search for their own identity and try to affirm it. It is thus that the people of a country are roused by nationalism; make their efforts to distinguish their manners and customs, religious and social institutions and culture and civilization. Identity remains one of the most urgent as well as the hotly disputed topics in literary and cultural studies. Amitav Ghosh, a world renowned novelist, was born in Kolkata (then Calcutta) in 1956. His father was an army officer that is why he got many chances to visit a number of countries including Sri Lanka, Bangladesh, Iran, Egypt and the United States. As an Indian and anthropologist Ghosh has been immensely influenced by the political and social milieu of the country. As an active anthropologist he never gives rest to his mind and thought. His long and deep contemplation about India, its people and its culture is widely reflected in his works. The Horrors of Colonialism: A Special Reference to Amitav Ghosh’s The Hungry Tide Dr. Ritu Sharma Dyal Singh College, Karnal Indian abroad are not just people abroad. They are bundles of attitudes, convictions, anxieties, shared notions, fears and anticipations, all of which are Indians in their very texture. The links of Indian with India when in abroad is many times stronger than that of many other nationalities in similar situations. “Members of the family” for the Indians abroad includes different kinds of relatives with considerable and pronounced detachment in attitude and affection generally maintains a strong sense of belonging to India. Two aspects about Indian Diaspora need to be emphasized: first, though abroad, Indian Diaspora is the product of Indian socio- political-cultural complex; second, it is not insulated against what goes on taking place in the mother country. Indian Diaspora continued and still goes on unabated, including skilled workers, businessmen, professionals, scientists and men and women of different cultures. The first generation of Diaspora has made their mark in many fields of activities in so many countries of the world. “The story of Indian diaspora is one of the sweat and toil often washed with tears, of achievements despite impediments, of educational advancements and economic progress, of political success at times crushed by cruel coups. The descendants of this diaspora have also inevitably produced work of literature illustrative of their history and heritage, of their own awareness of their society and its problems, its achievements, its limitations and frustrations. Some of them have turned their attention to India to assess and understand the nature of their relationship with the land of their ancestors. They have explored their own Indianness in its uniqueness, its new geo-political and social context; its separation from the forces and factors that changed the face of India in the years following the emigration of their ancestors. The search for this identity goes on and finds their expression in their writings. International Journal of Social Science And Human Research ISSN(print): 2644-0679, ISSN(online): 2644-0695 Volume 05 Issue 07 July 2022 DOI: 10.47191/ijsshr/v5-i7-50, Impact factor- 5.871 Page No: 3127-3130 International Journal of Social Science A ISSN(print): 2644-0679, ISSN(online): 2644-0695 Volume 05 Issue 07 July 2022 DOI: 10.47191/ijsshr/v5-i7-50, Impact factor- 5.871 Page No: 3127-3130 and even between faiths.” The Hungry Tide reveals the characters’ quest to know the history about Lusibari, Hamilton House and Sir Daniel Hamilton using story telling method and flashback techniques. The Hungry Tide elaborates the conditions of resettlement which is harsh and alien. In 1978, a group of refugees fled from the Drandakarnya camp in Madhya Pradesh and came to the island of Morichjhapi in the Sundarbans with the intention of settling here. They cleared the land for agriculture and began to fish and farm. But their presence there alarmed the left front ministry and settlers were evicted in a brutal display of state power in May 1979. Many, like the girl Kusum, were killed in this massacre. Nirmal’s idealist hopes had been killed and he himself is shattered by the events and never recovers. After Morichjhapi massacre, the victory of Bengali refugees, when Nirmal, Horen and Rajon went to the house to see Kusum. They wanted to know about Kusum and her struggle. When the question is asked about her conditions, she again asked question and made everyone to think about this massacre: “It was terrible to see Kusum; her bones protruded from her skin, like the ribs of drum and she was too weak to rise from her mat. Fokir, young as he was, appeared to have weathered the siege in better health and it was he who was looking after his mother.”4 Nirmal and Neelima came to Sundarbans after their marriage when Nirmal’s revolutionary ideas became too dangerous in Calcutta. In starting, their settlement in Lusibari was very difficult. Working as headmaster when he approaches near retirement, he feels that his wife was poorly spent because he never lived up to his revolutionary ideas. He secretly envies his wife for achieving what she did and he is overwhelmed with a sense of uselessness for having achieved practically nothing for all his bluster about revolution. The conflict of Nirmal Bose has been given as “Life as he approaches retirement the true tragedy of a routinely spent life is that its wastefulness doesn't become apparent till it’s too late.” The feeling of alienation was in the behaviour of Nirmal felt by Nilima, his wife. He became changed after his visit to Morichjhapi. After 30 years of the death of Nirmal Bose, when Piyali and Kanai reached Lusibari, Neelima got herself involved in Piyali. The Horrors of Colonialism: A Special Reference to Amitav Ghosh’s The Hungry Tide Amitav Ghosh’s famous novel The Hungry Tide was published in 2004 and was awarded one of the Best Works in English Fiction in Hutch Cross Book Award. The Hungry Tide has been divided into two parts: Part I - The Ebba: Bhata, Part II: The Flood: Jowar. The Hungry Tide is about migration, cultural dispersion and assimilation, globalisation and its circuits of movement and exchange. It transports the reader into a place and time about which people know very little. IJSSHR, Volume 05 Issue 07 July 2022 Page 3127 www.ijsshr.in and even between faiths.” While discussing with Piyali, Neelima feels alienation of her own life. The Hungry Tide explores the quest of Nilima Bose after marriage. In Lusibari, Nilima made a women union and everyone calls her Mashima. At the age of seventy, she called up Kanai to show the packet left by her husband for him. She was closely linked with Kusum as well as Kanai. She achieved success and respect in Lusibari but always felt uneasiness in her husband's presence. The Hungry Tide also touches upon the quest of Kusum who had to depend on women union after her father's death. But her aspirations make her strong. She wants to be educated herself and to be a nurse for Lusibari's hospital. The Hungry Tide also touches upon the quest of Kusum who had to depend on women union after her father's death. But her aspirations make her strong. She wants to be educated herself and to be a nurse for Lusibari's hospital. At the age of forty, when Kanai returned to Lusibari, to meet Nilima, he was surprised to see Moyna, Kusum's daughter-in-law’s quest to win life Even Kanai feels concerned about the struggle Moyna has undergone in her life: The Hungry Tide also touches upon the quest of Kusum who had to depend on women union after her father's death. But her aspirations make her strong. She wants to be educated herself and to be a nurse for Lusibari's hospital. At the age of forty, when Kanai returned to Lusibari, to meet Nilima, he was surprised to see Moyna, Kusum's daughter-in-law’s quest to win life. Even Kanai feels concerned about the struggle, Moyna has undergone in her life: “Just think of life she’s led, said Kanai. She’s struggled to educate herself against heavy odds. Now she is well on her way to become a nurse. She knows what she wants - for herself and her family - nothing is going to keep her from pursuing it. She is ambitious, she’s tough and she's going to go a long way.”(146) At the age of forty, when Kanai returned to Lusibari, to meet Nilima, he was surprised to see Moyna, Kusum's daughter-in-law’s quest to win life. Even Kanai feels concerned about the struggle, Moyna has undergone in her life: quest to win life. The Horrors of Colonialism: A Special Reference to Amitav Ghosh’s The Hungry Tide The Hungry Tide tells a very contemporary story of adventure, love, identity and history; set in one of the most fascination regions on the earth. Off the easternmost coast of India, in the Bay of Bengal lies the immense labyrinth of tiny islands known as the Sundarbans. For settlers, life is extremely precarious. Without warning, tidal floods rise and surge over the land, leaving devastation in their wake. In this place, the lives of three people from different worlds collide. Piyali Roy is a young marine biologist of Indian descent but stubbornly American. Her Journey begins with a disaster when she is thrown from a boat into crocodile - infested waters. Rescue comes in the form of a young fisherman Fokir. Piyali engages Fokir to help with her research and finds a translator in Kanai Dutt, a businessman from Delhi whose idealistic aunt and uncle are long time settlers in the Sundarbans; Kanai comes to visit his widowed aunt and to review some writings left behind by his uncle. Both Piyali and Kanai disturb the delicate balance of settlement life and set in motion a fateful cataclysm. From this moment, the tide begins to turn. The Hungry Tide is a prophetic novel of remarkable insight, beauty and humanity. Marshal Zeringue explores that The Hungry Tide is a story about the 1979 massacre of refugees by government forces who had settled the village of Morichjhapi. Through Nirmal’s journal, Amitav Ghosh tells us about this historical event as well as the search of identity of the refugee settlers from the island of Morichjhapi in the Sundarbans and the massacre done by left front government of West Bengal in 1979. The story of Morichjhapi occupies a central place in The Hungry Tide by which Amitabh Ghosh wants to explore human struggle, conflict and desire to re-settlement. To each, the Sundarbans represents something different but for all they become, “a proliferation of small worlds........a meeting not just of many rivers, but a roundabout people can use to pass in many .... from country to country and even between faiths.” 3 and even between faiths.” 3 he Horrors of Colonialism: A Special Reference to Amitav Ghosh’s The Hungry Tide The Hungry Tide explores the character of Kanai Dutt, an interpreter, who runs a successful translation business in Delhi. In his childhood, he had been in Lusibari as a teenager. After rustication from school for his pride and arrogance, he had been sent by his parents. He is now been summoned by Nilima because of a packet left for Kanai by her late husband, Nirmal which has just been found some twenty years after his death. The packet now left for Kanai contains important information. As Kanai read the pages, its narrative of past events at the end of Nirmal’s life revolves around Kusum, her son Fokir and the catastrophic struggle of the dispossessed to form a new society on the island of Morichjhapi. This is a life which Kanai fails to understand. His wealth, servants and pride have no values here, while he himself feels superior to Fokir. He tries to seduce Piyali but begins to realise something extraordinary about that diasporic Indian Marine Biologist. Fokir’s eye always shows his quest for life for, his livelihood, for his parents. He has not seen ever his mother. While his journey to Nirmal, Nirmal acquainted his quest and explained it. Fokir’s quest can be felt in his conversation, his notes, his music and his lifestyle. Piya feels herself secure with Fokir. She imagines about Fokir’s life. But she feels sympathy for Fokir when she comes to know about his life. Both had similar childhood. Both had grown up without mother even Fokir’s conditions were much poor. She doesn’t understand his language, but feels grief and isolation in his music. At the end of the novel, both Kanai and Piyali return to Lusibari to live with Kanai’s aunt Nilima. They both returned due to their personal and professional quest. Kanai wants to rewrite from memory, his uncle’s journal which was lost during a storm and which contains records of local culture and Morichjhapi Massacre; Piyali wants to continue her research on dolphins by partnering with Nilima’s NGO so that international organisations can contribute funds to help them both. The Hungry Tide, thus, relates local, national and global levels of consciousness with each other. Piyali’s research is situated between the local and the global,while Kanai’s re-writing mediates between the local and the nation. he Horrors of Colonialism: A Special Reference to Amitav Ghosh’s The Hungry Tide The Hungry Tide emphasizes the positive contributions of professional dedication in contrast to Nirmal’s revolutionary romanticism and his alienation felt in the tide cou The Hungry Tide is about migration, cultural dispersion and assimilation, globalisation and its circuits of movement and exchange. The Hungry Tide transports the reader to a remote time and place and gives people information and feeling about something they knew very little about. One remark holds views about it as an “armchair safari” in the Sundarbans, the tidal country between India and Bangladesh. These terms “tourism” and “safari” suggest an anachronism and irreducible difference in which the people and the places are depicted. There is some sort of pre-modern wilderness and they appear to be remote and cut off from “our” modernity “over here”. The issues of social class barriers appear in The Hungry Tide twice over across two generations - in the refugees’ revolt of which Kusum is a part and in the friendship that develops across the cross lines between Fokir, her illiterate son and the cosmopolitan outsider Piya. The Hungry Tide is the work produced by Amitav Ghosh at the peak of his power. It is geographically quite narrow. It is limited to the Sundarban islands in the Bay of Bengal and perhaps by extension Bengal. It is conceptually limited - basically to conceptual plots. First it explores the plight of displaced people especially group of refugees from Bangladesh who found themselves in a confrontation with the Indian state in 1979. The other conceptual question is how humans share a complex and dangerous system with animals. Amitav Ghosh has discovered yet another new territory, summoning a singular place from its history, language and myth and bringing it to life. Yet the achievement of The Hungry Tide is in its acceleration of a particle and more unknowable Jungle the human heart. Amitav Ghosh presents a ruthless appraisal of the horrors of colonialism. and even between faiths.” Even Kanai feels concerned about the struggle, Moyna has undergone in her life: “Just think of life she’s led, said Kanai. She’s struggled to educate herself against heavy odds. Now she is well on her way to become a nurse. She knows what she wants - for herself and her family - nothing is going to keep her from pursuing it. She is ambitious, she’s tough and she's going to go a long way.”(146) The Hungry Tide also expresses Piyali’s alienation in India during her visit. She is a cytologist who is of Indian parents but remains stubbornly American in orientation and temperament. She is the daughter of Bangla parents who had immigrated to Seattle. She is a woman used to solitude and rigors of the life of a scientist working in a field. She decides to work in an area where she knows neither of customs not the language. She can survive for some days on just energy bars and ovation. Her life quest is to create a body of knowledge on the river dolphin which is on the verge of extinction. She perceives herself to have been overlooked by life and loneliness is slowly closeting her gradually. She creates an aura of self - sufficiency around her to keep away from unwanted IJSSHR, Volume 05 Issue 07 July 2022 Page 3128 www.ijsshr.in 1) Sarva Daman Singh, “Diaspora: Essays on Culture and Identity,” The Indian Diaspora, 96. 2) K.A.Mingoue, Nationalism, (London: 1) Sarva Daman Singh, “Diaspora: Essays on Culture and Identity,” The Indian Diaspora, 96. 2) K A Mi N ti li (L d he Horrors of Colonialism: A Special Reference to Amitav Ghosh’s The Hungry Tide intrusion in her life. The Hungry Tide reveals Piyali’s search for marine life. She is not able to understand language of that region but she decides to come here with some cards. With her cards, she starts her search. Ghosh touches upon the quest of Piyali: “When communication was possible, she would show them to fishermen andboatmen and ask questions about sightings, abundance, behaviour, seasonal distribution and so on. When there was no one to translate she would hold up the cards and wait for a response. This often worked; they would recognise the animal and point her to places where they were commonly seen” (32) The Hungry Tide explorers Piyali’s quest to know about her identity. In her life with her parents, she hadn't listen a single word about Indian Heritage. Her quest for love brings her to Fokir when she feels jealous of his wife .She feels herself alienated but for safety Fokir sacrificed his life The Hungry Tide explorers Piyali’s quest to know about her identity. In her life with her parents, she hadn't listen a single word about Indian Heritage. Her quest for love brings her to Fokir when she feels jealous of his wife .She feels herself alienated but for safety, Fokir sacrificed his life. Ghosh adds that according to Piyali, her life had been complicated. She explained her life to Nilima which underlines her feeling of alienation and rootlessness in her life: “You see, my father's parents were Bengalis who’d settled in Burma- they came to India as R f d i th S d W ld W H i d d l t f th h ll th th i b t i i t d Ghosh adds that according to Piyali, her life had been complicated. She explained her life to Nilima which underlines her feeling of alienation and rootlessness in her life: “You see, my father's parents were Bengalis who’d settled in Burma- they came to India as Refugees, during the Second World War.Having moved around a lot, my father has all these theories about immigrants and refugees.He believes that Indian- Bengalis in particular don't travel well, because their eyes are always turned backwards, towards n we move to America, he decided he was not going to make that mistake, he was going to try to fit in it. 2) K.A.Mingoue, Nationalism, (London: 1) Sarva Daman Singh, “Diaspora: Essays on Culture and Identity,” The Indian Diaspora, 96. REFERENCES IJSSHR, Volume 05 Issue 07 July 2022 IJSSHR, Volume 05 Issue 07 July 2022 www.ijsshr.in www.ijsshr.in Page 3129 Page 3129 The Horrors of Colonialism: A Special Reference to Amitav Ghosh’s The Hungry Tide 3) Marshal Zerinuge, Review: “Campaign For The American Reader: The Hungry Tide (27August 2006). 4) Methuen,1969) 25. Amitav Ghosh, The Hungry Tide (UK: Harper Collins Publishers, 2004),260. (All the subsequent citations from the text are from this edition only and the page nos. have been given in parentheses after the quotations.) There is an Open Access article, distributed under the term of the Creative Commons Attribution – Non Commercial 4.0 International (CC BY-NC 4.0) (https://creativecommons.org/licenses/by-nc/4.0/), which permits remixing, adapting and building upon the work for non-commercial use, provided the original work is properly cited. The Horrors of Colonialism: A Special Reference to Amitav Ghosh’s The Hungry Tide 3) Marshal Zerinuge, Review: “Campaign For The American Reader: The Hungry Tide (27August 2006). 4) Methuen,1969) 25. Amitav Ghosh, The Hungry Tide (UK: Harper Collins Publishers, 2004),260. (All the subsequent citations from the text are from this edition only and the page nos. have been given in parentheses after the quotations.) he Horrors of Colonialism: A Special Reference to Amitav Ghosh’s The Hungry Tide The Horrors of Colonialism: A Special Reference to Amitav Ghosh’s The Hungry Tide 3) Marshal Zerinuge, Review: “Campaign For The American Reader: The Hungry Tide (27August 2006). 4) Methuen,1969) 25. Amitav Ghosh, The Hungry Tide (UK: Harper Collins Publishers, 2004),260. (All the subsequent citations from the text are from this edition only and the page nos. have been given in parentheses after the quotations.) IJSSHR, Volume 05 Issue 07 July 2022 www.ijsshr.in Page 3130 There is an Open Access article, distributed under the term of the Creative Commons Attribution – Non Commercial 4.0 International (CC BY-NC 4.0) (https://creativecommons.org/licenses/by-nc/4.0/), which permits remixing, adapting and building upon the work for non-commercial use, provided the original work is properly cited. There is an Open Access article, distributed under the term of the Creative Commons Attribution – Non Commercial 4.0 International (CC BY-NC 4.0) IJSSHR, Volume 05 Issue 07 July 2022 Page 3130 Page 3130 www.ijsshr.in
https://openalex.org/W4310128755	https://archimer.ifremer.fr/doc/00832/94388/101714.pdf	English	null	Hot Vents Beneath an Icy Ocean: The Aurora Vent Field, Gakkel Ridge, Revealed	Oceanography	2,023	cc-by	11,694	A SHORT HISTORY OF GAKKEL RIDGE EXPLORATIONit Forty-five years after the discovery of hydrothermal vents (Corliss et al., 1979), research into these unique hab- itats and their rich submarine ecosys- tems has brought about revolutionary findings in biology, chemistry, and geo- physics. Understanding how these dis- tinctive ecosystems are supported by sunlight-independent microbial primary productivity based on chemosynthesis changed the way we understand life on Earth (Van Dover et al., 2018). They have inspired our understanding of the ori- gin of life on Earth (Martin et al., 2008) and are now influencing the choice of exploration targets aimed at the discov- ery of extraterrestrial life in our solar system (Hand and German, 2018; Hand et al., 2020). The exotic faunal commu- nities at active hydrothermal vents are also of high interest given their physio- logical adaptations and the high degree of endemicity, and for their potential in providing marine genetic resources of use in biomedicine, cosmetics, and bio- fuels, among others (Van Dover et al., 2018). In addition, interest in the poten- tial for mineral resources in hydrother- mal vent deposits has greatly increased in the last two decades, and exploration licenses for such resources have been granted for national and international waters (Jones et al., 2020). Si th di f d h d Current data on vent communities globally has identified 11 biogeographic provinces, but their delineation is still being debated (Rogers et al., 2012). Until now, the vent faunal communities of the ice-covered Gakkel Ridge in the Central Arctic Ocean remained unexplored because of their remote and climatologi- cally challenging location. This study puts the Aurora Vent Field of the Gakkel Ridge on the global map of chemosynthetic- based ecosystems, providing an initial overview of the vent field and the eco- system it supports.h Building on the results of the AMORE 2001 expedition (Edmonds et al., 2003), in 2014, R/V Polarstern expedition PS86 AURORA aimed to study geophysical, geological, geochemical, and biological processes at hydrothermal vents on the Gakkel Ridge, with a focus on the southern segment (Boetius, 2015). In this region, the spreading rate is 14.5–13.5 mm yr–1, (slightly faster than the average rate for the overall ridge), and the ridge axis floor The Gakkel Ridge (Figure 1a) extends 1,800 km from the northern end of the Lena Trough off Northeast Greenland (81°N) to near the Siberian shelf at 87°N. REGULAR ISSUE FEATURE HOT VENTS BENEATH AN ICY OCEAN THE AURORA VENT FIELD, GAKKEL RIDGE, REVEALED By Eva Ramirez-Llodra, Claudio Argentino, Maria Baker, Antje Boetius, Carolina Costa, Håkon Dahle, Emily M. Denny, Pierre-Antoine Dessandier, Mari H. Eilertsen, Benedicte Ferre, Christopher R. German, Kevin Hand, Ana Hilário, Lawrence Hislop, John W. Jamieson, Dimitri Kalnitchenko, Achim Mall, Giuliana Panieri, Autun Purser, Sofia P. Ramalho, Eoghan P. Reeves, Leighton Rolley, Samuel I. Pereira, Pedro A. Ribeiro, Muhammed Fatih Sert, Ida H. Steen, Marie Stetzler, Runar Stokke, Lissette Victorero, Francesca Vulcano, Stig Vågenes, Kate Alyse Waghorn, and Stefan Buenz Oceanography \| Vol. 36, No. 1 6 HOT VENTS BENEATH AN ICY OCEAN THE AURORA VENT FIELD, GAKKEL RIDGE, REVEALED By Eva Ramirez-Llodra, Claudio Argentino, Maria Baker, Antje Boetius, Carolina Costa, Håkon Dahle, Emily M. Denny, Pierre-Antoine Dessandier, Mari H. Eilertsen, Benedicte Ferre, Christopher R. German, Kevin Hand, Ana Hilário, Lawrence Hislop, John W. Jamieson, Dimitri Kalnitchenko, Achim Mall, Giuliana Panieri, Autun Purser, Sofia P. Ramalho, Eoghan P. Reeves, Leighton Rolley, Samuel I. Pereira, Pedro A. Ribeiro, Muhammed Fatih Sert, Ida H. Steen, Marie Stetzler, Runar Stokke, Lissette Victorero, Francesca Vulcano, Stig Vågenes, Kate Alyse Waghorn, and Stefan Buenz R/V Kronprins Haakon is being positioned here on an ice floe to drift toward the Aurora Vent Field on Gakkel Ridge, Arctic Ocean. © REV Ocean Oceanography \| Vol. 36, No. 1 6 ABSTRACT. Evidence of hydrothermal venting on the ultra-slow spreading Gakkel Ridge in the Central Arctic Ocean has been available since 2001, with first visual evi- dence of black smokers on the Aurora Vent Field obtained in 2014. But it was not until 2021 that the first ever remotely operated vehicle (ROV) dives to hydrothermal vents under permanent ice cover in the Arctic were conducted, enabling the collection of vent fluids, rocks, microbes, and fauna. In this paper, we present the methods employed for deep-sea ROV operations under drifting ice. We also provide the first description of the Aurora Vent Field, which includes three actively venting black smokers and dif- fuse flow on the Aurora mound at ~3,888 m depth on the southern part of the Gakkel Ridge (82.5°N). The biological communities are dominated by a new species of coccu- linid limpet, two small gastropods, and a melitid amphipod. The ongoing analyses of Aurora Vent Field samples will contribute to positioning the Gakkel Ridge hydrother- mal vents in the global biogeographic puzzle of hydrothermal vents. rate (E.T. Baker et al., 1996). This hypoth- esis was revisited after exploration of the Southwest Indian Ridge showed that even ultra-slow spreading ridges could host abundant submarine venting (German et al., 1998). Technological and method- ological challenges of working at great depth in regions of permanent sea ice cover have constrained the exploration of the Gakkel Ridge. In 2001, Edmonds et al. REGULAR ISSUE FEATURE HOT VENTS BENEATH AN ICY OCEAN THE AURORA VENT FIELD, GAKKEL RIDGE, REVEALED By Eva Ramirez-Llodra, Claudio Argentino, Maria Baker, Antje Boetius, Carolina Costa, Håkon Dahle, Emily M. Denny, Pierre-Antoine Dessandier, Mari H. Eilertsen, Benedicte Ferre, Christopher R. German, Kevin Hand, Ana Hilário, Lawrence Hislop, John W. Jamieson, Dimitri Kalnitchenko, Achim Mall, Giuliana Panieri, Autun Purser, Sofia P. Ramalho, Eoghan P. Reeves, Leighton Rolley, Samuel I. Pereira, Pedro A. Ribeiro, Muhammed Fatih Sert, Ida H. Steen, Marie Stetzler, Runar Stokke, Lissette Victorero, Francesca Vulcano, Stig Vågenes, Kate Alyse Waghorn, and Stefan Buenz (2003) obtained first evidence of hydrothermal venting on nine to twelve discrete locations along the Gakkel Ridge during the InterRidge two-icebreaker (R/V Polarstern and USCGC Healy) Arctic Mid-Ocean Ridge Expedition (AMORE; Figure 1a). Continued explo- ration during the Arctic Gakkel Vents (AGAVE) expedition in 2007 provided evidence of explosive volcanism at 85°N and demonstrated that large-scale pyro- clastic activity is possible along even the deepest portions of the global mid-ocean ridge volcanic system (Sohn et al., 2008). Seismic studies suggest substantial mag- matic activity, serpentinization, and fluid flow at this slowest of all Earth’s ridge sys- tems (Michael et al., 2003; Schlindwein and Schmid, 2016). Between 2002 and 2010, the ChEss program aimed to improve understanding of the global bio- geography of chemosynthetic-based eco- systems (M.C. Baker et al., 2010). Based on the increasing evidence of hydro- thermal venting along the Gakkel Ridge, the ChEss program identified a num- ber of poorly investigated regions where research efforts should focus. The Gakkel Ridge was recognized as one of the miss- ing pieces of the global biogeographic puzzle (Ramirez-Llodra et al., 2007). Building on the results of the AMORE 2001 expedition (Edmonds et al., 2003), i 2014 R/V P l t diti PS86 investigated (Beaulieu et al., 2015). To date, exploration has yielded an inventory of 722 confirmed high-temperature vent sites, with a further 720 high-temperature vents inferred from water column data, as reported in the InterRidge Vents Database in September 2022 (Beaulieu and Szafranski, 2020). There may be hun- dreds of additional active hydrothermal systems and their associated faunal com- munities yet to be discovered worldwide along the unexplored branches and sec- tions of the global mid-ocean ridge sys- tem, particularly along the least explored slow and ultra-slow spreading ridges (Beaulieu et al., 2015). March 2023 \| Oceanography 7 A SHORT HISTORY OF GAKKEL RIDGE EXPLORATIONit It was initially predicted to host an extremely low number of active sites based on the assumption that hydrother- mal flux scaled directly with spreading Since the discovery of deep-sea hydro- thermal vents in 1977, just over 30% of the global mid-ocean ridge system has been March 2023 \| Oceanography 7 March 2023 \| Oceanography 7 7 Recorder) instrument attached to the dredge revealed evidence for a turbid- ity anomaly consistent with a nearby source of active black smoker venting at a depth of 2,800–3,400 m (Edmonds et al., 2003; Michael et al., 2003). During the PS86 AURORA expedition, CTD profil- ing, coupled with water column chemis- try, revealed further evidence for ongoing hydrothermal activity on the Aurora mound (Boetius, 2015; German et al., 2022a). Seabed surveys with the Ocean Floor Observation System (OFOS) deep- tow camera across the summit from north to south revealed deep rifts through the thick sedimented seafloor across the base of the volcanic mound. This imag- ing, paired with CTD data, led to the first imaging of an active black smoker on Gakkel Ridge at 82°53.83'N, 6°15.32'W, at ~3,900 m depth, on what was named the Aurora Vent Field (AVF; Boetius, 2015; German et al., 2022a). The OFOS surveys showed that the Aurora mound has steep vertical basalt walls intermixed with lower angle, sediment-draped steps. The top of the mound is flat and sediment covered, and the observed fauna con- sisted of high abundances of filter feeders, mostly glass sponges and anemones, and at least two species of shrimp. Ophiuroids, swimming polychaetes, and crustaceans (potentially isopods) were also observed. At the active vent site, bacterial mats and small gastropods and amphipods were observed (Boetius, 2015). The physico- chemical and microbiological character- ization of the huge buoyant vent plume hovering above the AVF showed evidence for venting fluids enriched in meth- ane, and possibly hydrogen, fueling high microbial activity in the plume (German et al., 2022a; Massimiliano Molari, Max Planck Institute for Marine Microbiology, pers. comm., 2022). Due to the lack of a deep-diving remotely operated vehicle (ROV), however, no physical samples of fluids, rocks, microbes, or animals could be collected from the vent field. at 4,200 m depth is bounded by steep rift valley walls and punctuated by a series of axial volcanic ridges and smaller vol- canic mounds (Michael et al., 2003). Edmonds et al. A SHORT HISTORY OF GAKKEL RIDGE EXPLORATIONit (2003) inferred the pres- ence of an active hydrothermal vent site from chemical data and assigned to a small (1.5–2 km in diameter) volcanic mound rising approximately 400 m from the seafloor, at depths between 4,300 m and 3,850 m (Figure 1b,c). A dredge from south to north across this mound recov- ered components of a sulfide chimney in addition to abundant pillow basalts. In parallel, in situ sensor data from a MAPR (Miniature Autonomous Plume a c b Gakkel Ridge Greenland Svalbard FIGURE 1. (a) Map of the Gakkel Ridge in the Central Arctic Ocean with known hydrothermal plume signals indicated by yellow stars (from Edmond et al., 2003) and the Aurora Vent Field marked with a red star. (b) A red triangle locates the Aurora Vent Field within the Aurora mound based on pre- vious bathymetry from the AMORE and AURORA/AWI expeditions. (c) The Aurora Vent Field (red triangle) is shown against multibeam bathymetry of the Aurora mound based on bathymetry from the HACON19 and HACON21 expeditions. a b Gakkel Ridge Greenland Svalbard a b Gakkel Ridge Greenland Svalbard c FIGURE 1. (a) Map of the Gakkel Ridge in the Central Arctic Ocean with known hydrothermal plume signals indicated by yellow stars (from Edmond et al., 2003) and the Aurora Vent Field marked with a red star. (b) A red triangle locates the Aurora Vent Field within the Aurora mound based on pre- vious bathymetry from the AMORE and AURORA/AWI expeditions. (c) The Aurora Vent Field (red triangle) is shown against multibeam bathymetry of the Aurora mound based on bathymetry from the HACON19 and HACON21 expeditions. c c In 2019, the Hot Vents in an Ice- Covered Ocean (HACON19) cruise on R/V Kronprins Haakon returned to the Aurora mound, with the aim of conduct- ing a multidisciplinary survey of the sea- floor ecosystems centered around the coordinates of the black smoker iden- tified in 2014 by the PS86 Aurora team (Boetius, 2015). This cruise obtained new visual data of the AVF with the towed Ocean Floor Observation and Bathymetry System (OFOBS; Purser et al., 2019; German et al., 2022a), con- firming the presence of at least three black smokers colonized by sparse fauna com- posed of mostly gastropods and amphi- pods (Bünz et al., 2019). In addition, a wealth of samples on the sedimented sur- face of the Aurora mound were collected, FIGURE 1. Oceanography \| Vol. 36, No. 1 8 A SHORT HISTORY OF GAKKEL RIDGE EXPLORATIONit (a) Map of the Gakkel Ridge in the Central Arctic Ocean with known hydrothermal plume signals indicated by yellow stars (from Edmond et al., 2003) and the Aurora Vent Field marked with a red star. (b) A red triangle locates the Aurora Vent Field within the Aurora mound based on pre- vious bathymetry from the AMORE and AURORA/AWI expeditions. (c) The Aurora Vent Field (red triangle) is shown against multibeam bathymetry of the Aurora mound based on bathymetry from the HACON19 and HACON21 expeditions. Oceanography \| Vol. 36, No. 1 8 Oceanography \| Vol. 36, No. 1 8 8 using coring equipment as well as the Woods Hole Oceanographic Institution hybrid ROV/AUV Nereus Under Ice (NUI), which collected sponges for tax- onomic analyses. However, technological challenges and ice conditions prevented NUI from reaching the vent field and col- lecting samples (Bünz et al., 2019). used to successfully dive on and sample deep hydrothermal vents under drifting ice with an ROV, providing an operational baseline for future robotic explorations of the deep Arctic. The second part of the paper provides a preliminary description of the Aurora Vent Field and its biologi- cal communities. open-water leads and pressure ridges between ice floes greatly influences the ability, time, and effort needed to reach the study area. When the vessel has reached the study area, the first step in planning ROV or OFOBS dives is to determine and pre- dict ice-drift velocity and direction in order to position the vessel within the context of current and developing ice conditions. Once the ROV is launched, the ship will no longer be able to reposi- tion, so it must be located “upstream” of the dive target. The aim is for the ROV to reach the seafloor ahead of the vent field/ study site, while the vessel drifts with the ice floe toward the study site. To deter- mine vessel positioning, we used satel- lite images that provided a regional over- view of sea ice conditions (i.e., ice floe sizes, their distribution, and the presence of open water or thin ice leads). In addi- tion, 24 hr predictions of regional ice drift velocity and direction provided daily by Drift+Noise Polar Services GmbH/AWI aided in positioning the vessel (SIDFEx, 2022). ROV DEEP-SEA EXPLORATION UNDER ICE The ocean surface above the Aurora mound is permanently covered by sea ice (Figure 2a). The maximum sea ice extent in the Arctic occurs at the end of winter, generally in March, and the min- imum extent is normally observed in September, at the end of the summer season, so mid-August to mid-October is the most suitable time window for oceanographic operations on the Gakkel Ridge. Reaching a specific study site in an ice-covered ocean is the first challenge to overcome. Ice floe size, coupled with the speed and direction of ice drift, will deter- mine the time it takes for a specific subsea location to be accessible. The speed and direction of ice drift varies within and between days, driven by tidal forces and wind conditions, creating an ever-chang- ing icescape for which we have limited predictability on the order of 6–12 hours (Boetius, 2015). The presence of thin-ice/ Later in 2021, the HACON21 cruise wrote the latest chapter in the explora- tion of Gakkel Ridge, contributing to the UN Decade of Ocean Science for Sustainable Development through the Challenger 150 programme (Howell et al., 2020). A multidisciplinary team sailed aboard R/V Kronprins Haakon from Longyearbyen (Svalbard) on September 29 to complete the first ROV survey and sampling of hydrother- mal vents under permanent ice cover in the Arctic (Bünz et al., 2021). Below, we describe the methodological approach b b FIGURE 2. (a) R/V Kronprins Haakon navigates a thin-ice lead between ice floes above the Aurora Vent Filed. © REV Ocean/L. Hislop (b) This satel- lite image shows the regional ice situation and the ice drift predictions (red dots) modeled by Drift+Noise Polar Services GmbH/AWI. The darker areas are open water or thin ice. The gray/white coloring indicates thicker ice. The yellow star indicates the position of the Aurora Vent Field. (c) ROV Aurora Borealis is recovered through the moonpool of R/V Kronprins Haakon along with its 1,000 m tether management system (TMS). a c a FIGURE 2. (a) R/V Kronprins Haakon navigates a thin-ice lead between ice floes above the Aurora Vent Filed. © REV Ocean/L. Hislop (b) This satel- lite image shows the regional ice situation and the ice drift predictions (red dots) modeled by Drift+Noise Polar Services GmbH/AWI. The darker areas are open water or thin ice. The gray/white coloring indicates thicker ice. The yellow star indicates the position of the Aurora Vent Field. A SHORT HISTORY OF GAKKEL RIDGE EXPLORATIONit These ice maps and ice-drift mod- els covered an area of 360 km2 around the Aurora mound and were useful for predicting when open-water leads would be positioned above the vent field (Figure 2b). With a range of six nautical miles, the vessel’s ice radar aided naviga- tion among ice floes. Between June and September 2021, the Joint Arctic Scientific Mid-Ocean Ridge Insight Expedition (JASMInE) car- ried out the first wide-angle reflection/ refraction seismic experiment along the Gakkel Ridge, from 75° to 102°, to map lithospheric structure (Ding et al., 2022). Preliminary results contribute to the understanding of oceanic crustal forma- tion and episodic magmatism in this end- member of global oceanic crustal accre- tion (Ding et al., 2022). ROV DEEP-SEA EXPLORATION UNDER ICE (c) ROV Aurora Borealis is recovered through the moonpool of R/V Kronprins Haakon along with its 1,000 m tether management system (TMS). FIGURE 2. (a) R/V Kronprins Haakon navigates a thin-ice lead between ice floes above the Aurora Vent Filed. © REV Ocean/L. Hislop (b) This satel- lite image shows the regional ice situation and the ice drift predictions (red dots) modeled by Drift+Noise Polar Services GmbH/AWI. The darker areas are open water or thin ice. The gray/white coloring indicates thicker ice. The yellow star indicates the position of the Aurora Vent Field. (c) ROV Aurora Borealis is recovered through the moonpool of R/V Kronprins Haakon along with its 1,000 m tether management system (TMS). March 2023 \| Oceanography 9 March 2023 \| Oceanography 9 fly faster than the boat drifts with the ice. This allows the ROV to reach the target before the vessel drifts over it, providing additional time for the ROV to work on the seafloor on a small target such as a vent field (Figure 3). Operations through the moonpool protected the ROV and its TMS from ice contact and ensured that an open area was always available for the recovery of the ROV. The oper- ational methodology described below aims at maximizing seafloor time on a small target (in this case, the active area of the Aurora Vent Field, only about 75 m2) at great depth (4,000 m) under drifting ice. If the goal was to conduct a survey in a region without a precise geo- graphic target (e.g., a biological or sedi- mentological transect in a region, to sam- ple opportunistically), vessel positioning and ice drift velocity and direction would be less critical. ahead of the vessel toward the vent field. Drift speeds up to 0.3 knots provided suf- ficient bottom time (1–2.5 hr) for the ROV to work on the seafloor while sta- tionary at a target location. Operations between 0.3 knots and 0.6 knots were feasible but challenging, and seafloor time at the study area was very lim- ited (20–40 min). Above 0.6 knots, the risk of operations was considered too high and the ROV was not launched. If the distance between launch and study area was too large, the probability of the ice drift changing direction during the ROV’s descent, taking the vessel away from the target area, was higher, particu- larly with slower ice drift <0.1 knot. ROV DEEP-SEA EXPLORATION UNDER ICE If the launch position was too close to the vent field, there was not sufficient time for the ROV to reach the seafloor before the ves- sel drifted beyond the study site and the ROV had to be recovered. Of the 14 times ROV Aurora was launched over the AVF, the dives had to be aborted on six occa- sions due to a change in ice drift direction and/or speed that took the vessel off-track from the vent field. On one occasion, the drift direction changed from south to southeast, and then east-northeast within hours, which resulted in the ROV being launched and recovered four times before we were able to complete a successful dive. ROV Operations ROV Aurora is a Kystdesign SUPPORTER 32-type ROV (Figure 2c) owned and operated by REV Ocean. It has a 6,000 m depth range and is fully equipped with a state-of-the-art science skid. Importantly for under ice operations, Aurora is con- figured to operate as part of a two- bodied system that comprises a separate tether management system (TMS) called Borealis, with an additional 1,000 m of neutrally buoyant tether. For protec- tion against adverse interactions with sea ice, the ROV was deployed through the moonpool of the Norwegian ice- breaker R/V Kronprins Haakon. The Aurora Borealis system descended as a unit toward the seafloor, which per- mitted a rapid descent (0.8–1 m s–1) of the vehicle while the vessel was drift- ing with the ice, avoiding tension on the ROV’s neutrally buoyant optical tether (Figure 3). When the vehicle was at 50–100 m above seafloor, depending on topography, the winch was stopped and the ROV deployed from its TMS. Safe ROV operations to the Arctic deep sea- floor were only possible during this expe- dition because of the two-bodied config- uration of the ROV and the availability of a moonpool for deployment and recovery operations. The soft umbilical of the TMS enables the ROV, once out of its TMS, to For the purpose of studying the AVF, once a suitable position for the vessel was determined, the speed and accurate direc- tion of the ice floe were assessed by posi- tioning the vessel against the ice floe and drifting with it for 15 minutes. Timing for ROV deployment could then be based on the drift speed. ROV DEEP-SEA EXPLORATION UNDER ICE ROV Aurora was launched at a distance from the vent field that allowed the vehicle to reach the sea- floor upstream from the study area and fly Geological and Geochemical Settingsh FIGURE 3. Operational sequence for ROV dives on small deep seafloor targets under drifting ice. The Aurora Vent Field is located on the southwest part of the Aurora mound (Figure 1b,c). It consists of pillow basalts and active and inactive vents, and encom- passes an area of ~140 m × 100 m. There are steep-sided inactive hydrothermal edifices up to several meters high, indi- vidual actively venting chimneys, and abundant hydrothermal crust and chim- ney debris that, along with the pillows, are variably covered by as much as ~3 m of sediment. Current hydrothermal activity is restricted to near the center of the AVF (82°53'49''N, 6°15'21''W) and consists primarily of three isolated, vigorously FIGURE 3. Operational sequence for ROV dives on small deep seafloor targets under drifting ice. Oceanography \| Vol. 36, No. 1 10 Oceanography \| Vol. 36, No. 1 10 10 of Enceladus, slightly south of a line between Enceladus and Hans Tore vents. This black smoker rises on a relatively flat seafloor and consists of black smoker fluid venting from five individual exit ori- fices at the base or sides of a partially col- lapsed chimney structure. Both Enceladus and Ganymede are characterized by thin walls, resulting in frequent collapse and regrowth, as evidenced by their relatively short heights and ramparts of abundant collapsed chimney debris. venting black smoker chimneys that occur in a ~10 m diameter cluster. These three active black smokers have been named Hans Tore, Enceladus, and Ganymede (Figure 4a,c,e). The Hans Tore vent was observed with OFOBS during HACON19 (Bünz et al., 2019). It was named in memory of Professor Hans Tore Rapp (Figure 4b), colleague and friend from the University of Bergen, who passed away in 2020. Prof. Rapp had been involved in the 2014 AURORA PS86 and HACON19 missions as fauna expert and was a key figure in the development of the HACON project. The two other smokers are named after “ocean world” moons in the solar system, some of which may host hydrothermal activity with the potential to harbor associated chemo- synthetic-based life. Saturn’s ice-covered moon Enceladus (Figure 4d) was chosen because its ocean is suspected to be many tens of kilometers deep and predicted to host hydrothermal activity (Cable et al., 2021). Geological and Geochemical Settingsh Ganymede, an ice-covered satellite of Jupiter (Figure 4f), is the largest moon in the solar system and may have the largest saline ocean—an ocean that may be implicated in altering its polar aurora (Saur et al., 2015), providing a fitting con- nection to this locale. The rocks collected are generally dark greenish-gray and composed primarily of chalcopyrite, pyrite, sphalerite, and barite. The sulfide minerals exhibit varying degrees of oxidation associated with pro- longed exposure to seawater (Figure 6a). Samples were also collected from several low-lying orange-brown oxidized iron- rich chimneys associated with lower- temperature diffuse flow, especially those surrounding the rim of the Hans Tore vent crater. The formation of these extremely friable iron-rich deposits has been associ- ated with microbial activity (Johannessen et al., 2020). Post-cruise analyses of the The chimneys and larger hydrothermal accumulations at the AVF were sampled with the ROV manipulator (Figure 5a). FIGURE 4. Aurora Vent Field black smokers and their names: (a) Hans Tore vent. (b) Professor Hans Tore Rapp during the HACON cruise in 2019. (c) Enceladus black smoker. (d) Enceladus moon. Credit: NASA, Cassini Mission (e) Ganymede black smoker. (f) Ganymede moon. Credit: NASA, Juno Mission a c e b d f b a a c d c d The Hans Tore vent (Figure 4a), found at 3,883 m depth, features a tall (at least 2 m), narrow chimney vigorously emit- ting black smoke fluid from its summit and a second, 1 m tall active chimney next to it. They sit in the middle of a 2 m diameter, 1 m deep circular crater, and black smoker fluid issues from the bot- tom of the crater through two small chim- neys. Diffuse, lower-temperature vent- ing occurs around the rim of the crater, and small oxidized iron-rich chimneys are evident at the rim. At the Enceladus vent (Figure 4c), ~10 m southwest of Hans Tore, a single 1.5 m tall chimney structure emits black vent fluid along its entire length and from its top. It is sur- rounded by a forest of inactive chimneys at 3,887 m depth.h e f f e FIGURE 4. Aurora Vent Field black smokers and their names: (a) Hans Tore vent. (b) Professor Hans Tore Rapp during the HACON cruise in 2019. (c) Enceladus black smoker. (d) Enceladus moon. Credit: NASA, Cassini Mission (e) Ganymede black smoker. (f) Ganymede moon. March 2023 \| Oceanography 11 Geological and Geochemical Settingsh Credit: NASA, Juno Mission The Ganymede vent (Figure 4e) is located at 3,884 m depth, ~5 m northeast March 2023 \| Oceanography 11 11 2012) that will enable first estimates of fluid mass, chemical, and heat fluxes from the vents. The ongoing work will be criti- cal for deciphering the types of chemical compositions venting at Aurora and how they support the ecosystem. The data will also provide information on the water- rock interactions between hydrothermal fluids and the underlying crustal substrate (e.g., Reeves et al., 2011, 2014), allowing direct comparison of the chemical energy “landscape” of AVF fluids (e.g., Dahle et al., 2015) with other vent sites along the global mid-ocean ridge system. paleo-plume events recorded in the sed- imentary record therefore enables us to provide spatial and temporal con- straints on vent activity and the contri- bution of seafloor vent metal exports to deep ocean budgets (Antonelli et al., 2017). Hydrothermal plumes also trans- port trace elements, nutrients, dissolved gases, and vent-derived biomass that can support heterotrophic deep-sea commu- nities (Cathalot et al., 2021; Levin et al., 2016). At the AVF, the dispersing plume was identified from water-mass turbid- ity measured during CTD casts. The buoyant plume was located in the bot- tom layer between 3,000 m and 4,000 m depth during the AURORA (Boetius, 2015; German et al., 2022), HACON19, and HACON21 cruises. Dissolved meth- ane concentrations in water samples col- lected from ROV-mounted Niskin bottles during HACON21 (Figure 5c) at ~10 m directly above one of the vents were measured using headspace gas chro- matography (e.g., Sert et al., 2020). In these samples, elevated dissolved meth- ane in the near vent buoyant plume (~102–103 nM) decreases dramatically to 30 nmol/L in the immediately over- lying non-buoyant plume (German et al., 2022a), 1–10 nmol/L in the more distal plume (Sert et al., 2022), and eventually to background concentrations (~<0.5 nM). This shows that strong methane gradi- ents that prevail in the vicinity of the ris- ing buoyant plume could support chemo- synthetic microbial activity in the water column (Anantharaman et al., 2013). In buoyant and non-buoyant plumes, meth- ane dilution, oxidation, and methanogen- esis may occur simultaneously (Ver Eecke et al., 2012), contributing to deep-sea bio- geochemical cycling. Geological and Geochemical Settingsh rock samples will be integrated with sed- iment geochemistry data sets and used to characterize the composition, age, and evolution of the vent field and to assess the relationship between the composition and age of the deposits and the animals and microbes that colonize them. paleo-plume events recorded in the sed- imentary record therefore enables us to provide spatial and temporal con- straints on vent activity and the contri- bution of seafloor vent metal exports to deep ocean budgets (Antonelli et al., 2017). Hydrothermal plumes also trans- port trace elements, nutrients, dissolved gases, and vent-derived biomass that can support heterotrophic deep-sea commu- nities (Cathalot et al., 2021; Levin et al., 2016). At the AVF, the dispersing plume was identified from water-mass turbid- ity measured during CTD casts. The buoyant plume was located in the bot- tom layer between 3,000 m and 4,000 m depth during the AURORA (Boetius, 2015; German et al., 2022), HACON19, and HACON21 cruises. Dissolved meth- ane concentrations in water samples col- lected from ROV-mounted Niskin bottles during HACON21 (Figure 5c) at ~10 m directly above one of the vents were measured using headspace gas chro- matography (e.g., Sert et al., 2020). In these samples, elevated dissolved meth- ane in the near vent buoyant plume (~102–103 nM) decreases dramatically to 30 nmol/L in the immediately over- lying non-buoyant plume (German et al., 2022a), 1–10 nmol/L in the more distal plume (Sert et al., 2022), and eventually to background concentrations (~<0.5 nM). This shows that strong methane gradi- ents that prevail in the vicinity of the ris- ing buoyant plume could support chemo- synthetic microbial activity in the water column (Anantharaman et al., 2013). In buoyant and non-buoyant plumes, meth- ane dilution, oxidation, and methanogen- esis may occur simultaneously (Ver Eecke et al., 2012), contributing to deep-sea bio- geochemical cycling. The Enceladus and Ganymede black smoker “end member” fluids and their stable maximum temperatures were each successfully sampled with duplicate iso- baric gas-tight (IGT) samplers (Seewald et al., 2002; Figure 5b) for comprehensive inorganic-organic geochemical and stable isotope characterization (Reeves et al., 2011, 2014); “paired” chimney material was also examined. Geological and Geochemical Settingsh Sufficient static video footage (several minutes) of at least one of the vents was also taken to allow for parti- cle image velocimetry (Mittelstaedt et al., Hydrothermal fluids discharged at the seafloor form neutrally buoyant mid-water plumes that disperse hori- zontally along isopycnals, transporting and redistributing metals across entire basins (Resing et al., 2015). Correlating FIGURE 5. Sampling on the Aurora Vent Field. (a) Rock collection with the manipulator on the flanks of the Hans Tore vent. (b) Isobaric gas-tight (IGT) sampling at Ganymede. (c) Plume sampling with Niskin bottles on the Hans Tore vent. (d) Blade core sampling on the side of the Enceladus black smoker. (e) Sampling bacterial mats with the biosyringe on the rim of the Hans Tore vent. (f) Suction sampling for amphipods on the flanks of the Hans Tore vent. a c e b d f a b b a d c c d c e f Oceanography \| Vol. 36, No. 1 12 Biological Communities of the Aurora Vent Field Deciphering geo-biological connec- tions in hydrothermal systems requires detailed analyses of the composition of fluids along with analyses of micro- bial community structure and function- ing. Samples for microbiological analyses were obtained from active and inactive chimneys, low-temperature diffuse flow sites, and background sediments. The rocks collected for geological studies (Figure 5a) were firstly sub-sampled on board for their microbiota. The blade core was used to sample undisturbed sediment samples (Figure 5d), and bio- syringes were used to sample microbial mats (Figure 5e). Ongoing geochem- ical and metagenomic-based commu- nity analyses will shed further light on the AVF microbial community structure and functioning and the biogeochemical relationships between sediment substrate (grain size, mineralogy, and geochemical composition) and microbial and faunal communities. The results will reveal to what extent the unique setting of the AVF stimulates development of unique micro- bial assemblages and ecosystems. and oxidized compounds in seawater (e.g., sulfate, nitrate, oxygen) (Van Dover, 2000). The availability of different chem- ical energy sources between and within vent systems form chemical energy land- scapes that shape microbial communities and possibly those of microbial primary production grazers (Dahle et al, 2015). Deciphering geo-biological connec- tions in hydrothermal systems requires detailed analyses of the composition of fluids along with analyses of micro- bial community structure and function- ing. Samples for microbiological analyses were obtained from active and inactive chimneys, low-temperature diffuse flow sites, and background sediments. The rocks collected for geological studies (Figure 5a) were firstly sub-sampled on board for their microbiota. The blade core was used to sample undisturbed sediment samples (Figure 5d), and bio- syringes were used to sample microbial mats (Figure 5e). Ongoing geochem- ical and metagenomic-based commu- nity analyses will shed further light on the AVF microbial community structure and functioning and the biogeochemical relationships between sediment substrate (grain size, mineralogy, and geochemical composition) and microbial and faunal communities. The results will reveal to what extent the unique setting of the AVF stimulates development of unique micro- bial assemblages and ecosystems. Samples of macro- and megafauna were obtained from chimney debris collected FIGURE 6. Samples from the Aurora Vent Field. (a) Representative chimney sample collected from the Enceladus black smoker, composed primarily of a mixture of sulfide minerals (chalcopy- rite, pyrite, sphalerite) and barite. (b) Rock hosting Cocculinid limpets and gastropods (scale bar = 5 mm). Biological Communities of the Aurora Vent Field Biological communities in hydrother- mal systems are typically driven by che- mosynthetic microorganisms acquir- ing energy from the oxidation of reduced compounds in hydrothermal fluids (e.g., sulfide, methane, hydrogen) FIGURE 5. Sampling on the Aurora Vent Field. (a) Rock collection with the manipulator on the flanks of the Hans Tore vent. (b) Isobaric gas-tight (IGT) sampling at Ganymede. (c) Plume sampling with Niskin bottles on the Hans Tore vent. (d) Blade core sampling on the side of the Enceladus black smoker. (e) Sampling bacterial mats with the biosyringe on the rim of the Hans Tore vent. (f) Suction sampling for amphipods on the flanks of the Hans Tore vent. Oceanography \| Vol. 36, No. 1 12 Oceanography \| Vol. 36, No. 1 12 12 kilometers away from the AVF are rep- resented by opportunistic species depen- dent on bacterial and phytodetrital matter, including low-energy species (Vanreusel et al., 2000). Meiofaunal community densities and diversity in hydrothermal- derived sediments were depleted when compared to communities within refer- ence sediments off vent. Benthic fora- minifera in these hydrothermal-derived sediments, however, include organic- walled allogromids that seem to tolerate reduced and low amounts of organic car- bon microhabitats. These initial results suggest a critical role of the meiofauna in the vicinity of vents for biodiversity and potential bio-indicators for ecosystem health in vent fields. with the ROV manipulators (Figure 5a), from the sediment surface collected with the suction sampler (Figure 5f), and from sponge aggregations at the vent periph- ery (Figure 6f). Preliminary analyses show an increased presence of molluscs, crustaceans, and polychaetes in the AVF compared to the surrounding rock fields. Within the molluscs, a new species of cocculinid limpet, Cocculina aurora sp. nov. (Chen et al., 2022; Figure 6b) and two species of gastropods of the fami- lies Rissoidae and Skeneidae (Figure 6c) were the most abundant taxa found on the rocks. Samples collected with the suc- tion sampler targeted amphipods, tenta- tively identified as belonging to the family Melitidae (Figure 6d,e), the second most dominant group at the active vent field after the molluscs. and oxidized compounds in seawater (e.g., sulfate, nitrate, oxygen) (Van Dover, 2000). The availability of different chem- ical energy sources between and within vent systems form chemical energy land- scapes that shape microbial communities and possibly those of microbial primary production grazers (Dahle et al, 2015). Biological Communities of the Aurora Vent Field (c) Detail of the rock in B showing the high abundance of gastropods (scale bar = 5 mm). (d) Amphipods on the rim of the Hans Tore vent. (e) Close-up of an amphipod in the lab (scale bar = 5 mm). (f) Cladorhizid carnivorous sponges on the periphery of the Aurora Vent Field. a c e b d f b b a d f d Infauna from sediments recovered from the vent field with the blade core (Figure 5d) and from a reference site a few kilometers off vent with a ship-operated multicorer were sectioned at different intervals, sieved with 1,000-, 500-, 250-, 150-, and 32-micron mesh sieves and identified under a microscope in the lab- oratory. The infauna was composed, pre- dominantly, of meiofaunal taxa (32 µm to 1,000 µm), specifically free-living nema- todes and foraminifera. These groups are known to be a key link in the deep-sea food web, including in hydrothermal vent and cold seep environments (Zeppilli et al., 2018). Classical abyssal polar meio- fauna communities in sediments a few e FIGURE 6. Samples from the Aurora Vent Field. (a) Representative chimney sample collected from the Enceladus black smoker, composed primarily of a mixture of sulfide minerals (chalcopy- rite, pyrite, sphalerite) and barite. (b) Rock hosting Cocculinid limpets and gastropods (scale bar = 5 mm). (c) Detail of the rock in B showing the high abundance of gastropods (scale bar = 5 mm). (d) Amphipods on the rim of the Hans Tore vent. (e) Close-up of an amphipod in the lab (scale bar = 5 mm). (f) Cladorhizid carnivorous sponges on the periphery of the Aurora Vent Field. FIGURE 6. Samples from the Aurora Vent Field. (a) Representative chimney sample collected from the Enceladus black smoker, composed primarily of a mixture of sulfide minerals (chalcopy- rite, pyrite, sphalerite) and barite. (b) Rock hosting Cocculinid limpets and gastropods (scale bar = 5 mm). (c) Detail of the rock in B showing the high abundance of gastropods (scale bar = 5 mm). (d) Amphipods on the rim of the Hans Tore vent. (e) Close-up of an amphipod in the lab (scale bar = 5 mm). (f) Cladorhizid carnivorous sponges on the periphery of the Aurora Vent Field. The Ice-Covered Arctic as a Bridge to the Search for Life in Space? g to the Search for Life in Space? A particularly exciting aspect of the dis- covery of hydrothermal vents beneath an ice-covered ocean is its impact on humanity’s search for life beyond Earth. The collection and analysis of 18 full sea- ice cores ranging from 98 cm to 181 cm in length, as well as three gray ice cores rang- ing from 8 cm to 15 cm, served the dual purpose of advancing our understanding of sea-ice processes on Earth and provid- ing a useful analog for investigating the surface chemistry of distant ice-covered ocean worlds elsewhere in our solar sys- tem. On Earth, the seasonally variable sea-ice cover modulates the exchange of gases between the ocean and the atmo- sphere. During the winter period of max- imum coverage, and of sea-ice growth, gases such as carbon dioxide and meth- ane cannot escape directly to the atmo- sphere, but rather are captured in the ice matrix. Similarly, gases from the atmo- sphere cannot reach the ocean, as mixing is inhibited by the ice layer. In addition, some buoyant materials, such as organic lipids and other biological materials, can become entrained in the ice matrix. The seasonal exchange efficiency of gases in the Arctic, especially carbon dioxide, is a poorly constrained variable in our efforts to model and understand climate change. We conducted analyses of trapped car- bon dioxide and methane in many of the cores while onboard R/V Kronprins Haakon and have generated depth pro- files of gas concentration that show the detailed transition from atmospheric to oceanic concentrations of carbon dioxide and methane. In addition, we measured the 13C concentration in methane, which we can use to differentiate between bio- genic and abiogenic contributions to the A particularly exciting aspect of the dis- covery of hydrothermal vents beneath an ice-covered ocean is its impact on humanity’s search for life beyond Earth. h Although a detailed biogeographic analysis that includes the fauna from the Aurora Vent Field is underway, the cur- rently available observations provide insights into the position of the Gakkel Ridge vent communities in global vent biogeography. Six biogeographic regions of vent fauna defined by Bachraty et al. (2009) were increased to 11 regions of biogeography when the data were reana- lyzed, incorporating the vent fields of the Southern Ocean (Rogers et al., 2012). However, none of these studies included data from Arctic latitudes. Biological Communities of the Aurora Vent Field March 2023 \| Oceanography 13 March 2023 \| Oceanography 13 13 pillow lavas, and basaltic ridges and out- crops in the proximity of the vent field were covered by dense aggregations of the hexactinellid sponges Caulophacus arcticus and Asconema megaatrialia. These species are common inhabitants of deeper areas in the Nordic Seas (Roberts et al., 2018), and the unusually high den- sities found at the Aurora mound com- pared to non-venting mounds on the Gakkel Ridge (Boetius, 2015) suggest that the higher productivity around the active vent field provides increased food supply that can support higher sponge density. Aggregations of sponges and dead stalks of C. arcticus may have a crucial facilitat- ing role for other fauna, since bythocarid shrimps, anemones, crinoids, and several species of isopods and amphipods seem to be more abundant in areas covered by hexactinellid sponge habitats. depths under drifting ice, which heavily constrains our capacity to develop robust management measures and monitoring procedures for a pristine region prior to the expected increase in industrial activ- ity as the Central Arctic Ocean opens to human activities. At the Hans Tore vent, amphipods and small gastropods can be seen on the rim and outer slopes of the crater. At Enceladus and Ganymede, amphi- pods are evident on the chimney walls, whereas gastropods are mostly pres- ent on chimney debris at the base of the black smokers. Suction sampling at the Hans Tore and Ganymede vents also retrieved a few specimens of polychaetes and some empty polychaete tubes. The AVF vent fauna, dominated by small gas- tropods and amphipods, is similar, on a higher taxonomic level, to the faunal com- munities of the Loki’s Castle Vent Field at 73.5°N on the Mohns Ridge (Pedersen et al., 2010). Further work on the taxon- omy, connectivity, and biogeography of the fauna of the AVF is ongoing in order to clarify the links between the AVF fauna and that of other vent fields. In contrast to the Mohns Ridge vents, sedimented areas with diffuse venting were not observed, which explains the restricted spatial dis- tribution of vent-adapted fauna close to the black smokers. Basalt outcrops within the vent field host dense aggregations of carnivorous sponges belonging to the family Cladorhizidae (Figure 6f). Oceanography \| Vol. 36, No. 1 14 Biological Communities of the Aurora Vent Field Even though these organisms are typical rep- resentatives of the background fauna, they can thrive at higher densities in the vicinity of active vents by possibly taking advantage of the food-enriched environ- ment driven by the hydrothermal activity, while tolerating increased abiotic stress (Levin et al., 2016; Georgieva et al., 2020). The Ice-Covered Arctic as a Bridge to the Search for Life in Space? Connection of this work to distant ice-covered moons, such as Jupiter’s Europa, is based on the knowledge that Europa’s ice shell is essentially ~10 km of sea ice (Figure 7). Ocean salts, carbon dioxide, and other materials have been observed spectroscopically on the sur- face of Europa (Hand et al., 2007; Hand and Carlson, 2015; Trumbo et al., 2019). The best analogous systems on Earth to Europa’s ice are the sea ice of the Arctic and the Antarctic. While the scale of ice thickness is much different, the pro- cesses by which gases and organic mate- rials, including microbes, are incorpo- rated into sea ice on Earth can inform the development of missions and instru- ments designed to search for such mate- rials either remotely or with a landed spacecraft on Europa. To this end, our team processed seven ice cores for micro- biology primarily to determine whether any thermophiles that may have been transported via hydrothermal plumes are captured in the ice cores. This is an interesting microbial ecology question in its own right, but it also carries interest- ing implications for the search for bio- signatures on Europa, as the seafloor there could be hydrothermally active, and plumes could deliver material to the ice-water interface. FIGURE 7. While the physical scale and thickness of the ice differs greatly between Earth’s sea ice and that of Europa’s ice shell, both icy environments serve as a window into the oceans hidden below. On Earth, we can study the ice, ocean, and seafloor to reveal any processes that provide connections across these interfaces. On Europa, we are, at least for the near future, limited to using the surface chemistry of the ice as our primary means for understanding the ocean and seafloor chemistry and geology. Image courtesy NASA/JPL and the 2017 Europa Lander Science Definition Team Report (Hand et al., 2017) of photosynthesis on Earth, but some of the microbes detected in the Aurora vent plume relate to microaerophilic or even anaerobic types (Massimiliano Molari, Max Planck Institute for Marine Microbiology, pers. comm., 2022). The intellectual challenge in pursuing this line of research becomes more compel- ling when considering that a significant proportion of the exoplanets that have now been discovered orbiting other stars may also be ocean worlds (Quick et al., 2020). A next critical step in that explora- tion will begin soon, with the 2023 launch of the European Space Agency’s JUpiter ICy moons Explorer (JUICE) expedi- tion to investigate Europa and Ganymede and the 2024 launch of NASA’s Europa Clipper mission to study that body’s hab- itability. In the longer term, planning has already begun for future landed missions to search for evidence of life on both Europa (Hand et al., 2022) and Enceladus (MacKenzie et al., 2021). of photosynthesis on Earth, but some of the microbes detected in the Aurora vent plume relate to microaerophilic or even anaerobic types (Massimiliano Molari, Max Planck Institute for Marine Microbiology, pers. comm., 2022). The intellectual challenge in pursuing this line of research becomes more compel- ling when considering that a significant proportion of the exoplanets that have now been discovered orbiting other stars may also be ocean worlds (Quick et al., 2020). A next critical step in that explora- tion will begin soon, with the 2023 launch of the European Space Agency’s JUpiter ICy moons Explorer (JUICE) expedi- tion to investigate Europa and Ganymede and the 2024 launch of NASA’s Europa Clipper mission to study that body’s hab- itability. In the longer term, planning has already begun for future landed missions to search for evidence of life on both Europa (Hand et al., 2022) and Enceladus (MacKenzie et al., 2021). The Ice-Covered Arctic as a Bridge to the Search for Life in Space? Our obser- vations of the Aurora Vent Field sug- gest faunal similarities with the Loki’s Castle Vent Field at 73°N (Pedersen et al., 2010) in terms of the presence of amphi- pods and gastropods as main groups, but also show differences such as the lack of siboglinid tubeworms and the presence of the new species of limpet Cocculina aurora sp. nov. (Chen et al., 2022). The evolution and genetic connectivity of fauna from the deep Central Arctic Ocean is a major unknown, and the data from the HACON project will contrib- ute to addressing it. 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REFERENCES Muhammed Fatih Sert is PhD Candidate, CAGE, Department of Geoscience, UiT - The Arctic University of Norway, Tromsø, Norway. Ida H. Steen is Professor, Department of Biological Sciences & Centre for Deep Sea Research, University of Bergen, Bergen, Norway. Marie Stetzler is PhD Candidate, CAGE, Department of Geoscience, UiT - The Arctic University of Norway, Tromsø, Norway. Runar Stokke is Researcher, Department of Biological Sciences & Centre for Deep Sea Research, University of Bergen, Bergen, Norway. Lissette Victorero is Postdoctoral Researcher, Norwegian Institute for Water Research, Oslo, Norway. Francesca Vulcano is PhD Candidate, Department of Biological Sciences & Centre for Deep Sea Research, University of Bergen, Bergen, Norway. Stig Vågenes is Head Engineer, Department of Biological Sciences & Centre for Deep Sea Research, University of Bergen, Bergen, Norway, and REV Ocean, Lysaker, Norway. Kate Alyse Waghorn is Postdoctoral Fellow, and Stefan Buenz is Team Leader, both at the CAGE, Department of Geoscience, UiT - The Arctic University of Norway, Tromsø, Norway. Saur, J., S. Duling, L. Roth, X. Jia, D.F. Strobel, P.D. Feldman, U.R. Christensen, K.D. Retherford, M.A. McGrath, F. Musacchio, and others. 2015. The search for a subsurface ocean in Ganymede with Hubble Space Telescope observations of its auro- ral ovals. Journal of Geophysical Research: Space Physics 120:1,715– 1,737, http://doi.org/10.1002/ 2014JA020778. ACKNOWLEDGMENTS ACKNOWLEDGMENTS We would like to thank the captains, officers, and crew of R/V Kronprins Haakon and R/V Polarstern, the teams of ROV Aurora, ROV/AUV NUI, and OFOBS, as well as all participants in the cruises for their invaluable contributions while at sea. The HACON project was funded by the Norwegian Research Council (grant # 274330). 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Autun Purser is Senior Researcher, Alfred Wegener Institute, Helmholtz Centre for Polar & Marine Research, Bremerhaven, Germany. Sofia P. Ramalho is Assistant Researcher, Centre for Environmental and Marine Studies & Biology Department, University of Aveiro, Campus Universitário de Santiago, Aveiro, Portugal. Eoghan P. Reeves is Associate Professor, Department of Earth Science & Centre for Deep Sea Research, University of Bergen, Bergen, Norway. Leighton Rolley is Science Systems Manager, REV Ocean, Lysaker, Norway. Samuel I. Pereira is PhD Candidate, Department of Earth Science & Centre for Deep Sea Research, University of Bergen, Bergen, Norway. Pedro A. Ribeiro is Researcher, Department of Biological Sciences & Centre for Deep Sea Research, University of Bergen, Bergen, Norway. ARTICLE CITATION Vanreusel, A., L. Clough, K. Jacobsen, W. Ambrose, J. Jivaluk, V. Ryheul, R. Herman, and M. Vincx. 2000. 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Proceedings of the National Academy of Sciences of the United States of America 109:13,674–13,679, https://doi.org/10.1073/pnas.1206632109. AUTHORS i AUTHORS Eva Ramirez-Llodra (eva.ramirez@niva.no) is Science Coordinator, REV Ocean, Lysaker, and Senior Scientist, Norwegian Institute for Water Research, Oslo, Norway. Claudio Argentino is Postdoctoral Fellow, Centre for Arctic Gas Hydrate, Environment and Climate (CAGE), Department of Geoscience, UiT - The Arctic University of Norway, Tromsø, Norway. Maria Baker is Executive Director at Deep-Ocean Stewardship Initiative, University of Southampton, Southampton, UK. Antje Boetius is Professor, Alfred Wegener Institute, Helmholtz Centre for Polar & Marine Research, Bremerhaven, Germany; Max Planck Institute for Marine Microbiology, Bremen, Germany; and Center for Marine Environmental Sciences (MARUM), University of Bremen, Germany. Carolina Costa is a gradu- ate student in the Centre for Environmental and Marine Studies & Biology Department, University of Aveiro, Campus Universitário de Santiago, Aveiro, Portugal. Håkon Dahle is Professor, and Emily M. Denny is PhD Candidate, both in the Department of Biological Sciences & Centre for Deep Sea Research, University of Bergen, Bergen, Norway. Pierre-Antoine Dessandier is Postdoctoral Fellow, CAGE, Department of Geoscience, UiT - The Arctic University of Norway, Tromsø, Norway, and Researcher, University of Brest, CNRS, Ifremer, Plouzané, France. Mari H. Eilertsen is Postdoctoral Fellow, Department of Biological Sciences & Centre for Deep Sea Research, University of Bergen, Bergen, Norway. Benedicte Ferre is Team Leader, CAGE, Department of Geoscience, UiT - The Arctic University of Norway, Tromsø, Norway. Christopher R. German is Senior Scientist, Woods Hole Oceanographic Institution, Woods Hole, MA, USA. 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https://openalex.org/W4393866879	https://www.frontiersin.org/journals/digital-health/articles/10.3389/fdgth.2024.1359771/pdf	English	null	Accuracy, concurrent validity, and test–retest reliability of pressure-based insoles for gait measurement in chronic stroke patients	Frontiers in digital health	2,024	cc-by	7,132	Accuracy, concurrent validity, and test–retest reliability of pressure-based insoles for gait measurement in chronic stroke patients EDITED BY Paola Saccomandi, Polytechnic University of Milan, Italy REVIEWED BY Yumeng Yao, University of Shanghai for Science and Technology, China Alessandra Angelucci, Polytechnic University of Milan, Italy CORRESPONDENCE Chris A. Easthope chris.awai@llui.org †These authors have contributed equally to this work and share ﬁrst authorship RECEIVED 21 December 2023 ACCEPTED 11 March 2024 PUBLISHED 03 April 2024 CITATION Neumann S, Bauer CM, Nastasi L, Läderach J, Thürlimann E, Schwarz A, Held JPO and Easthope CA (2024) Accuracy, concurrent validity, and test–retest reliability of pressure- based insoles for gait measurement in chronic stroke patients. Front. Digit. Health 6:1359771. Saskia Neumann 1,2†, Christoph M. Bauer 3† , Luca Nastasi 1,2, Julia Läderach 2, Eva Thürlimann 4, Anne Schwarz 4, Jeremia P. O. Held 4 and Chris A. Easthope 1,2 Saskia Neumann 1,2†, Christoph M. Bauer 3† , Luca Nastasi 1,2, Julia Läderach 2, Eva Thürlimann 4, Anne Schwarz 4, Jeremia P. O. Held 4 and Chris A. Easthope 1,2* 1DART, Lake Lucerne Institute, Vitznau, Switzerland, 2Cereneo Foundation, Vitznau, Switzerland, 3Therapy Science Group, Lake Lucerne Institute, Vitznau, Switzerland, 4Vascular Neurology and Neurorehabilitation, University of Zurich, Zurich, Switzerland Introduction: Wearables are potentially valuable tools for understanding mobility behavior in individuals with neurological disorders and how it changes depending on health status, such as after rehabilitation. However, the accurate detection of gait events, which are crucial for the evaluation of gait performance and quality, is challenging due to highly individual-speciﬁc patterns that also vary greatly in movement and speed, especially after stroke. Therefore, the purpose of this study was to assess the accuracy, concurrent validity, and test–retest reliability of a commercially available insole system in the detection of gait events and the calculation of stance duration in individuals with chronic stroke. Neumann S, Bauer CM, Nastasi L, Läderach J, Thürlimann E, Schwarz A, Held JPO and Easthope CA (2024) Accuracy, concurrent validity, and test–retest reliability of pressure- based insoles for gait measurement in chronic stroke patients. Front. Digit. Health 6:1359771. d i 10 3389/fd th 2024 1359771 COPYRIGHT © 2024 Neumann, Bauer, Nastasi, Läderach, Thürlimann, Schwarz, Held and Easthope. 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. TYPE Original Research PUBLISHED 03 April 2024 DOI 10.3389/fdgth.2024.1359771 stroke, reliability and validity, gait disorders, spatiotemporal gait parameters, wearables, rehabilitation, gait, pressure insoles Frontiers in Digital Health 1 Introduction (29), various walking patterns, including gait asymmetry, heel walking, and a low heel pressure strategy, were assessed in individuals in different rehabilitation stages after stroke during a 10-min walking trial. The insole system consists of eight force-sensitive resistors per insole, which collects data at a frequency of 100 Hz that were further integrated into a personalized self-management rehabilitation system designed for gait relearning. Similarly, a shoe-based sensor system (SmartShoe), which includes ﬁve force-sensitive resistors in each ﬂexible insole, has demonstrated acceptable validity for the monitoring of physical activity and common activities of daily life in individuals who are at least three months post-stroke (22). Wearables have the potential to serve as valuable tools across the entire patient pathway, by providing assessments of gait and mobility of individuals with stroke both in clinical and home settings. While standardized clinical assessments capture a snapshot of a patient’s capacity within a controlled clinical setting, wearables present the opportunity to collect additional information about the quality and quantity of movement, as well as a patient’s performance in day-to-day activities (10, 11). This approach provides patients, clinicians, and researchers with the ability to continuously monitor the patient pathway, document progress, and gain insights into the capacity– performance relationship (12). It also assesses whether progress made in rehabilitation can be successfully transferred to daily life (13). A systematic review of the application of wearables in gait rehabilitation of individuals with stroke revealed that accelerometers, activity monitors, and pressure sensors are frequently employed, particularly in hospital-based and inpatient settings. Commonly evaluated gait parameters include gait speed, cadence, step count, and duration of activity (14). Most metrics for the evaluation of gait performance and quality center around the accurate detection of two main gait events: the foot strike (FS; when the foot ﬁrst touches the ground), and the foot off (FO; when the foot ﬁrst leaves the ground) (15). These events, which are well-established and well-described anchors for the calculation of gait quality features, are also relevant for measures of gait quantity, such as the number of steps (16). Therefore, as a ﬁrst principle, a robust, reliable, and accurate detection of gait events is paramount in a gait measuring system to make it ﬁt for purpose. 1 Introduction With over 12.2 million new cases worldwide, stroke is a signiﬁcant global health concern and one of the leading causes of disability in the western world (1, 2). Individuals with stroke suffer from motor impairments that substantially affect their gait function (3). In comparison to their healthy counterparts, individuals with stroke frequently display reduced mobility, which negatively inﬂuences their participation in everyday activities (4, 5). Therefore, rehabilitation goals commonly focus on enhancing activities such as mobility and participation in daily life (6). One of the main aims of stroke rehabilitation is to achieve each patient’s potential for recovery. This includes patient-tailored counseling to maintain and increase physical activity and self-training outside the therapeutic setting (7). This patient-tailored counseling involves a continuous cycle of identiﬁcation of a patient’s trajectory, personalized and precise therapy, and continuous monitoring of progress during the entire patient pathway (8, 9). Piezoresistive insoles have been validated in a population with neurological conditions aside from stroke, demonstrating their capability to accurately identify 94.5% of all steps, with recall and precision exceeding 0.98 when compared to a Vicon motion capture system (23). In healthy populations, piezoresistive pressure sensors exhibit accuracy levels ranging from an Intraclass correlation coefﬁcient (ICC) of 0.86–0.97, depending on walking speed (24). The FeetMe® Monitor system, validated in individuals with chronic stroke, exhibits an ICC > 0.77 and test–retest reliability of ICC > 0.9 (18). In gait rehabilitation, pressure-based insoles have already been used at different stages of the patient pathway, for example, as a supplementary tool for automatically assessing gait or for identifying activity levels and gait patterns (25–27). David et al. (28) evaluated the rehabilitation progress of 35 individuals in the acute and chronic phase of stroke using 90 s of level walking and the Timed Up and Go Test. Gait parameters, including spatiotemporal (e.g., stride time, step time), kinematic (e.g., angle at FS/FO), and gait cycle events, were assessed using the insole system eSHOE. Before and after four weeks of inpatient rehabilitation, the insoles, which are equipped with a triaxial accelerometer; a triaxial gyroscope; a triaxial magnetometer; and pressure sensors under the big toe, metatarsal heads Ⅰand Ⅴ, and heel, were used to monitor rehabilitation progress. In a study by Munoz- Organero et al. Accuracy, concurrent validity, and test–retest reliability of pressure-based insoles for gait measurement in chronic stroke patients No use, distribution or reproduction is permitted which does not comply with these terms. Methods: Pressure insole data were collected from 17 individuals with chronic stroke during two measurement blocks, each comprising three 10-min walking tests conducted in a clinical setting. The gait assessments were recorded with a video camera that served as a ground truth, and pressure insoles as an experimental system. We compared the number of gait events and stance durations between systems. Results and discussion: Over all 3,820 gait events, 90.86% were correctly identiﬁed by the insole system. Recall values ranged from 0.994 to 1, with a precision of 1 for all measurements. The F1 score ranged from 0.997 to 1. Excellent absolute agreement (Intraclass correlation coefﬁcient, ICC = 0.874) was observed for the calculation of the stance duration, with a slightly longer stance duration recorded by the insole system (difference of −0.01 s). Bland– Altmann analysis indicated limits of agreement of 0.33 s that were robust to changes in walking speed. This consistency makes the system well-suited for individuals post-stroke. The test–retest reliability between measurement timepoints T1 and T2 was excellent (ICC = 0.928). The mean difference in stance duration between T1 and T2 was 0.03 s. We conclude that the insole system is valid for use in a clinical setting to quantitatively assess continuous walking in individuals with stroke. Frontiers in Digital Health 01 frontiersin.org Neumann et al. 10.3389/fdgth.2024.1359771 capture the force interactions between the foot surface area and the ground, presenting an intuitive, straightforward, and reliable method for gait event detection (19). Pressure-based measurement systems such as pressure mats and foot switches have been used as a silver standard for validation and algorithm development studies (20, 21). Various techniques, including piezoresistive, resistive, capacitive, and piezoelectric methods, can be used as mechanisms for sensing plantar force. Different sensor technologies come with distinct sets of advantages and disadvantages. Piezoresistive and resistive methods have a broad force measurement range at a budget-friendly cost but involve the challenges of low repeatability and susceptibility to temperature and humidity. Conversely, capacitive and piezoelectric methods offer increased force sensitivity, but they are susceptible to changes in temperature and interference from electromagnetic sources (22). Frontiers in Digital Health 2.3 Measurement system A Stappone insole system (version 1.0, stapp one, Vienna, Austria) collected plantar pressure with 12 resistive textile pressure sensors in speciﬁc zones under the foot. Additionally, it comprised a triaxial accelerometer, a battery, onboard buffer storage, and a Bluetooth Low Energy 4.2 emitter for each insole. Once the data was collected, parameters were processed onboard the system using a proprietary adaptive thresholding algorithm. The sampling frequency was set to 100 Hz. Raw data for acceleration, plantar pressure, and gait event timings (FS and FO) were recorded and transferred via Bluetooth to the Stappone software (version 0.9.5.6, stapp one, Vienna, Austria) after each trial. The pressure calibration of the insoles was automatically adjusted by the software during postprocessing after each measurement was taken. The insole data were analyzed using an internal threshold-based algorithm for gait event detection. The sensor system was lightweight and could be worn for 36 h before needing a recharge. To address these limitations in validity and reliability of spatiotemporal parameters in individuals with stroke, the purpose of this study was to assess the accuracy, concurrent validity, and test–retest reliability of a commercially available insole system in the detection of gait events and the calculation of stance duration in individuals with chronic stroke. This was accomplished by comparing the data collected from the insoles with a camera- based reference system during continuous walking in a 10-min walking test (10mWT). 2.1 Participants Individuals with stroke were recruited from the University Hospital Zurich research database. Inclusion criteria were age ≥ 18 years, chronic stage of stroke (≥six months since the incident), and ability to walk without assistance on a level surface for at least six minutes (Functional Ambulation Category, FAC ≥3). The use of assistive devices was allowed. Potential participants were excluded if they had gait deﬁcits unrelated to stroke or were unable to understand verbal instructions. Seventeen individuals with chronic stroke were recruited for this project. The study was approved by the cantonal ethics committee (BASEC-Nr. Req-2019-00565, Ethics Committee Zurich, Zurich, Switzerland). All participants provided written informed consent after being informed about the study procedure. Measurement signals were synchronized for each trial using an audio output that was generated by the insoles when the measurement was started. The GoPro was connected to the audio output using a microphone adapter, which recorded the start signal as a sound burst. During postprocessing, this burst was identiﬁed with a threshold algorithm and veriﬁed manually. 2.2 Experimental protocol The participants were invited to attend a single measurement event consisting of two sessions of approximately one hour each. After obtaining informed consent, demographic information and outcome measures were collected to characterize the sample. This included age, gender, time since stroke, type of stroke, use of walking aids, 10mWT, gait speed, six-minute walk test, and Motricity Index. All participants were equipped with insoles placed within their personal footwear while performing two measurement blocks (timepoints T1 and T2), each with three repetitions of the 10mWT. A break of at least 30 min separated the two measurement blocks; during this break, the measurement system was removed from the shoe and charged. Post-break, system mounting was performed from scratch, facilitating the evaluation of test–retest reliability while minimizing the impact of day-to-day variation. All gait tests were recorded with both measurement systems, which were started and synchronized before each test. 2 Materials and methods A GoPro Hero 8 camera (GoPro, Inc., San Mateo CA, USA) served as the reference system because, unlike optoelectronic systems, it does not suffer from the uncertainty associated with human gait labeling (15). The use of manual labeling can lead to inaccuracies due to human error. To minimize these errors, the four-eyes principle was introduced. The camera was securely mounted on a rolling trolley at the height of the feet to capture all trials from a low vantage point with minimal occlusions (Figure 1). The sampling frequency was set to 120 Hz with a resolution of 1,080 pixels and no lens correction. During postprocessing, the gait events (FS and FO) were manually labeled frame by frame by one rater, and the timing was calculated as the time elapsed since the start signal. 1 Introduction This has proven challenging over past decades of research, especially in populations with neurological conditions, in which gait patterns are extremely individual- speciﬁc and both gait patterns and movement speed are highly variable (17). Compounding this, movement patterns and speeds both change over the course of rehabilitation. The major challenge of using wearables for gait measurement in individuals with stroke lies in the precise and consistent measurement of the gait patterns typically associated with hemiparesis. These patterns include decreased stance time on the Pressure sensors integrated into insoles are adaptable to various shoe types and contain a standardized arrangement of pressure sensors to ensure both validity and comfort (18). Pressure insoles Frontiers in Digital Health 02 frontiersin.org Neumann et al. Neumann et al. 10.3389/fdgth.2024.1359771 affected side of the body, longer stance time on the unaffected side, and reduced stride frequency and step length, and all contribute to an asymmetrical walking pattern with ﬂuctuating walking accelerations (30, 31). Despite the widespread use of wearables in research, their application for gait analysis involves limitations in the validity and reliability of spatiotemporal parameters and gait events in individuals with stroke (14). 2.5 Statistical analysis two-sided t-test (α < 0.05) was employed to detect statistically signiﬁcant differences between the systems. Furthermore, the agreement of the stance durations between the two measurement systems was characterized using Bland– Altman plots. Such plots have a strong history in the comparison of reference systems to experimental systems in the medical ﬁeld (32, 33). Their utility lies in the fact that they not only compare means over the given range of the test population but also enable the identiﬁcation of ﬁxed and variable bias individually. Both features are important in judging whether a system is sufﬁciently precise for a given application and population. The accuracy of the event detection was assessed with the F1 score to balance precision and recall. Precision was deﬁned as the total number of true positive (TP) identiﬁcations by the insole system and the reference system divided by the sum of the TPs and the false positives (FPs; events detected by the insole system but not by the reference system). Precision ¼ TP (TP þ FP) Finally, the ICC2,1 was used to examine the agreement between the two measurement systems. The ICC2,1 was calculated using the following equation: Recall was deﬁned as the number of TPs divided by the sum of the TPs and the false negatives (FNs; events identiﬁed by the reference system but not by the insole system). ICC (2, 1) ¼ MSR MSE MSR þ (k 1)MSE ICC (2, 1) ¼ MSR MSE MSR þ (k 1)MSE Recall ¼ TP (TP þ FN) Recall ¼ TP (TP þ FN) where MSR = mean square for rows, MSE = mean square for error, and k = number of raters/measurements (34). According to Fleiss, an ICC ≥0.75 is considered “excellent,” 0.40 ≤ICC < 0.75 is considered “moderate to good,” and ICC < 0.40 is considered “poor” (35). The ICC was applied, as it provides a measure- independent metric of agreement that allows for comparison between different metrics and measurement systems. The F1 score was used to balance precision and recall and was deﬁned as: F1 score ¼ 2 Precision Recall Precison þ Recall To evaluate the concurrent validity of the insoles, the differences between the stance durations measured by the insole system and by the reference system were analyzed. Normality of the distributions of stance duration was checked by visual inspection of QQ-plots by two investigators. 2.4 Data preprocessing The insole system and the reference system were used to assess the stance duration for both the affected and the unaffected side. The accuracy, concurrent validity, and test– retest reliability of stance duration were analyzed from the steps taken in the six 10mWTs per participant. Stance duration, deﬁned as the time between FS and the subsequent FO of the same foot, was calculated for every step. The stance duration was calculated once based on the insole timings and once based on the video reference timings. The timings of FS and FO were calculated as the time elapsed since the starting signal of the video. To ensure correspondence between FS and FO, e.g., that both belonged to the same step on the same side, a plausibility check was performed to remove all out-of-distribution values. Frontiers in Digital Health 03 frontiersin.org Neumann et al. 10.3389/fdgth.2024.1359771 FIGURE 1 Experimental setup for the 10mWT. Participants completed three repetitions of the 10mWT while wearing the insole system within their personal footwear. As the participants walked, the GoPro camera mounted on the rolling trolley was guided by an assistant walking alongside to capture the gait events during the 10mWT`s. FIGURE 1 Experimental setup for the 10mWT. Participants completed three repetitions of the 10mWT while wearing the insole system within their personal footwear. As the participants walked, the GoPro camera mounted on the rolling trolley was guided by an assistant walking alongside to capture the gait events during the 10mWT`s. 3.1 Gait event detection accuracy Of all 3,820 gait events, 90.86% were correctly identiﬁed by the insole system. A cumulative confusion matrix illustrates gait event detection for continuous walking in all participants by summation (Figure 2). The data analysis showed that 91 right FSs, 86 right FOs, 87 left FSs, and 85 left FOs were not identiﬁed as gait events by the insole system. Further investigation showed that all missing events corresponded to either the ﬁrst FO or the last FS of the trial. To 2.5 Statistical analysis For conﬂicting ratings, a consensus was reached through discourse. Subsequently, a paired In a last comparison, we evaluated test–retest reliability for each system by comparing stance duration data from T1 with data obtained at T2. Differences were identiﬁed using a paired two-sided t-test after a visual check for normality of the distributions using QQ-plots. The ICC2,1 (34) was again applied to permit comparability of reliability to other systems and measures. Frontiers in Digital Health Frontiers in Digital Health 04 frontiersin.org 10.3389/fdgth.2024.1359771 Neumann et al. TABLE 1 Characteristics of the study participants (n = 17). seven participants were using walking aids outdoors, only ﬁve used walking aids during the trials. Descriptive information on the participants’ characteristics is provided in Table 1. TABLE 1 Characteristics of the study participants (n = 17). Characteristic Value Age [years ± SD] 65.5 ± 11.2 Female 7 Time since stroke [months ± SD] 54.7 ± 40.4 Hemiparetic side Right Left 6 11 Occasional use of walking aids [%] 41.2 Functional ambulation categories 4 (independently on even ground) 5 (independently anywhere) 8 9 Gait velocity during 10MWT [m/s ± SD] Test Re-test 0.90 ± 0.37 0.90 ± 0.37 0.92 ± 0.38 Community ambulators (gait speed > 0.8 m/s [%] 58.8 Distance in 6-min walking test [m] 370.4 ± 164.5 Motoricity index (max 100 pts) Ankle dorsal ﬂexion [pts ± SD] Knee extension [pts ± SD] Hip ﬂexion [pts ± SD] 40.3 ± 20.0 24.1 ± 6.6 26.5 ± 3.9 24.6 ± 4.0 Of the 17 participants, one participant was excluded from the analysis due to a measurement system failure. The data from six additional 10mWT trials were not usable due to failure of either synchronization or recording initiation. Additionally, two trials were excluded due to a scrambled gait event sequence proposed by the internal algorithm of the insole system (e.g., multiple consecutive FOs from the same side). Consequently, a total of 88 10mWT trials from 16 participants were included in this analysis. The dataset comprised 3,820 gait events, encompassing 1910 FSs and FOs, covering 995 steps on the right side and 915 steps on the left side. 3 Results Seventeen individuals with chronic stroke, seven women and ten men, were recruited for the study. The average age was 65.5 years, and the mean time since stroke was 54.7 months. While FIGURE 2 Cumulative confusion matrix for continuous walking. The rows of the table correspond to the total number of gait events detected by the reference system. The columns correspond to the total number of gait events detected during continuous walking by the insole system. FIGURE 2 Cumulative confusion matrix for continuous walking. The rows of the table correspond to the total number of gait events detected by the reference system. The columns correspond to the total number of gait events detected during continuous walking by the insole system. Frontiers in Digital Health Frontiers in Digital Health 05 frontiersin.org Neumann et al. 10.3389/fdgth.2024.1359771 FIGURE 3 Bland-Altman plot for the comparison of the insole system with the reference system. The mean of the differences is indicated by the solid black line, and 95% limits of agreement are indicated by the dashed lines. FIGURE 3 Bland-Altman plot for the comparison of the insole system with the reference system. The mean of the differences is indicated by the solid black line, and 95% limits of agreement are indicated by the dashed lines. FIGURE 3 Bland-Altman plot for the comparison of the insole system with the reference system. The mean of the differences is indicated by the solid black line, and 95% limits of agreement are indicated by the dashed lines. simulate continuous walking, these gait events were excluded from further analysis. Recall values ranged from 0.994 to 1, with a precision of 1 for all measurements. The F1 score ranged from 0.997 to 1, as shown in Figure 2. 0.33 s (Figure 3). The correlation analysis between the insole and the video reference system was robust, with an excellent ICC2,1 value of 0.874. To enhance the visual clarity of our analysis, we have provided two alternative visualizations of the data reported by the Bland– Altman plot. Speciﬁcally, we show the difference in stance duration between both systems categorized by affected vs. unaffected side (Figure 4). Furthermore, we depict differences in stance duration of three categories of ambulators (Figure 5): limited community ambulators (<0.8 m/s), community ambulators (0.8–1.4 m/s), and those able to cross the street safely (>1.4 m/s) (36). 3.2 Stance duration concurrent validity Concurrent validity of stance duration was determined through comparison of the insole-derived timings and the video reference measurements. FP events were excluded from the analysis. Sixteen left (1.96% of a total of 815) and 17 right (1.94% of a total of 878) stance durations were excluded as outliers. No systematic reason was identiﬁed for the causation of these outliers. 4.1 Gait event detection accuracy The insole system correctly identiﬁed 90.86% of the 3,820 recorded gait events. This result aligns with a study that correctly identiﬁed 94.5% of all steps in a population with neurological conditions by employing insoles equipped with a piezoresistive device (23). Both recall and precision were >0.98. These rates are comparable to or exceed those reported in studies using accelerometers in individuals with stroke (37, 38). The insole system failed to detect 9.14% of the gait events. Analysis revealed that the initial FOs at the start of the trial and the ﬁnal FSs at the end of the trial were consistently missed because participants initiated and concluded trials in a standing position. Simulating continuous walking by removing the ﬁrst FOs and the last FSs from the analysis resulted in a detection error rate of only 0.2%. Among these erroneously detected gait events, eight were FSs detected before the participant started walking, suggesting potential algorithm sensitivity to weight shifts without actual foot movement (39). 3.3 Stance duration test–retest reliability The normality of the distributions of stance duration data was conﬁrmed by both raters using the QQ-plots. The t-test performed on the stance durations calculated by the insoles and the reference system revealed no signiﬁcant difference (p = 0.06). The stance durations recorded by the insole system tended to be slightly longer than those recorded by the reference system (difference of −0.01 s). The limits of agreement showed a deviation of insole measurements of up to The normality of the distributions of stance duration data between T1 and T2 was conﬁrmed by both raters using the QQ- plots. The paired, two-sided t-test indicated no signiﬁcant difference (p = 0.44) between the two measurement blocks, T1 and T2. The test–retest reliability between measurement timepoints T1 and T2 was excellent, with an ICC2,1 of 0.928. The mean difference in stance duration between T1 and T2 was 0.03 s. Frontiers in Digital Health 06 frontiersin.org Neumann et al. 10.3389/fdgth.2024.1359771 FIGURE 4 Boxplot representation of the difference in stance duration [ms] between the video system and the insole system for the affected and unaffected side. FIGURE 5 Boxplot representation of the difference in stance duration [ms] between the video system and the insole system for both sides together. We show how the stance duration difference compares between different levels of walking ability. FIGURE 5 Boxplot representation of the difference in stance duration [ms] between the video system and the insole system for both sides together. We show how the stance duration difference compares between different levels of walking ability. FIGURE 4 Boxplot representation of the difference in stance duration [ms] between the video system and the insole system for the affected and unaffected side. FIGURE 4 Boxplot representation of the difference in stance duration [ms] between the video system and the insole system for the affected and unaffected side. 4.2 Stance duration concurrent validity The validity of the stance duration measurement was excellent. In comparison to the FeetMe® Monitor system (ICC > 0.77), which was validated in individuals with chronic stroke over three 8-min trials (18), the utilized insole system demonstrated superior performance. Our results align with the accuracy of the ZeroWire® footswitch system, which has four piezoresistive pressure sensors on each foot and was used to record stance time in healthy participants; its accuracy ranged from an ICC of 0.86 to 0.97, depending on walking speed (24). Due to the increased variability and decreased walking speed that are often found in pathological gait, Inertial Measurement Units (IMU) often report reduced accuracy at low walking speeds of 0.4 m/s or severe deviations from normal human gait (40, 41). This is reﬂected in the lower accuracy of IMU sensors used to detect stance duration in individuals with stroke (42). While we did not record any individuals walking at less than 0.4 m/s, the insole system performed remarkably well for 0.5 m/s–0.6 m/s speeds, which are associated with long stance durations (43). This consistency indicates that the system should function well, even for slow- walking individuals post-stroke. The aim of this study was to assess the accuracy, concurrent validity, and -reliability of an insole system in individuals with stroke-related gait deﬁcits during a 10mWT, measured across two sessions in a clinical setting. We targeted a wide functional range of patients to reﬂect the clinical reality of rehabilitation. Ethics statement consistent with ﬁndings for the FeetMe® Monitor system (ICC > 0.9) (18). The difference between T1 and T2 can be attributed to typical variation in stance duration (44). These ﬁndings indicate that the measures are repeatable over time, even if the insole is removed and reinstalled in the shoe. It is important to note that a short pressure calibration was performed after each introduction of the system into the shoe to compensate for changes in lacing pressure. The study involving humans was approved by the Ethics committee Zurich, Zurich, Switzerland (BASEC-Nr. Req-2019- 00565). The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study. 4.3 Stance duration test–retest reliability The high level of agreement between the two measurement timepoints, T1 and T2, as indicated by the ICC2,1 of 0.92, is Frontiers in Digital Health 07 frontiersin.org Neumann et al. 10.3389/fdgth.2024.1359771 Funding The author(s) declare ﬁnancial support was received for the research, authorship, and/or publication of this article. Funding was graciously provided by the P&K Foundation. 5 Conclusion 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. In conclusion, the insole system is accurate, valid, and reliable for detecting gait events and calculating stance duration during continuous walking of individuals with stroke, both within and across sessions. Hence, the current model of the insole system can be used in a clinical setting to quantitatively measure gait in such individuals. Acknowledgments Our ﬁndings suggest that the insole system is applicable for assessing stance duration in individuals post-stroke. However, this conclusion is limited to clinical settings and continuous, straight walking. Further research is necessary to determine whether these ﬁndings can be transferred to a real-world environment and various activities of daily life. Thank you to all participants for their time and engagement in this study, and especially to Andreas Mursch-Radlgruber for his technical support throughout the study. Author contributions SN: Formal Analysis, Writing – original draft, Writing – review & editing, Visualization. CB: Supervision, Writing – original draft, Writing – review & editing. JL: Data curation, Investigation, Methodology, Writing – original draft, Writing – review & editing. LN: Formal Analysis, Writing – original draft, Writing – review & editing. ET: Investigation, Writing – original draft, Writing – review & editing. AS: Investigation, Methodology, Writing – original draft, Writing – review & editing. JH: Conceptualization, Supervision, Writing – original draft, Writing – review & editing. CA: Conceptualization, Funding acquisition, Supervision, Writing – original draft, Writing – review & editing. Alterations in walking patterns or the ability to walk can serve as a valuable source of information regarding patient trajectories throughout the rehabilitation pathway and beyond. Gait strategies are inﬂuenced by various clinical factors, including neurological, cardiovascular, and musculoskeletal conditions; cardiovascular and metabolic diseases; age-related changes; and trauma (9). Wearables have found extensive application in research, and their utilization is expanding into clinical settings. They can provide information about spatiotemporal, kinematic, and kinetic parameters. Nevertheless, caution is advised in the application of these devices, as they are speciﬁcally designed to extract certain parameters in particular patient populations. Therefore, these applications need to undergo a validation process in patients with speciﬁc diseases or conditions in clinical settings, as well as in real-world conditions if they are employed in such environments (45). Most importantly, the information gathered using these sensors must align with clinical endpoints and have clinical relevance (46). This ensures that such information can be effectively used by clinicians to provide valuable additional insights into individual health conditions and to facilitate clinical decision-making. Frontiers in Digital Health Data availability statement 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. The datasets presented in this article are not readily available due to ethical and legal limitations, as they contain identiﬁable patient information. Requests to access the datasets should be directed to the corresponding author. 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https://openalex.org/W2265199486	https://biblio.ugent.be/publication/7902140/file/7902318	English	null	Riemann-Hilbert problems for monogenic functions in axially symmetric domains	Boundary value problems	2,016	cc-by	6,752	© 2016 He et al. 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, pro- vided 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. R ES EA RCH Open Access Fuli He1, Min Ku2, Uwe Kähler2, Frank Sommen3 and Swanhild Bernstein4 Fuli He1, Min Ku2, Uwe Kähler2, Frank Sommen3 and Swanhild Bernstein4 Correspondence: kumin0844@163.com 2CIDMA, Department of Mathematics, University of Aveiro, Aveiro, 3810-193, Portugal Full list of author information is available at the end of the article Abstract We consider Riemann-Hilbert boundary value problems (for short RHBVPs) with variable coeﬃcients for axially symmetric monogenic functions deﬁned in axial symmetric domains. This is done by constructing a method to reduce the RHBVPs for axially symmetric monogenic functions deﬁned in four-dimensional axial symmetric domains into the RHBVPs for analytic functions deﬁned over the complex plane. Then we derive solutions to the corresponding Schwarz problem. Finally, we generalize the results obtained to null-solutions of (D – α)φ = 0, α ∈R, where R denotes the ﬁeld of real numbers. MSC: Primary 30E25; 35Q15; 31A25; 31B20; secondary 31B10; 35J56; 35J58 Keywords: quaternion analysis; generalized Cauchy-Riemann operator; axial symmetry; Riemann-Hilbert boundary value problems; variable coeﬃcients He et al. Boundary Value Problems ( 2016) 2016:22 DOI 10.1186/s13661-016-0530-x Correspondence: kumin0844@163.com 2CIDMA, Department of Mathematics, University of Aveiro, Aveiro, 3810-193, Portugal Full list of author information is available at the end of the article 1 Introduction The classic theory of Riemann-Hilbert boundary value problems (for short RHBVPs) for analytic functions is closely connected with the theory of singular integral equations and has a wide range of applications in other ﬁelds, such as the theory of elasticity, quantum mechanics, statistical physics, the theory of orthogonal polynomials, and asymptotic anal- ysis (cf. [–]). Over the years this type of boundary value problems has been systemat- ically investigated by many authors, e.g., [–, –]. Therefore, it is natural to attempt to generalize this type of boundary value problem to higher dimensions. This will be not only a purely theoretical question, since such problems are closely linked to the physical applications like transport problems or problems in hydro-dynamic mechanics. To our knowledge, there are two principal ways to generalize complex analysis to higher dimensions, the theory of several complex variables and Cliﬀord analysis. The latter, in particular quaternionic analysis, is the theory of so-called monogenic functions and a reﬁnement of real harmonic analysis in the sense that the principal operator in Cliﬀord analysis, the Dirac or generalized Cauchy-Riemann operator, factorizes the higher Laplace operator. As principal references for this theory we mention [–]. Furthermore, in the context of Cliﬀord analysis RHBVPs, particularly Riemann boundary value problems, with constant coeﬃcients were widely discussed; see, e.g., [–]. Here, a direct application of the properties of the Cauchy integral operator and existing power series expansions allows one to represent the solution in terms of integral operators and Taylor series expansions. This kind of Riemann boundary value problems is also closely connected with other types He et al. Boundary Value Problems ( 2016) 2016:22 Page 2 of 11 Page 2 of 11 of boundary value problems for diﬀerent partial diﬀerential equations in higher dimen- sions; see, e.g., [–]. However, there are almost no results with respect to RHBVPs with variable coeﬃcients even after more than years of research in this direction. The ob- stacles lie in the following facts: while monogenic functions retain many of the properties of analytic function, there are two drawbacks. First, the product of monogenic functions is in general not monogenic and second the composition of monogenic is in general not monogenic. While the latter property is not so important for our problem at hand the for- mer is a major problem. 1 Introduction Moreover, even in the simplest case of quaternionic analysis, we have several major problems, such as the lack of a function which has the same properties as the logarithmic function of one complex variable. These make it very diﬃcult to solve RHBVPs with variable coeﬃcients for monogenic functions in higher dimensions by di- rectly employing classic methods from complex analysis. But, as a particular interesting case, it was ﬁrst introduced by Fueter in the case of quaternionic analysis in [] that the product of monogenic functions of axial type is still a monogenic function of axial type, and later on this class was studied in detail by Sommen et al. (cf. [, ]). This makes this class more similar to the classic case of analytic functions in the plane and, therefore, if one wants to generalize any property or fact from complex analysis to quaternionic or Cliﬀord analysis, the standard approach is to see if it can be done for axially monogenic functions. So, instead of trying to solve this problem directly, we are going to show that RHBVPs with variable coeﬃcients for monogenic functions with axial symmetry can be solved in the context of quaternionic analysis. This represents an initial and very crucial step to solve RHBVPs for monogenic functions in the future. Our idea is to transform a RHBVP with variable coeﬃcients for monogenic functions with axial symmetry in Rinto a RHBVP for analytic functions over the complex plane, use the classic results from complex analysis, and transfer it back. In this way we can obtain the solution in an explicit form. As a special case we present the solution to the Schwarz problem for monogenic functions with axial symmetry. Furthermore, we adapt the ap- proach above and generalize to the case of null-solutions to (D – α)φ = , α ∈R, where R denotes the ﬁeld of real numbers (see Section .). Here, the main contribution that we emphasize lies in constructing a way to solve a RHBVP with variable coeﬃcients in four-dimensional spaces. Moreover, by using the possibility to decompose a monogenic functions into a sum of monogenic functions with axial symmetry, we plan to extend this idea to the case of RHBVPs with variable coeﬃcients for monogenic functions. The paper is organized as follows. In Section , we will recall the necessary facts about quaternion analysis. 1 Introduction In Section , we will focus on RHBVPs with variable coeﬃcients for monogenic functions with axial symmetry. We will derive the solution to the RHBVP with boundary data belonging to a Hölder space in terms of an integral representation. More- over, we give the solutions to the Schwarz problem for monogenic functions and null- solutions to (D – α)φ = , α ∈R with axial symmetry, where R denotes the ﬁeld of real numbers. 2 Preliminaries Good introductions to Cliﬀord algebras, Quaternions, and Cliﬀord analysis can be found in [–, , ]. Let {e,e,e,e} be the standard basis of H. These basic vectors satisfy eiej + ejei = –δi,j, i,j = ,,, ee= e, ee= e, ee= e, He et al. Boundary Value Problems ( 2016) 2016:22 He et al. Boundary Value Problems ( 2016) 2016:22 Page 3 of 11 Page 3 of 11 where δ denotes the Kronecker delta, and e= denotes the identity element of the algebra of quaternions H. Thus H is a real linear, associative, but non-commutative algebra. An arbitrary x ∈H can be written as x = x+xe+xe+xe≜x+x, where Sc(x) ≜x and Vec(x) ≜x are the scalar and vector part of x ∈H, respectively. Elements x ∈Rcan be identiﬁed with quaternions x ∈H. The conjugation is deﬁned by ¯x =  j=xj¯ej with ¯e= e and ¯ej = –ej, j = ,,, and hence, xy = ¯y¯x. The algebra of quaternions possesses an inner product (y,x) = Sc(y¯x) = (y¯x)for all x,y ∈H. The corresponding norm is \|x\| = ( j=\|xj\|)  = √(x,x). Any element x ∈H\{} is in- vertible with inverse element x–≜¯x\|x\|–, i.e., xx–= x–x = . Furthermore, we can intro- duce the set [x] = y : y = Sc(x) + I\|x\|,I ∈S , where S= {x ⊂R: \|x\| = }. In this paper we will consider the generalized Cauchy-Riemann operator D =  j=ej∂xj in R. The generalized Cauchy-Riemann operator factorizes the Laplacian in the following sense: DD =  j=∂ xj = , where denotes the Laplacian in R. j j We also need to deﬁne axially symmetric open sets. Deﬁnition .(Axially symmetric open set) Let be a non-empty open subset of R. We say that is axially symmetric if for any x ∈, the subset [x] is contained in ⊂R. Remark The unit ball of Rand the upper half space R + = {x ∈R\|x> } are examples of axially symmetric domains. Let be an axially symmetric domain of Rwith smooth boundary ∂. An H-valued function φ =  j=φjej is continuous, Hölder continuous, p-integrable, continuously dif- ferentiable and so on if all components φj have that property. The corresponding func- tion spaces, considered as either right-Banach or right-Hilbert modules, are denoted by C(,H), Hμ(,H) (< μ ≤), Lp(,H) (< p < +∞), C(,H), respectively. 2 Preliminaries Applying Fueter’s theorem (see [, ]) a function of axial type is given by ) = A(x,r) + ωB(x,r), () () φ(x) = A(x,r) + ωB(x,r), () φ(x) = A(x,r) + ωB(x,r), where x = x+ x = x+ rω ∈R, r = \|x\|, ω ∈{x =  j=xjej : \|x\| = ,xj ∈R (j = ,,)}, A(x,r), and B(x,r) are scalar-valued functions. It should be mentioned that in [] a function of axial type is also called a function with axial symmetry. Throughout this paper any functions deﬁned on ∪∂ ⊂Rwith values in H are supposed to be of axial type unless otherwise stated. Deﬁnition .A function φ ∈C(,H) is called (left-) monogenic if and only if Dφ = . A monogenic function of axial type is called axially monogenic. The set of all axially mono- genic functions deﬁned in forms a right-module, denoted by M(,H). Deﬁnition .For a function of axial type φ : →H, we deﬁne the real part as Reφ = A. Remark In [, ] it is shown that the equation Dφ = for functions of axial type is equivalent to a special kind of Vekua system []. He et al. Boundary Value Problems ( 2016) 2016:22 Page 4 of 11 In dimension two, i.e., the case of R,= {x = x+ xe: e = –,xj ∈R (j = ,)} ∼= C, the ﬁeld of complex numbers, we have Reφ = Sc(φ), which exactly corresponds to the usual understanding in complex analysis. In the following D ⊂C+ is the projection of the axially symmetric sub-domain ⊂Rinto the (x,r)-plane, where C+ is the upper half of the (x,r)-plane. 3.1 A lemma Let us start with the following lemma. Lemma .Let A(D,C) denote the set of analytic functions deﬁned in D ⊂C+. Then there exists an injective mapping ψ between A(D,C)/kerϕ and M(,H), where ϕ is a surjective mapping from A(D,C) onto M(,H) given by A(D,C) →M(,H), f = u(x,y) + iv(x,y) →φ = A(x,r) + ωB(x,r), A(D,C) →M(,H), f = u(x,y) + iv(x,y) →φ = A(x,r) + ωB(x,r), where A(x,r) + ωB(x,r) = u(x,r) + ωv(x,r) , x = x+ rω ∈R, and D is the projection of onto the (x,r)-plane. In fact the surjective map ϕ is coming from Fueter’s theorem [, , ]. In fact the surjective map ϕ is coming from Fueter’s theorem [, , ]. Proof Let the map ψ be given by A(D,C)/kerϕ →M(,H), f + ˆg →φ, A(D,C)/kerϕ →M(,H), f + ˆg →φ, where kerϕ = {f : ϕ(f ) = ,f ∈A(D,C)}, f = u(x,r) + iv(x,r), φ = A(x,r) + ωB(x,r), ˆg = u(x,r) + iv(x,r), and ˆg ∈kerϕ. Then the map ψ is an injective mapping between A(D,C)/kerϕ and M(,H), where u, vare deﬁned similarly to u, v, respectively, and M(,H) is the same as that in Section . The result follows. □ where kerϕ = {f : ϕ(f ) = ,f ∈A(D,C)}, f = u(x,r) + iv(x,r), φ = A(x,r) + ωB(x,r), ˆg = u(x,r) + iv(x,r), and ˆg ∈kerϕ. Then the map ψ is an injective mapping between A(D,C)/kerϕ and M(,H), where u, vare deﬁned similarly to u, v, respectively, and M(,H) is the same as that in Section . The result follows. □ 3 Riemann-Hilbert boundary value problems In this section we discuss RHBVPs with variable coeﬃcients for monogenic functions. In this case we will restrict ourselves to functions which have axial symmetry and are deﬁned over axial domains of R. We ﬁrst solve the RHBVP in Rby transforming it into a RHBVP for complex analytic functions. In the case of boundary values belonging to a Hölder space we represent its solution in terms of an explicit integral representation formula. As a corollary we obtain the solution to the corresponding Schwarz problem in quaternion analysis. 3.2 RHBVPs for monogenic functions The idea behind this lemma allows us to link RHBVPs for axially monogenic functions with RHBVPs for analytic functions over the complex plane. Let us start the discussion with the following problem. Page 5 of 11 Page 5 of 11 He et al. Boundary Value Problems ( 2016) 2016:22 Problem I Find a function φ ∈C(,H) of axial type which satisﬁes the condition (⋆) Dφ(x) = , x ∈, Re{λ(t)φ(t)} = g(t), t ∈∂, (⋆) Dφ(x) = , x ∈, Re{λ(t)φ(t)} = g(t), t ∈∂, (⋆) Re{λ(t)φ(t)} = g(t), t ∈∂, where g : ∂ →R and λ is a R-valued function deﬁned on ∂. where g : ∂ →R and λ is a R-valued function deﬁned on ∂. Let us ﬁrst transform the above problem to a RHBVP for analytic functions. The result is given in the following theorem. Theorem .The Riemann-Hilbert boundary value problem (⋆) is equivalent to the fol- lowing problem: Find a complex-valued analytic function h, such that ∂¯zh(z) = , z ∈D ⊂C+, Re{λ(z)h(z)} = r g(z), z ∈∂D ⊂C+, ∂¯zh(z) = , z ∈D ⊂C+, Re{λ(z)h(z)} = r g(z), z ∈∂D ⊂C+, Re{λ(z)h(z)} = r g(z), z ∈∂D ⊂C+, where z = x+ ir, ∂¯z =  (∂x+ i∂r), h = ∂rf , f , and r given as in Lemma ., D is a domain of C+ with boundary ∂D given as the projection of ⊂Rinto the complex plane, and λ,g : ∂D →R are both scalar-valued functions. Proof From Lemma .we have complex-valued functions f = u + iv ∈A(D,C) and ˆg = u+ iv∈A(D,C) with ˆg ∈kerϕ, such that ψ(f + ˆg) = φ. Hence, ϕ ◦ψ–(φ) = f , where ψ– is the inverse of ψ on its range and ϕ ◦ψ–denotes the composition of ϕ and ψ–. Adapting the boundary condition we see that the Riemann-Hilbert boundary value problem (⋆) is equivalent to the case (⋇) ∂¯zf (z) = , z ∈D ⊂C+, Re{λ(t)u}(t) = g(t), t ∈∂D ⊂C+. (⋇) Re{λ(t)u}(t) = g(t), t ∈∂D ⊂C+. Using the Laplacian operator = ∂ x + ∂ r+  r ∂r +  r∂ω –  r∂ ω, we have u =  r ∂ru. Now, let h = ∂rf then we have ∂¯zh(z) = , z ∈D since f is analytic in D. 3.2 RHBVPs for monogenic functions Hence, our boundary value problem (⋇) reduces to the case Using the Laplacian operator = ∂ x + ∂ r+  r ∂r +  r∂ω –  r∂ ω, we have u =  r ∂ru. Now, let h = ∂rf then we have ∂¯zh(z) = , z ∈D since f is analytic in D. Using the Laplacian operator = ∂ x + ∂ r+  r ∂r +  r∂ω –  r∂ ω, we have u =  r ∂ru. Now, let h = ∂rf then we have ∂¯zh(z) = , z ∈D since f is analytic in D. Hence, our boundary value problem (⋇) reduces to the case Hence, our boundary value problem (⋇) reduces to the case ∂¯zh(z) = , z ∈D ⊂C+, Re{  r λ(z)h(z)} = g(z), z ∈∂D ⊂C+. □ □ The above theorem allows us to study the solvability of our original RHBVP by studying the equivalent RHBVP over the complex plane. Theorem .Suppose g ∈Hμ(∂,R), and D = {z : \|z – a\| < } ⊂C+ with z = x+ ir, a = a+ ia∈C+ being the corresponding domain of C+ with boundary ∂D. (i) If λ ∈Hμ(∂,R), and λ ̸= for arbitrary z ∈∂D then the Riemann-Hilbert boundary value problem (⋆) is solvable and its solution is given by Theorem .Suppose g ∈Hμ(∂,R), and D = {z : \|z – a\| < } ⊂C+ with z = x+ ir, a = a+ ia∈C+ being the corresponding domain of C+ with boundary ∂D. φ(x) = Re(f ) x,\|x\| + ω Im(f ) x,\|x\| , x ∈, Page 6 of 11 He et al. Boundary Value Problems ( 2016) 2016:22 where Re(f ) and Im(f ) denote the real and imaginary part of the complex-valued function f , respectively. f itself is given by f (z) =  π z a ∂D ˜g(ζ) ζ – ξ dζ dξ + +∞ n= tn(z – a)n, z ∈D, with ˜g = λ–r g, tn ∈C. g g (ii) Suppose λ = ˆλ with (x) = m i=(x – ˆαi)νi, ˆαi ∈∂ and νi ∈N. 3.2 RHBVPs for monogenic functions He et al. Boundary Value Problems ( 2016) 2016:22 Page 7 of 11 Value Problems ( 2016) 2016:22 Page 7 of 11 Page 7 of 11 He et al. Boundary Value Problems ( 2016) 2016:22 Page 7 of 11 Hence, the unique solution to the boundary value problem (⋇⋇) is expressed by f (z) =  π z a ∂D ˜g(ζ) ζ – ξ dζ dξ + +∞ n= tn(z – a)n, z ∈D, () () where tn ∈C. where tn ∈C. We end up with the solution to the Riemann-Hilbert boundary value problem (⋆) in the form φ(x) = Re(f ) x,\|x\| + ω Im(f ) x,\|x\| , x ∈. φ(x) = Re(f ) x,\|x\| + ω Im(f ) x,\|x\| , x ∈. In the second case (ii) we map again to D and λ = ˆλ with (x) = m i=(x – ˆαi)νi where ˆαi ∈∂ becomes λ(z) = (z)ˆλ(z), z ∈∂D, where (z) = m i=(z – αi)νi, αi ∈∂D and ˆλ ̸= , z ∈∂D. By transforming the boundary value problem in the same way as in the ﬁrst case we arrive at the problem ∂¯z H(z) = , z ∈C+\∂D, H+(z) = H–(z) + ˆλ–(z) r g(z), z ∈∂D, ∂¯z H(z) = , z ∈C+\∂D, H+(z) = H–(z) + ˆλ–(z) r g(z), z ∈∂D, where H = H, z ∈C+\∂D, and H±, z ∈∂D are the boundary values of H on ∂D from D and C+\(D ∪∂D), respectively. where H = H, z ∈C+\∂D, and H±, z ∈∂D are the boundary values of H on ∂D from D and C+\(D ∪∂D), respectively. y Since ˆλ ∈Hμ(∂D,R), we have ˆg ≜ˆλ–r g =  i ˆλ–(z – ¯z)g ∈Hμ(∂D,R). This allows us to write the solution to the boundary value problem (⋇⋇) in the form Since ˆλ ∈Hμ(∂D,R), we have ˆg ≜ˆλ–r g =  i ˆλ–(z – ¯z)g ∈Hμ(∂D,R). This allows us to write the solution to the boundary value problem (⋇⋇) in the form f (z) =  π z a ∂D  (ξ) ˆg(ζ) ζ – ξ dζ dξ + +∞ n= z a ln(ξ – a)n (ξ) dξ, z ∈D, () () where ln ∈C. where ln ∈C. 3.2 RHBVPs for monogenic functions Furthermore, if ˆλ ∈Hμ(∂,R) and ˆλ ̸= for arbitrary x ∈∂ then the Riemann-Hilbert boundary value problem (⋆) is solvable, and its solution is given again by  (ii) Suppose λ = ˆλ with (x) = m i=(x – ˆαi)νi, ˆαi ∈∂ and νi ∈N. Furthermore, if ˆλ ∈Hμ(∂,R) and ˆλ ̸= for arbitrary x ∈∂ then the Riemann-Hilbert boundary value problem (⋆) is solvable, and its solution is given again by φ(x) = Re(f ) x,\|x\| + ω Im(f ) x,\|x\| , x ∈, where f is given by f (z) =  π z a ∂D  (ξ) ˆg(ζ) ζ – ξ dζ dξ + +∞ n= z a ln(ξ – a)n (ξ) dξ, z ∈D, with ˆg = ˆλ–r g, ln ∈C. with ˆg = ˆλ–r g, ln ∈C. with ˆg = ˆλ–r g, ln ∈C. Proof Since D = {z : \|z – a\| < } ⊂C+ is the projection of into (x,r)-plane from Theo- rem .we see that the problem (⋆) is equivalent to the problem Proof Since D = {z : \|z – a\| < } ⊂C+ is the projection of into (x,r)-plane from Theo- rem .we see that the problem (⋆) is equivalent to the problem (⋇⋇) ∂¯zh(z) = , z ∈D, Re{λ(z)h(z)} = r g(z), z ∈∂D. Consider now the function Consider now the function H(z) = h(z), z ∈D ⊂C+, h∗(z) = ¯h(/z), z ∈C+\D ∪∂D, and let us study each case separately. and let us study each case separately. and let us study each case separately. For the case (i) we can proceed in the following way. Since λ,g ∈Hμ(∂,R) with λ ̸=  for arbitrary x ∈∂ we can recast our problem as the following Riemann boundary value problem: (ℵ) ∂¯zH(z) = , z ∈C+\∂D, H+(z) = H–(z) + λ–(z) r g(z), z ∈∂D, (ℵ) H+(z) = H–(z) + λ–(z) r g(z), z ∈∂D, where H± are the boundary values of H on ∂D from D and C+\(D ∪∂D), respectively. Because of λ,g ∈Hμ(∂D,R) we have ˜g ≜λ–r g =  iλ–(z – ¯z)g ∈Hμ(∂D,R). Therefore, the solution to the boundary value problem (ℵ) is given by H(z) =  πi ∂D ˜g(ζ) ζ – z dζ + +∞ n= ln(z – a)n, z ∈D, where ln ∈C. where ln ∈C. 3.2 RHBVPs for monogenic functions Therefore, the solution to the Riemann-Hilbert boundary value problem (⋆) is given by φ(x) = Re(f ) x,\|x\| + ω Im(f ) x,\|x\| , x ∈. This ﬁnishes the proof. □ s ﬁnishes the proof. □ This ﬁnishes the proof. □ As a special case of Problem I, we can consider the following Schwarz problem. Schwarz problem Find a function φ ∈C(,H), which satisﬁes the system Schwarz problem Find a function φ ∈C(,H), which satisﬁes the system (♯) Dφ(x) = , x ∈, Re{φ(t)} = g(t), t ∈∂, where g : ∂ →R is a R-valued function deﬁned on ∂. From Theorem .we can deduce the following theorem. From Theorem .we can deduce the following theorem. Theorem .If g ∈Hμ(∂,R) then the Schwarz problem (♯) is solvable, and its solution is given by φ(x) = Re(f ) x,\|x\| + ω Im(f ) x,\|x\| , x ∈, φ(x) = Re(f ) x,\|x\| + ω Im(f ) x,\|x\| , x ∈, φ(x) = Re(f ) x,\|x\| + ω Im(f ) x,\|x\| , x ∈, Page 8 of 11 Page 8 of 11 Page 8 of 11 He et al. Boundary Value Problems ( 2016) 2016:22 where f is given by f (z) =  π z a ∂D ˜g(ζ) ζ – ξ dζ dξ + +∞ n= tn(z – a)n, z ∈D, with ˜g= r g, tn ∈C. Remark Theorem .actually gives us a method to solve the RHBVPs with variable coeﬃcients for axially monogenic functions in Rby transferring it to the study of the corresponding RHBVP for analytic functions over the complex plane. When the space dimension is , then trivially RHBVP (⋆) reduces to the RHBVP for analytic functions on the complex plane [, , ]. Moreover, when the coeﬃcient λ is a constant equal to for all x ∈∂ our RHBVP (♯) is just the Schwarz problem for analytic functions over the complex plane [, , ]. Remark In fact, Theorem .gives us explicit solutions to the Schwarz problem for a special type of Vekua system deﬁned over the complex plane [, ]. As far as we know, in general it is not easy to get explicit but non-formal analytic solutions of this system. 3.3 RHBVPs for null-solutions to (D – α)φ = 0, α ∈R The above approach can be adapted and generalized to other cases. In the following we will discuss RHBVPs with variable coeﬃcients for functions of axial type deﬁned over an axial domain of Rwhich are null-solutions to the equation (D –α)φ = , α ∈R. We begin with the following extension of Problem I. Problem II Find a function φ ∈C(,H) of axial type which satisﬁes the condition (⋆⋆) (D – α)φ = , α ∈R,x ∈, Re{λ(t)φ(t)} = g(t), t ∈∂, (⋆⋆) (D – α)φ = , α ∈R,x ∈, Re{λ(t)φ(t)} = g(t), t ∈∂, where α is understood as αI, with I being the identity operator, g : ∂ →R, and λ is a R-valued function deﬁned on ∂. where α is understood as αI, with I being the identity operator, g : ∂ →R, and λ is a R-valued function deﬁned on ∂. Theorem .Suppose g ∈Hμ(∂,R), and D = {z : \|z – a\| < } ⊂C+ with z = x+ ir, a = a+ ia∈C+ being the corresponding domain of C+ with boundary ∂D. (i) If λ ∈Hμ(∂,R), and λ ̸= for arbitrary z ∈∂D then the Riemann-Hilbert boundary value problem (⋆) is solvable and its solution is given by φ(x) = eαx Re(f ) x,\|x\| + ω Im(f ) x,\|x\| , x ∈, φ(x) = eαx Re(f ) x,\|x\| + ω Im(f ) x,\|x\| , x ∈, where Re(f ) and Im(f ) denote the real and imaginary part of the complex-valued function f , respectively. f itself is given by where Re(f ) and Im(f ) denote the real and imaginary part of the complex-valued function f , respectively. f itself is given by f (z) =  π z a ∂D e–αxg(ζ) ζ – ξ dζ dξ + +∞ n= tn(z – a)n, z ∈D, with ˜g = λ–r g, tn ∈C. with ˜g = λ–r g, tn ∈C. He et al. Boundary Value Problems ( 2016) 2016:22 Page 9 of 11 (ii) Suppose λ = ˆλ with (x) = m i=(x – ˆαi)νi, ˆαi ∈∂, and νi ∈N. 3.3 RHBVPs for null-solutions to (D – α)φ = 0, α ∈R Furthermore, if ˆλ ∈Hμ(∂,R) and ˆλ ̸= for arbitrary x ∈∂ then the Riemann-Hilbert boundary value problem (⋆) is solvable, and its solution is given again by (ii) Suppose λ = ˆλ with (x) = m i=(x – ˆαi)νi, ˆαi ∈∂, and νi ∈N. Furthermore, if ˆλ ∈Hμ(∂,R) and ˆλ ̸= for arbitrary x ∈∂ then the Riemann-Hilbert boundary value problem (⋆) is solvable, and its solution is given again by φ(x) = eαx Re(f ) x,\|x\| + ω Im(f ) x,\|x\| , x ∈, φ(x) = eαx Re(f ) x,\|x\| + ω Im(f ) x,\|x\| , x ∈, φ(x) = eαx Re(f ) x,\|x\| + ω Im(f ) x,\|x\| , x ∈, where f is given by f (z) =  π z a ∂D  (ξ) e–αxg(ζ) ζ – ξ dζ dξ + +∞ n= z a ln(ξ – a)n (ξ) dξ, z ∈D, with ˆg = ˆλ–r g, ln ∈C. Proof Since (D – α)φ = D(e–αxφ), α ∈R, then Proof Since (D – α)φ = D(e–αxφ), α ∈R, then (D – α)φ =  is equivalent to D e–αxφ = , α ∈R. () (D – α)φ =  is equivalent to D e–αxφ = , α ∈R. () () Therefore, problem (⋆⋆) is equivalent to the case Therefore, problem (⋆⋆) is equivalent to the case Therefore, problem (⋆⋆) is equivalent to the case D(e–αxφ) = , α ∈R,x ∈, Re{λ(t)e–αxφ(t)} = e–αxg(t), t ∈∂, where g : ∂ →R and λ is a R-valued function deﬁned on ∂. where g : ∂ →R and λ is a R-valued function deﬁned on ∂. Noting that D = {z : \|z – a\| < } ⊂C+ with z = x+ ir, a = a+ ia∈C+ is the projection of onto the (x,r)-plane, associating with g ∈Hμ(∂,R), we have e–αxg ∈Hμ(∂,R). Noting that D = {z : \|z – a\| < } ⊂C+ with z = x+ ir, a = a+ ia∈C+ is the projection of onto the (x,r)-plane, associating with g ∈Hμ(∂,R), we have e–αxg ∈Hμ(∂,R). 3.3 RHBVPs for null-solutions to (D – α)φ = 0, α ∈R Noting that D = {z : \|z – a\| < } ⊂C+ with z = x+ ir, a = a+ ia∈C+ is the projection of onto the (x,r)-plane, associating with g ∈Hμ(∂,R), we have e–αxg ∈Hμ(∂,R). We apply Theorem .. (i) If λ ∈Hμ(∂,R), and λ ̸= for arbitrary z ∈∂D then the Riemann-Hilbert boundary value problem (⋆) is solvable and its solution is given by (i) If λ ∈Hμ(∂,R), and λ ̸= for arbitrary z ∈∂D then the Riemann-Hilbert boundary value problem (⋆) is solvable and its solution is given by φ(x) = eαx Re(f ) x,\|x\| + ω Im(f ) x,\|x\| , x ∈, φ(x) = eαx Re(f ) x,\|x\| + ω Im(f ) x,\|x\| , x ∈, where Re(f ) and Im(f ) denote the real and imaginary part of the complex-valued function f , respectively. f itself is given by where Re(f ) and Im(f ) denote the real and imaginary part of the complex-valued function f , respectively. f itself is given by f (z) =  π z a ∂D e–αxg(ζ) ζ – ξ dζ dξ + +∞ n= tn(z – a)n, z ∈D, with ˜g = λ–r g, tn ∈C. with ˜g = λ–r g, tn ∈C. g g (ii) Suppose λ = ˆλ with (x) = m i=(x – ˆαi)νi, ˆαi ∈∂, and νi ∈N. Furthermore, if ˆλ ∈Hμ(∂,R) and ˆλ ̸= for arbitrary x ∈∂ then the Riemann-Hilbert boundary value problem (⋆) is solvable, and its solution is given again by φ(x) = eαx Re(f ) x,\|x\| + ω Im(f ) x,\|x\| , x ∈, φ(x) = eαx Re(f ) x,\|x\| + ω Im(f ) x,\|x\| , x ∈, where f is given by f (z) =  π z a ∂D  (ξ) e–αxg(ζ) ζ – ξ dζ dξ + +∞ n= z a ln(ξ – a)n (ξ) dξ, z ∈D, □ = ˆλ–r g, ln ∈C. The result follows. □ □ with ˆg = ˆλ–r g, ln ∈C. The result follows. with ˆg = ˆλ–r g, ln ∈C. The result follows. with ˆg = ˆλ–r g, ln ∈C. The result follows. □ with ˆg = ˆλ–r g, ln ∈C. The result follows. □ He et al. Author details 1 Author details 1School of Mathematics and Statistics, Central South University, Changsha, 410083, P.R. China. 2CIDMA, Department of Mathematics, University of Aveiro, Aveiro, 3810-193, Portugal. 3Cliﬀord Research Group, Department of Mathematical Analysis, Ghent University, Ghent, Belgium. 4TU Bergakademie Freiberg, Institute of Applied Analysis, Freiberg, 09599, Germany. 3.3 RHBVPs for null-solutions to (D – α)φ = 0, α ∈R Boundary Value Problems ( 2016) 2016:22 Page 10 of 11 Remark When the space dimension is , Problem II reduces to the Riemann-Hilbert boundary value problem for meta-analytic functions deﬁned over the complex plane [, ]. Similar to the Schwarz problem in the previous subsection, let λ = in Problem II, we can take it in account in the following problem. Problem III Find a function φ ∈C(,H) which satisﬁes the system Problem III Find a function φ ∈C(,H) which satisﬁes the system (D – α)φ = , α ∈R,x ∈, Re{φ(t)} = g(t), t ∈∂, (D – α)φ = , α ∈R,x ∈, Re{φ(t)} = g(t), t ∈∂, where α is understood as αI, with I being the identity operator, and g : ∂ →R is a R-valued function deﬁned on ∂. where α is understood as αI, with I being the identity operator, and g : ∂ →R is a R-valued function deﬁned on ∂. Thanks to Theorem .we can derive the following theorem. Thanks to Theorem .we can derive the following theorem. Theorem .If g ∈Hμ(∂,R) then Problem III is solvable, and its solution is given by Theorem .If g ∈Hμ(∂,R) then Problem III is solvable, and its solution is given by φ(x) = eαx Re(f ) x,\|x\| + ω Im(f ) x,\|x\| , x ∈, φ(x) = eαx Re(f ) x,\|x\| + ω Im(f ) x,\|x\| , x ∈, where f is given by where f is given by f (z) =  π z a ∂D eαxg(ζ) ζ – ξ dζ dξ + +∞ n= tn(z – a)n, z ∈D, with ˜g= r g, tn ∈C. Remark If the boundary data are of Hölder class, we consider the Riemann-Hilbert boundary value problems I, II, III with variable coeﬃcients for monogenic functions of axial type in Rand null-solutions to (D –α)φ = , α ∈R. Note that Problem II reduces to Problem I when α equals while Problem III reduces to Problem II when λ equals . Fol- lowing the same argument, all results can be extended to the Riemann-Hilbert boundary value problems of monogenic functions of axial type in R, with boundary data belonging to Lp (< p < +∞). Competing interests Th h d l h Competing interests The authors declare that they have no competing interests. Received: 9 October 2015 Accepted: 12 January 2016 Received: 9 October 2015 Accepted: 12 January 2016 No. SFRH/BPD/74581/2010. The authors cordially thank two anonymous referees for their valuable comments, which lead to the improvement of this paper. No. SFRH/BPD/74581/2010. The authors cordially thank two anonymous referees for their valuable comments, which lead to the improvement of this paper. Acknowledgements g This work was supported in part by the Portuguese Foundation for Science and Technology (‘FCT-Fundação para a Ciência e a Tecnologia’) through the CIDMA - Center for Research and Development in Mathematics and Applications, within project UID/MAT/04106/2013, and by the Postdoctoral Foundation from FCT (Portugal) under Grant Page 11 of 11 Page 11 of 11 He et al. Boundary Value Problems ( 2016) 2016:22 References 1. Muskhelishvili, NI: Singular Integral Equations. Noordhoﬀ, Leyden (1977) 2. Lu, J-K: Boundary Value Problems for Analytic Functions. World S 3. Fokas, AS: A Uniﬁed Approach to Boundary Value Problems. SIAM, Philadelphia (2008) 4. Balk, MB: On Poly-Analytic Functions. Akademie Verlag, Berlin (1991) 4. Balk, MB: On Poly-Analytic Functions. Akademie Verlag, Berlin (1991) 5. 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https://openalex.org/W3156890144	https://baas.aas.org/pub/2021n4i052/download/pdf	Latin	null	Returning Samples from Enceladus for Life Detection	null	2,021	cc-by	7,350	Returning Samples from Enceladus for Life Detection Primary author: Marc Neveu - NASA Goddard Space Flight Center, Greenbelt, MD / University of Maryland, College Park, MD - Phone: +1 301-614-6872 - Email: marc.f.neveu@nasa.gov Co-authors: Co-authors: Ariel Anbar - Arizona State University, Tempe, AZ Alfonso Davila - NASA Ames Research Center, Moffett Field, CA Daniel P. Glavin - NASA Goddard Space Flight Center, Greenbelt, MD Shannon M. MacKenzie - Johns Hopkins Applied Physics Laboratory, Laurel, MD Charity Phillips-Lander - Southwest Research Institute, San Antonio, TX Brent Sherwood - Blue Origin, Seattle, WA, USA Yoshinori Takano - Japan Agency for Marine-Earth Science & Technology, Yokosuka, Japan Peter Williams - Arizona State University, Tempe, AZ Hajime Yano - Institute of Space and Astronautical Science, Japan Aerospace Exploration Agency, Sagamihara, Japan Co-signers: Jose Aponte - NASA Goddard Space Flight Center, Greenbelt, MD Ellen Czaplinski - University of Arkansas, Fayetteville, AR Simon Friesen - University of Waterloo, Ontario, Canada Kosuke Fujishima - Earth Life Science Institute, Tokyo Institute of Technology, Tokyo, Japan Julie Huber - Woods Hole Oceanographic Institution, Woods Hole, MA Kim Reh - Jet Propulsion Laboratory, California Institute of Technology, CA Christophe Sotin - Jet Propulsion Laboratory, California Institute of Technology, CA Wataru Takahagi - The University of Tokyo, Bunkyo, Japan Bethany Theiling - NASA Goddard Space Flight Center, Greenbelt, MD Vassilissa Vinogradoff - CNRS, Aix-marseille University, PIIM, LAM, France Ariel Anbar - Arizona State University, Tempe, AZ Alfonso Davila - NASA Ames Research Center, Moffett Field, CA Daniel P. Glavin - NASA Goddard Space Flight Center, Greenbelt, MD Shannon M. MacKenzie - Johns Hopkins Applied Physics Laboratory, Laurel, MD Charity Phillips-Lander - Southwest Research Institute, San Antonio, TX Brent Sherwood - Blue Origin, Seattle, WA, USA Yoshinori Takano - Japan Agency for Marine-Earth Science & Technology, Yokosuka, Japan Peter Williams - Arizona State University, Tempe, AZ Hajime Yano - Institute of Space and Astronautical Science, Japan Aerospace Exploration Agency, Sagamihara, Japan Co-signers: Technology developments are needed for sample collection and preservation, as well as for the implementation of Restricted Earth Return planetary protection policy for cold samples. 5. Technology developments are needed for sample collection and preservation, as well as for the implementation of Restricted Earth Return planetary protection policy for cold samples. 6. The ease of access of ocean material through the plume allows one to bypass the sequence of missions to “fly by, orbit, land, rove, and return samples”. 7. The considerations discussed here could apply to other worlds when hints of erupted material are confirmed to be samples of a subsurface ocean, e.g., Europa (Roth et al. 2014), Ceres (Küppers et al. 2014), or Triton (Kirk et al. 1995). 7. The considerations discussed here could apply to other worlds when hints of erupted material are confirmed to be samples of a subsurface ocean, e.g., Europa (Roth et al. 2014), Ceres (Küppers et al. 2014), or Triton (Kirk et al. 1995). Co-signers: A Saturn orbiter flying about ten times through Enceladus’ plume minimizes the mission duration to ≈ 15 years, but the effect of collection at ≥ 1 km/s must (and can) be minimized. 5. Technology developments are needed for sample collection and preservation, as well as for the implementation of Restricted Earth Return planetary protection policy for cold samples. 6. The ease of access of ocean material through the plume allows one to bypass the sequence of missions to “fly by, orbit, land, rove, and return samples”. 7. The considerations discussed here could apply to other worlds when hints of erupted material are confirmed to be samples of a subsurface ocean, e.g., Europa (Roth et al. 2014), Ceres (Küppers et al. 2014), or Triton (Kirk et al. 1995). Returning Samples from Enceladus for Life Detection - Key points: Returning Samples from Enceladus for Life Detection - Key points: 1. This white paper complements the Planetary Mission Concept Study (PMCS) report “Flagship Concepts for Astrobiology at Enceladus”, which does not address sample return. 1. This white paper complements the Planetary Mission Concept Study (PMCS) report “Flagship Concepts for Astrobiology at Enceladus”, which does not address sample return. 2. Sample return allows adaptation of analyses to prior findings, access to the full diversity of existing and future interrogation techniques, and time and resources to assess the validity of results. Previous experience has shown that all three aspects are crucial to life detection. 2. Sample return allows adaptation of analyses to prior findings, access to the full diversity of existing and future interrogation techniques, and time and resources to assess the validity of results. Previous experience has shown that all three aspects are crucial to life detection. 3. Sample return may require substantially less plume sample (milligrams = a few flybys) than in situ investigations (grams) to perform life detection. 3. Sample return may require substantially less plume sample (milligrams = a few flybys) than in situ investigations (grams) to perform life detection. 4. A Saturn orbiter flying about ten times through Enceladus’ plume minimizes the mission duration to ≈ 15 years, but the effect of collection at ≥ 1 km/s must (and can) be minimized. 4. A Saturn orbiter flying about ten times through Enceladus’ plume minimizes the mission duration to ≈ 15 years, but the effect of collection at ≥ 1 km/s must (and can) be minimized. 5. 1. Why return samples from Enceladus for life detection? A world ripe for life detection. Saturn’s tiny moon Enceladus has a subsurface ocean that sources plumes of water vapor and ice vented to space from its south polar terrain (SPT). The vented material provides the strongest evidence to date of a habitable world beyond Earth (Fig. 1) with liquid water, the major elements for life H, C, N, O, simple and complex organic compounds, bioavailable energy, and clement physicochemical conditions (Postberg et al. 2018). This offers the chance to A world ripe for life detection. Saturn’s tiny moon Enceladus has a subsurface ocean that sources plumes of water vapor and ice vented to space from its south polar terrain (SPT). The vented material provides the strongest evidence to date of a habitable world beyond Earth (Fig. 1) with liquid water, the major elements for life H, C, N, O, simple and complex organic compounds, bioavailable energy, and clement physicochemical conditions (Postberg et al. 2018). This offers the chance to search for life in the plume. Figure 1. Enceladus is farthest along the roadmap to ocean worlds (Hendrix, Hurford et al. 2019). A scientific breakthrough. A positive detection could provide the first definitive evidence for life beyond Earth, with a likely independent emergence given the dearth of material exchange between Enceladus and the inner Solar System (Worth et al. 2013). This would suggest biology is universal and provide a second data point to understand its nature. The alternative result could be the first habitat where signs of life are not detected, constraining the detectability of biospheres beyond Earth (fl term in the Drake equation; Burchell 2006). gy y g Figure 1. Enceladus is farthest along the roadmap to ocean worlds (Hendrix, Hurford et al. 2019). Figure 1. Enceladus is farthest along the roadmap to ocean worlds (Hendrix, Hurford et al. 2019). g A positive detection could provide the first definitive evidence for life beyond Earth, with a likely independent emergence given the dearth of material exchange between Enceladus and the inner Solar System (Worth et al. 2013). This would suggest biology is universal and provide a second data point to understand its nature. The alternative result could be the first habitat where signs of life are not detected, constraining the detectability of biospheres beyond Earth (fl term in the Drake equation; Burchell 2006). Co-signers: Co-signers: Jose Aponte - NASA Goddard Space Flight Center, Greenbelt, MD Ellen Czaplinski - University of Arkansas, Fayetteville, AR Simon Friesen - University of Waterloo, Ontario, Canada Kosuke Fujishima - Earth Life Science Institute, Tokyo Institute of Technology, Tokyo, Japan Julie Huber - Woods Hole Oceanographic Institution, Woods Hole, MA Kim Reh - Jet Propulsion Laboratory, California Institute of Technology, CA Christophe Sotin - Jet Propulsion Laboratory, California Institute of Technology, CA Wataru Takahagi - The University of Tokyo, Bunkyo, Japan Bethany Theiling - NASA Goddard Space Flight Center, Greenbelt, MD Vassilissa Vinogradoff - CNRS, Aix-marseille University, PIIM, LAM, France Co-signers: Jose Aponte - NASA Goddard Space Flight Center, Greenbelt, MD Ellen Czaplinski - University of Arkansas, Fayetteville, AR Simon Friesen - University of Waterloo, Ontario, Canada Kosuke Fujishima - Earth Life Science Institute, Tokyo Institute of Technology, Tokyo, Japan Julie Huber - Woods Hole Oceanographic Institution, Woods Hole, MA Kim Reh - Jet Propulsion Laboratory, California Institute of Technology, CA Christophe Sotin - Jet Propulsion Laboratory, California Institute of Technology, CA Wataru Takahagi - The University of Tokyo, Bunkyo, Japan Bethany Theiling - NASA Goddard Space Flight Center, Greenbelt, MD Vassilissa Vinogradoff - CNRS, Aix-marseille University, PIIM, LAM, France This white paper is an edited version of an eponymous paper by the same authors accepted for publication in Frontiers in Astronomy and Space Sciences: Neveu M, Anbar AD, Davila AF, Glavin DP, MacKenzie SM, Phillips-Lander CM, Sherwood B, Takano Y, Williams P and Yano H (2020) Returning Samples From Enceladus for Life Detection. Front. Astron. Space Sci. 7:26. doi: 10.3389/fspas.2020.00026. That publication is an open-access article distributed under the terms of a CC BY license authorizing use, distribution or reproduction provided it is cited and authors and copyright holders credited, as is done here. Returning Samples from Enceladus for Life Detection - Key points: 1. This white paper complements the Planetary Mission Concept Study (PMCS) report “Flagship Concepts for Astrobiology at Enceladus”, which does not address sample return. 2. Sample return allows adaptation of analyses to prior findings, access to the full diversity of existing and future interrogation techniques, and time and resources to assess the validity of results. Previous experience has shown that all three aspects are crucial to life detection. 3. Sample return may require substantially less plume sample (milligrams = a few flybys) than in situ investigations (grams) to perform life detection. 4. 1. Why return samples from Enceladus for life detection? evidence for life beyond Earth, with a likely independent emergence given the dearth of material exchange between Enceladus and the inner Solar System (Worth et al. 2013). This would suggest biology is universal and provide a second data point to understand its nature. The alternative result could be the first habitat where signs of life are not detected, constraining the detectability of biospheres beyond Earth (fl term in the Drake equation; Burchell 2006). y Earth, with a likely independent emergence given the dearth of material exchange between Enceladus and the inner Solar System (Worth et al. 2013). This would suggest biology is universal and provide a second data point to understand its nature. The alternative result could be the first habitat where signs of life are not detected, constraining the detectability of biospheres beyond Earth (fl term in the Drake equation; Burchell 2006). 1 Why sample return? Life detection mission concepts ranging from in situ (Lunine et al. 2015; MacKenzie et al. 2016; Eigenbrode et al. 2018), to orbiting or landing (MacKenzie et al. PMCS report), to sample return (Tsou et al. 2012; Sekine et al. 2014) have been proposed or discussed. Returning samples of ejected ocean material comes with challenges (Section 3), but uniquely enables investigations that can be adapted as results are obtained, are exquisitely sensitive, and leverage progress in measurement capabilities for decades to come. Life detection... In situ In returned samples Advantages • Contextual understanding • Sample minimally altered • Adaptable analyses • Unmatched instruments • Archival Drawbacks • Inflexible measurement capabilities • Limited space-proof instruments • Lack of contextual understanding • Sample alteration between collection and measurement Table 1. Relative merits of in situ and sample return missions for life detection. Why sample return? Life detection mission concepts ranging from in situ (Lunine et al. 2015; MacKenzie et al. 2016; Eigenbrode et al. 2018), to orbiting or landing (MacKenzie et al. PMCS report), to sample return (Tsou et al. 2012; Sekine et al. 2014) have been proposed or discussed. Returning samples of ejected ocean material comes with challenges (Section 3), but uniquely enables investigations that can be adapted as results are obtained, are exquisitely sensitive, and leverage progress in measurement capabilities for decades to come. Life detection... 1. Why return samples from Enceladus for life detection? In situ In returned samples Advantages • Contextual understanding • Sample minimally altered • Adaptable analyses • Unmatched instruments • Archival Drawbacks • Inflexible measurement capabilities • Limited space-proof instruments • Lack of contextual understanding • Sample alteration between collection and measurement Table 1. Relative merits of in situ and sample return missions for life detection. Adaptable analyses. The path to every confirmed or refuted claim of life detection in samples of ancient (e.g., Schopf 1993), extraterrestrial (McKay et al. 1996), or otherwise secluded environments (Priscu et al. 1999) has taken years of subsequent, initially unplanned analyses. This is the time needed for cross-validation by other research teams, refutation of claims, or follow-on hypothesis-driven measurements, which proceed at the pace of journal publications. In this context, in situ life detection presents the risk that the payload finds tantalizing clues that it cannot confirm. As versatile as flight instruments can be, their capabilities are frozen-in at the time of design, years ahead of the measurements, with a preconception of what could be measured. With returned samples, the next measurement can be devised based on the results of the previous one, with ample time to think through the path of analysis. Laboratory measurements can be adapted to sample properties, repeated, and checked with complementary techniques. The best instruments cannot be flown. For life detection, these include: 1. higher performance than can be flown at any given time (Table 2). 2. instruments that cannot be miniaturized, requiring a room or a facility (Fig. 2; Table 2). 3. complex wet chemistry protocols or sample preparation steps (Elsila et al. 2009), 4. a more diverse suite of techniques than could be accommodated on spacecraft. Figure 2. Example techniques to search for life that cannot be flown. A. Secondary ion mass spectrometer for isotopic measurements on small samples proximal to contamination, built for the Genesis mission. B. Synchrotron used, e.g., for micro X-ray diffraction. C. Matrix-assisted laser desorption / ionization-mass microscope. Archival. Archiving part of returned material enables analyses with techniques that cannot be fathomed at the time of mission design. Analyses of archived Apollo lunar samples keep on yielding insights with new means of investigation (Shearer & Borg 2006; Zeigler et al. 2019). The best instruments cannot be flown. For life detection, these include: The best instruments cannot be flown. For life detection, these include: The best instruments cannot be flown. 1. Why return samples from Enceladus for life detection? For life detection, these include: 1. higher performance than can be flown at any given time (Table 2). 2. instruments that cannot be miniaturized, requiring a room or a facility (Fig. 2; Table 2). 3. complex wet chemistry protocols or sample preparation steps (Elsila et al. 2009), 4. a more diverse suite of techniques than could be accommodated on spacecraft. Figure 2. Example techniques to search for life that cannot be flown. A. Secondary ion mass spectrometer for isotopic measurements on small samples proximal to contamination, built for the Genesis mission. B. Synchrotron used, e.g., for micro X-ray diffraction. C. Matrix-assisted laser desorption / ionization-mass microscope. samples proximal to contamination, built for the Genesis mission. B. Synchrotron used, e.g., for micro X-ray diffraction. C. Matrix-assisted laser desorption / ionization-mass microscope. Archival. Archiving part of returned material enables analyses with techniques that cannot be fathomed at the time of mission design. Analyses of archived Apollo lunar samples keep on yielding insights with new means of investigation (Shearer & Borg 2006; Zeigler et al. 2019). Archival. Archiving part of returned material enables analyses with techniques that cannot be fathomed at the time of mission design. Analyses of archived Apollo lunar samples keep on yielding insights with new means of investigation (Shearer & Borg 2006; Zeigler et al. 2019). 2 2. Measurement strategy, sample needed, and mission architectures The community has set a high bar: life must be the only possible explanation for the results (Neveu et al. 2018). To this end, life detection strategies target multiple attributes (potential “biosignatures”) that distinguish biological from abiotic properties or processes (e.g., Hand et al. 2017; Neveu et al. 2018; Glavin et al. 2019). If life is not found, these measurements are crucial to find out how far toward life chemistry has progressed, reconstruct the sample’s provenance (journey between synthesis and collection), and quantify how much biomass (if any) was expected. Example attributes, detection techniques, and corresponding amounts of solid sample needed (water ice with percent-level salts and organic compounds) are provided in Table 2. 1. Why return samples from Enceladus for life detection? Biosignature Example technique Capability on Earth Capability in situ Sample needed Over-representation of amino acids of high molar mass and structural isomeric preference Mass spectrometry with separation stage (e.g., capillary electrophoresis, gas or liquid chromatography) Abundances of amino acids of mass ≤ 500 atomic mass units (amu) at ≥ 1 pmol g-1 Abundances of amino acids of mass ≤ 500 amu at ≥ 1 pmol g-1 (ref. 1,2) 0.1 g (in situ) (ref. 2) Distribution of carbon chain length in long-chain hydrocarbons Relative abundances of hydrocarbons with mass ≤ 104 amu present at ≥ 1 pmol g-1 Relative abundances of hydrocarbons ≤ 500 amu at ≥ 1 pmol g-1 (ref. 1,2) 0.1 g (in situ) (ref. 2) Distribution of the number of types of operations needed to build each organic molecule Organic compounds ≤ 104 amu at ≥ 1 fmol g-1, mass resolution > 105-106 Organic compounds ≤ 1000 amu at ≥ 1 pmol g-1, mass resolution 1 amu (ref. 2) 0.1 g (in situ) (ref. 2) Enantiomeric excess in all chiral amino acids As above, plus relative molar abundances of enantiomers of amino acids with up to 7 C atoms at ppb levels (ref. 3,4) As above, plus relative molar abundances of enantiomers of amino acids at ppm levels (ref. 1,5) 0.5 mg (sample return) (ref. 6,7) 0.5 g (in situ) (ref. 2,7) Enantiomeric excess in all chiral sugar compounds (polyols) Liquid extraction, purification, derivatization, and gas chromatography (ref. 4,8) Relative abundances of enantiomers of sugars with up to 6 C atoms (ref. 4,8) Cannot currently be implemented (ref. 4) mg to g (sample return) (ref. 4,9) Polymers with a repeating charge in their backbone Nanopore sequencing Identify and sequence DNA, RNA, and other biopolymers agnostically (ref. 10) from ≥ 1 ng polymer (ref. 11) Not yet fully established (ref. 12) > 1 kg based on ~100 ng DNA (kg Earth ocean water)-1 (ref. 13). Enceladus biomass > 102 to times more dilute (ref. 14) Polymers of sugars, amino acids, or nucleotides Immunoassay Specific compounds at 10-18-10-12 g g-1 (ref. 15) Specific compounds at > 0.1-1 µg g-1 (ref. 16) > 1 mg (sample return); > 1-10 g (in situ), assuming 25 fg protein cell-1 (ref. 17) Matrix assisted laser desorption/ionization mass spectrometry (MALDI) Compounds at ≤ 10-11 g µm-2 (ref. 18). Imaging at µm scale (ref. 19). LDI only, < 103 amu (ref. 1. Why return samples from Enceladus for life detection? Inventory of species differing from abiotic thermodynamic equilibrium or steady state Same as Distribution of the number of types of operations needed to obtain each molecule in the pool of detected organic compounds and Co-located oxidants and reductants Table 2. Life detection capabilities and sample needed. Red: no capability; orange: in progress; bold: Earth capability exceeds that in situ by orders of magnitude. References listed at the end. Same as Distribution of the number of types of operations needed to obtain each molecule in the pool of detected organic compounds and Co-located oxidants and reductants Inventory of species differing from abiotic thermodynamic equilibrium or steady state Same as Distribution of the number of types of operations needed to obtain each molecule in the pool of detected organic compounds and Co-located oxidants and reductants Table 2. Life detection capabilities and sample needed. Red: no capability; orange: in progress; bold: Earth capability exceeds that in situ by orders of magnitude. References listed at the end. Milligrams (a few flybys) may be enough. The sample needed is partly constrained by the plume’s sub-percent organic content. It also hinges on the ill-constrained possible biomass density; in Table 2 a plausible 100 cells mL-1 is assumed (Hand et al. 2017; Cable et al. 2020). In situ, the sample needed is several grams. This excludes use of the nanopore and assumes measurements are repeated (Lorenz 2019). In returned samples, only milligrams may be needed (Table 2). Because a 1 m2 area flown through the plume at 50 km altitude is expected to collect on the order of 3 mg (Porco et al. 2017), in situ analyses require hundreds of flybys by a Saturn or Enceladus orbiter or a landed mission, whereas one flyby may suffice for a sample return. Mission architectures capable of returning milligrams of sample are compared in Table 3. SUN ORBITER 1 flyby (no contingency) SATURN ORBITER ≈ 15 flybys year-1 ENCELADUS ORBITER 2 flybys day-1 CATCHING LANDER SURFACE SAMPLING LANDER Collection rate 0.003 g m-2 in total 0.045 g m-2 year-1 2 g m-2 year-1 100 g m-2 y-1 near stripes 3.5 g day-1 Collection velocity ~km s-1 ~km s-1 250 m s-1 (orbital speed + plume particle speed; ref. 1). ≤ 150 m s-1 (ref. 1. Why return samples from Enceladus for life detection? 2) (< 10-mer). Sensitivity 1 ng g-1. > 10 mg (sample return) (ref. 18,19); > 1 g (in situ LDI) (ref. 2) based on laser spot sizes. Morphologies resembling microbial cells co-located with physical activity, textures, or compositions distinct from the environmental background Atomic force microscopy Atom by atom imaging, force spectroscopy nm-scale imaging, µm-scale field of view (ref. 20). No force spectroscopy yet (ref. 1,21). > 1 g (assuming 100 cells g-1) Optical microscopy µm-scale imaging, 0.5 mm field of view (FoV). UV fluorescence. µm-scale imaging, FoV 0.5 mm. UV fluorescence with UV light source (ref. 1,22,23). > 1 g (assuming 100 cells g-1) Digital holographic microscopy No motility measurements possible if no live cells Motion rate, direction. µm- imaging, FoV 0.5 mm (ref. 23) > 1 g (assuming 100 cells g-1) MALDI imaging µm-scale organic composition maps to 104-105 amu (ref. 19) No mapping capability < 1 ng (sub-mm grains) Atom probe tomography; e- microscopy / X-ray spectro. Elemental maps from µm to sub-nm scales (ref. 24,25) Cannot be flown < 1 ng (sub-mm grains) [Nanoscale-] secondary ion mass spectrometry (SIMS) Isotopic maps to sub-µm scales (ref. 25) Cannot be flown < 1 ng (sub-mm grains) 3 3 Isotopic fractionation in organic compounds and inorganic species Orbitrap mass spectrometry Isotopic ratios to 5‰ of C, N, S and if possible H and O in specific organic compounds at ≥ 0.1 µmol g-1 (≈ 10 ppm) Orbitrap being developed for flight (ref. 26). Wet chemistry/GC-c-IRMS not yet developed for flight. 0.1 g (for C), 0.25 g (for N) for 0.1-nmol limit of detection (ref. 1,27) Wet chemistry + Gas chromatography - combustion - isotope ratio mass spec. Inductively coupled plasma mass spectrometry, SIMS 54Fe/56Fe to 0.1‰ precision vs. Fe abundance (ref. 28). ICP-MS not yet developed for flight. SIMS cannot be flown. < 1 mg (sample return) Co-located oxidants and reductants X-ray diffractometry; Infrared / Raman spectro.; Imaging mass spec.; α-particle X-ray spectrometry; Laser-induced breakdown spec. Maps of organic and inorganic species < 104-105 amu at µm to nm-scale. Minerals at > 5 vol%. Organic and mineral crystal structures. Maps of organic and inorganic species < 500 amu (ref. 1) at sub-mm scales. Minerals at > 5 vol%. << 1 g (sample return); ≤ 1 g (in situ) (ref. 29,30). No sample needed for in situ infrared spectroscopy at close range. 1. Why return samples from Enceladus for life detection? 1) Fallback at ≤ 150 m s-1 experienced by material Age since ejection < 5 min (≲ 50 km/0.15 km s-1) < 5-10 min > 10 years Access to neg. control2 No Depends on understanding of individual jet sources No (unless mobile) Limited by reach/mobility Rationale for collection rate 3 (±2.5) mg m-2 per flyby at 50 km altitude (ref. 1) 3 (±2.5) mg m-2 per flyby at 50 km altitude (ref. 1). 2 flybys per day (ref. 3). > 0.1 mm year-1 near tiger stripes (ref. 4). 10 g per scoop every other day (ref. 5). Age based on fallback rate. Mission ∆v ≈0 ~3 km s-1 (ref. 6) ~4.5 km s-1 (2x ref. 5 ∆v budget in Saturn system) ~5 km s-1 (Orbiter ∆v + ≈2*∆v from/to halo orbit with semimajor axis 500 km) (refs. 3,7) Mission duration (launch to reentry) 25-34 years (ref. 8) 13-15 years possible (ref. 6) ≥ 26 years (ref. 8, 2x ref. 7) due to prolonged pump-down in the Saturn system (gravity assists from Titan, Rhea, Dione, Tethys; or electric propulsion) Table 3. Architecture trades. Simpler options have less time at Enceladus, do less contextual science, and collect samples at higher speed. References listed at the end. Traded quantities are mission duration, ∆v (fuel mass, cost), and sampling velocity (to minimize changes to the sample upon collection), whereas sampling altitude is set by navigation uncertainty. Missions are all longer than the ≈ 15 years for which spacecraft parts are qualified, save for the Saturn orbiter option. That option leads to the highest collection speeds (km s-1). Although gentler capture by orbiters and landers is preferred, even at km s-1 molecules up to small chains of monomers survive impact if encased in ice grains (Burchell et al. 2014) because Traded quantities are mission duration, ∆v (fuel mass, cost), and sampling velocity (to minimize changes to the sample upon collection), whereas sampling altitude is set by navigation uncertainty. Missions are all longer than the ≈ 15 years for which spacecraft parts are qualified, save for the Saturn orbiter option. That option leads to the highest collection speeds (km s-1). Although gentler capture by orbiters and landers is preferred, even at km s-1 molecules up to small chains of monomers survive impact if encased in ice grains (Burchell et al. 2014) because 4 much of the impact kinetic energy is consumed in sublimating the ice. 1. Why return samples from Enceladus for life detection? A few percent of (dead) cells can withstand ejection (Bywaters et al. 2020). Whether they withstand sampling at > 100 m s-1 is unknown. The merits of architectures are further discussed in the Enceladus PMCS report. much of the impact kinetic energy is consumed in sublimating the ice. A few percent of (dead) cells can withstand ejection (Bywaters et al. 2020). Whether they withstand sampling at > 100 m s-1 is unknown. The merits of architectures are further discussed in the Enceladus PMCS report. 3. Addressing challenges germane to sample return Samples are altered between their synthesis and their measurement (Fig. 3). Some alteration steps are natural (#1-3 in Fig. 3). Understanding them requires understanding the sample’s geological context, which on returned samples is only accessible at scales smaller than the sample. The other steps can be controlled. Steps #5-9 are specific to sample return. Figure 3. Steps altering samples between their synthesis in Enceladus’ ocean and their measurement in the laboratory. Understanding the geological context. An in situ study of plume material would establish a necessary baseline from which any alterations due to sample return can be identified. This makes an in situ mission (Lunine et al. 2015; Eigenbrode et al. 2018; MacKenzie et al. PMCS report) a logical precursor to sample return (Fig. 4) unless the in situ study is done by the sample return mission. The latter approach may be sensible given long trip times to Enceladus. 3. Addressing challenges germane to sample return Retaining compounds freed from ice upon impact requires deposition on secondary surfaces (Aksyonov & Williams 2001). These could be set up for robotic insertion into analytical Figure 4. Paths for the astrobiological exploration of Enceladus. Steps may be bypassed (dashed arrows) based on changing knowledge and the design and findings of future missions. Modified from Sherwood (2016). Figure 4. Paths for the astrobiological exploration of Enceladus. Steps may be bypassed (dashed arrows) based on changing knowledge and the design and findings of future missions. Modified from Sherwood (2016). Minimizing sample alteration between capture and measurement. The plume contains water and other volatile species silicate grains organic compounds up to (at least) ~103 amu and noble Minimizing sample alteration between capture and measurement. The plume contains water and other volatile species, silicate grains, organic compounds up to (at least) ~103 amu, and noble gases Each component may require specific handling We focus on plume solids (Table 2) Minimizing sample alteration between capture and measurement. The plum Minimizing sample alteration between capture and measurement. The plume contains water and other volatile species, silicate grains, organic compounds up to (at least) ~103 amu, and noble gases. Each component may require specific handling. We focus on plume solids (Table 2). Collection. Low-density aerogel can capture this material at km s-1 and preserve it (Sandford et al. 2006; Elsila et al. 2009; Carr et al. 2014). However, aerogel also traps organic contaminants. Both are dispersed in the aerogel matrix, complicating recovery and making aerogel a high-organic background material. Likewise, stacks of thin metal foils can capture particles relatively gently but share with aerogel the difficulty of extracting the sample. On solid metal (e.g., Al, Au, stainless steel, or Ti) collectors, contamination is more easily removed chemically or thermally. Retaining compounds freed from ice upon impact requires deposition on secondary surfaces (Aksyonov & Williams 2001). These could be set up for robotic insertion into analytical Collection. Low-density aerogel can capture this material at km s-1 and preserve it (Sandford et al. 2006; Elsila et al. 2009; Carr et al. 2014). However, aerogel also traps organic contaminants. Both are dispersed in the aerogel matrix, complicating recovery and making aerogel a high-organic background material. Likewise, stacks of thin metal foils can capture particles relatively gently but share with aerogel the difficulty of extracting the sample. 3. Addressing challenges germane to sample return Samples are altered between their synthesis and their measurement (Fig. 3). Some alteration steps are natural (#1-3 in Fig. 3). Understanding them requires understanding the sample’s geological context, which on returned samples is only accessible at scales smaller than the sample. The other steps can be controlled. Steps #5-9 are specific to sample return. Figure 3. Steps altering samples between their synthesis in Enceladus’ ocean and their measurement in the laboratory. Understanding the geological context. An in situ study of plume material would establish a necessary baseline from which any alterations due to sample return can be identified. This makes an in situ mission (Lunine et al. 2015; Eigenbrode et al. 2018; MacKenzie et al. PMCS report) a logical precursor to sample return (Fig. 4) unless the in situ study is done by the sample return mission. The latter approach may be sensible given long trip times to Enceladus. Figure 4. Paths for the astrobiological exploration of Enceladus. Steps may be bypassed (dashed arrows) based on changing knowledge and the design and findings of future missions. Modified from Sherwood (2016). Minimizing sample alteration between capture and measurement. The plume contains water and other volatile species, silicate grains, organic compounds up to (at least) ~103 amu, and noble gases. Each component may require specific handling. We focus on plume solids (Table 2). Figure 4. Paths for the astrobiological exploration of Figure 4. Paths for the astrobiological exploration of Enceladus. Steps may be bypassed (dashed arrows) based on changing knowledge and the design and findings of future missions. Modified from Sherwood (2016). Minimizing sample alteration between capture and measurement. The plume contains water and other volatile species, silicate grains, organic compounds up to (at least) ~103 amu, and noble gases. Each component may require specific handling. We focus on plume solids (Table 2). Collection. Low-density aerogel can capture this material at km s-1 and preserve it (Sandford et al. 2006; Elsila et al. 2009; Carr et al. 2014). However, aerogel also traps organic contaminants. Both are dispersed in the aerogel matrix, complicating recovery and making aerogel a high-organic background material. Likewise, stacks of thin metal foils can capture particles relatively gently but share with aerogel the difficulty of extracting the sample. On solid metal (e.g., Al, Au, stainless steel, or Ti) collectors, contamination is more easily removed chemically or thermally. 3. Addressing challenges germane to sample return During reentry, keeping the collector cold mitigates volatile loss but enhances contamination from the atmosphere and outgassing of the capsule if a hermetic seal cannot be maintained. In curation, besides contamination inputs, the sample can lose volatile components. Levels of the amino acid glycine derived from a volatile precursor decreased by a factor > 6 in Stardust foils stored at room temperature in a Class 5 cleanroom over 1000 days (Glavin et al. 2014). Contamination and volatile compound losses can be mitigated by sealing (Okazaki et al. 2017) and leak rate monitoring. Samples would likely be curated in conditions emulating the plume’s cold vacuum. Implementing Restricted Earth Return planetary protection policy. The COSPAR policy for Cat. V – Restricted missions (Kminek et al. 2017) is not quantitative. This allows flexibility in its implementation and means to check compliance but hampers preliminary design and costing. Quantitative requirements and means to check compliance could be specified for its provisions: “Unless the samples […] are steriliz[ed], the canister(s) […] shall be closed, with an appropriate verification process, and the samples shall remain contained during all mission phases through transport to a receiving facility where it (they) can be opened under containment.” A maximum leakage rate could be specified for particles of the size of the smallest known biological pathogens (10 to 15 nm for prions; Silveira et al. 2005), and tested with smaller tracers embedded in and monitored outside the sealed areas. Helium is commonly used, but its comparatively small van der Waals radius (0.14 nm) may be unnecessarily stringent. “The mission [...] must provide a method to ‘break the chain of contact’ with […] Enceladus. No uncontained hardware […] shall be returned to the Earth’s biosphere or the Moon. Isolation […] shall be provided during [all] transfer operations [...].” This could require that uncontained parts in contact with the plume be jettisoned prior to or sterilized during reentry (actively or passively by, e.g., ambient radiation). Maximum probabilities of impact of unconfined parts on the Earth or the Moon, or of microbial survival on such parts, could be specified. “For unsterilized samples returned to Earth, a program of life detection and biohazard testing, or a proven sterilization process, shall be undertaken as an absolute precondition for the controlled distribution of any portion of the sample.” The policy could specify at which mission stages this program could take place. 3. Addressing challenges germane to sample return On solid metal (e.g., Al, Au, stainless steel, or Ti) collectors, contamination is more easily removed chemically or thermally. Retaining compounds freed from ice upon impact requires deposition on secondary surfaces (Aksyonov & Williams 2001). These could be set up for robotic insertion into analytical 5 instruments upon return, minimizing exposure to Earth contaminants. They could also be patterned so the diffuse sample layer can be concentrated into small spots prior to analysis. instruments upon return, minimizing exposure to Earth contaminants. They could also be patterned so the diffuse sample layer can be concentrated into small spots prior to analysis. Preservation through reentry. In the return cruise, radiation accumulates and an increasing solar flux warms the capsule, but the highest thermal and mechanical stresses occur at reentry and (possibly impact) landing. Refractory (thermally tolerant) and gas (mechanically resilient) sample fractions may be separated prior to reentry to tailor stress mitigation for each (Lunine et al. 2018). However, there is no ready approach to preserving ices sensitive to both stresses. Thermal mitigations include passive or active cooling and/or phase change materials. Contamination mitigation includes cleaning the collector and capsule before launch, vacuum bakeout of the collector in space, protecting the collector from recontamination prior to and after sample collection, using witness materials that share the collector’s composition and geometry, and outgassing and exposing these to space radiation prior to sampling (e.g., Summons et al. 2014). During reentry, keeping the collector cold mitigates volatile loss but enhances contamination from the atmosphere and outgassing of the capsule if a hermetic seal cannot be maintained. In curation, besides contamination inputs, the sample can lose volatile components. Levels of the amino acid glycine derived from a volatile precursor decreased by a factor > 6 in Stardust foils stored at room temperature in a Class 5 cleanroom over 1000 days (Glavin et al. 2014). Contamination and volatile compound losses can be mitigated by sealing (Okazaki et al. 2017) and leak rate monitoring. Samples would likely be curated in conditions emulating the plume’s cold vacuum. Contamination mitigation includes cleaning the collector and capsule before launch, vacuum bakeout of the collector in space, protecting the collector from recontamination prior to and after sample collection, using witness materials that share the collector’s composition and geometry, and outgassing and exposing these to space radiation prior to sampling (e.g., Summons et al. 2014). 3. Addressing challenges germane to sample return (1999) Science 286:2141 • Roth et al. (2014) Science 343:171 • Sandford et al. (2006) Science 314:1720 • Schopf (1993) Science 260:640 • Sekine et al. 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(2014) Astrobiology 14:969 • Takano et al. (2014) Adv Sp Res 53:1135 • Tsou et al. 3. Addressing challenges germane to sample return A first option is within the capsule just after capture (Yano et al. 2016a). A second is post-recovery of the capsule on Earth but prior to breaking the seal, either by outfitting the capsule with optical waveguides or puncturable interfaces (Yano et al. 6 2016b), or by using non-contact techniques such as X-ray computed tomography (Takano et al. 2014; Zeigler et al. 2019). Life detection after opening the capsule but prior to distributing the sample could take place onboard a research ship confined in international waters (Takano et al. 2014). The policy could also list proven methods of sterilization. 2016b), or by using non-contact techniques such as X-ray computed tomography (Takano et al. 2014; Zeigler et al. 2019). Life detection after opening the capsule but prior to distributing the sample could take place onboard a research ship confined in international waters (Takano et al. 2014). The policy could also list proven methods of sterilization. Providing this detail either in the policy or in vetted documentation (e.g., NASA Procedural Requirement 8020.12D that implements the COSPAR policy), and allowing other approaches that equivalently achieve compliance, would advance astrobiology sample return. References: Aksyonov & Williams (2001) Rapid Comm Mass Spec 15:2001 • Burchell (2006) Int J Astrobio 5:243 • Burchell et al. (2014) Astrobio 14:473 • Cable et al. (2020) in Planetary Astrobiology, UA Press • Bywaters et al. (2020) Life 10:40. • Eigenbrode et al. (2018) COSPAR Sci Assy, F3.6-3-18 • Elsila et al. (2009) MaPS 44:1323 • Glavin et al. (2014) Intl. Workshop Instru Planet Missions, 1012 • Glavin et al. (2019) Chem Rev, in press. Hand et al. (2017) Europa Lander SDT Report • Hendrix, Hurford et al. (2019) Astrobio 19:1 • Kirk et al. (1995) in Neptune and Triton, UA Press, p. 949 • Kminek et al. (2017) COSPAR’s Planetary Protection Policy • Küppers et al. (2014) Nature 505:525 • Lorenz (2019) Nat Astron 3:466 • Lunine et al. (2015) EGU 17:14923 • Lunine et al. (2018) EPSC abstr. 122 • MacKenzie et al. (2016) Adv Sp Res 58:1117 • MacKenzie et al. PMCS Report • McKay et al. (1996) Science 273:924 • Neveu et al. (2018) Astrobio 18: 1375 • Okazaki et al. (2017) Sp Sci Rev 208:107 • Porco et al. (2017) Astrobio 17:876 • Postberg et al. (2018) in Enceladus and the Icy Moons of Saturn, UA Press, p. 129 • Priscu et al. 3. Addressing challenges germane to sample return (2012) Astrobiology 12:730 • Worth et al. (2013) Astrobio 13:1155 • Yano et al. (2016a) COSPAR Sci Assy, B0.6-8-16 • Yano et al. (2016b) COSPAR Sci Assy, B0.6-3-16 • Zeigler et al. (2019) Elements 15:286. Table 2: 1Hand et al. (2017). 2Goesmann et al. (2017) Astrobio 17:655. 3Koga & Naraoka (2017) Sci Rep 7:636. 4Glavin et al. (2019). 5Freissinet et al. (2010) J Chromato A 1217:731. 6Burton et al. (2013) MaPS 48:390. 7Assuming a ratio of amino acids to total (≈ dissolved) organic carbon of 1:200 (e.g., Lee & Bada 1975, EPSL 26:61). 8Cooper & Rios (2016) PNAS 113:E3322. 9Furukawa et al. (2019) PNAS 116:24440. 10Rezzonico (2014) Astrobio 14:344. 11Plesivkova et al. (2019) Wiley Int Rev: Forens Sci 1:e1323. 12Sutton et al. (2019) Sci Rep 9:5370. 13Collins et al. (2018) Comm Biol 1:185. 14Cable et al. (2020). 15Zhang et al. (2013) Adv Mat 25:3802. 16Parro et al. (2011) Astrobio 11:15. 17Zubkov et al. (1999) Appl Env Micr 65:3251. 18Cornett et al. (2007) Nat Meth 4:828. 19Guenther et al. (2010) Int J Mass Spec 294:7. 20Riedler et al. (2007) Sp Sci Rev 128:869. 21Bentley et al. (2016) Acta Astron 125:11. 22Hecht et al. (2008) JGR Planets 113:E00A22. 23Bedrossian et al. (2017) Astrobio 17:913. 24Branson et al. (2016) PNAS 113: 12934. 25Jin & Bose (2019) Sci Adv 5:p.eeav8106. 26Selliez et al. (2019) Planet Sp Sci 170:42. 27Elsila et al. (2009). 28Johnson et al. (2008) AREPS 36:457. 29e.g., Nurul Abedin et al. (2018) Appl Opt 57:62. 30e.g., Blake et al. (2012) Sp Sci Rev 170:341. Table 3: 1Porco et al. (2017). 2Lorenz (2019). 3Massarweh & Cappuccio (2020) doi: 10.2514/6. 2020-0467. 4Southworth et al. (2019) Icarus 319:33. 5Hand et al. (2017). 6Tsou et al. (2012). 7Spencer et al. (2010) V&V MCS Report. 8Sekine et al. (2014). 7
W4241608657.txt	http://www.nomos-elibrary.de/10.5771/0010-3497-2002-3-344.pdf	de		Bericht - Personalia - Chronik	Communicatio socialis	2,002	cc-by	7,679	Communicatio Socialis 35 (2002), Nr. 3: 344–366 Quelle: www.communicatio-socialis.de https://doi.org/10.5771/0010-3497-2002-3-344, am 14.07.2024, 00:36:21 Open Access – - https://www.nomos-elibrary.de/agb DURCH RECHERCHEN ENTHÜLLT lungsgeschichten über Sex-Skandale begonnen, als die Tageszeitung Boston Globe meldetete, dass der gegenwärtig wegen eines länger zurückliegenden pädophilen Missbrauchs eines Jungen angeklagte, inzwischen pensionierte Geistliche John Geoghan sich über 30 Jahre lang immer wieder an Jungen vergangen hatte_ Zwei Reporter der Zeitung hatten bei ihren Recherchen in Gerichtsakten herausgefunden, dass die Delikte des Priesters der erzbischöflichen Behörde bekannt waren, dass Geoghan aber auf Anweisung des seit langem amtierenden Ortsbischofs, Kardinal Bernhard Law, nach dem Bekanntwerden seiner Vergehen lediglich auf andere Stellen in der Seelsorge versetzt worden war, während die Opfer durch finanzielle Abfindungen zum Schweigen verurteilt worden waren. Als sich im Zuge weiterer Recherchen der Zeitung herausstellte, dass der Kardinal weitere Fälle von pädophilen Vergehen seiner Priester in gleieher Weise sowohl in der Öffentlichkeit als auch bei den betroffenen Gemeinden vertuscht hatte, versuchte das Erzbistum die Fälle zunächst herunterzuspielen und der Presse den Vorwurf zu machen, sie wolle nur der Kirche und speziell Kardinal Law schaden, zumal dieser als Vorsitzender der Pro Life-Kommission der US-Bischofskonferenz immer höchste moralische Ansprüche in der Öffentlichkeit gestellt hatte. Vor 20 Jahren, so verteidigte sich das Erzbistum, habe man nach medizinischwissenschaftlichen Erkenntnissen geglaubt, Pädophile könnten nach einer Therapie geheilt werden, und die finanzielle Abfindung sei zum Schutz der Missbrauchten vor öffentlichen Skandalen erfolgt. Die Kirche von Boston reagierte also mit dem üblichen Vorwurf an die Medien, sie inszenierten die Sex-Skandale aus Profitgier. Dann eskalierte die Sache jedoch in mehrfacher Hinsicht. Nachdem in Boston die Barriere des Schweigens gebrochen war, meldeten sich fast täglich in weiteren Bistümern Männer, die -meist vor 20 oder 30 Jahren - von Priestern belästigt worden waren und jetzt, oft gedrängt von geschäftstüchtigen Anwälten, finanzielle Entschädigungen verlangten. Kardinal Law geriet zusätzlich unter Beschuss, als Reporter des Boston Globe den Fall eines Priesters aufdeckten, der sich in Boston nicht nur als Seelsorger für Homosexuelle engagiert hatte, sondern auch selbst als "gay" outete. Kardinal Law hatte der von diesem Priester selbst erbetenen Versetzung in ein kalifornisches Bistum zugestimmt, ohne den zuständigen Ortsbischof über die Hintergründe zu informieren, und dem Priester sogar noch Unbedenklichkeit attestiert. In Kalifornien hatte der Priester mit seinem Freund ein Hotel betrieben, in dem vorwiegend Schwule und Lesben verkehrten. Selbst jetzt verteidigte der Kardinal sich noch - ebenso wie bei mehreren Gerichtsverhandlungen in Boston damit, nichts gewusst und in gutem Glauben gehandelt zu haben. Inzwischen hatten sämtliche Medien die Brisanz der Skandale enthttps://doi.org/10.5771/0010-3497-2002-3-344, am 14.07.2024, 00:36:21 Open Access – - https://www.nomos-elibrary.de/agb 345 GERTEL deckt, und nicht nur die Lokalpresse und die lokalen TV-Sender berichteten ausführlich über jeden neuen Fall und jede neue Wendung, sondern auch die nationalen Netzwerke und die großen Führungsblätter an der Ost- und Westküste, im Süden und im Mittleren Westen brachten täglich eine "katholische Story", und zwar auf der Titelseite: New York Timesund Washington Post, Chicago Tribune, St. Louis Post Dispatch und Los Angeles Times. Selbst die einzige in ganz Amerika verbreitete Boulevardzeitung USA Today stieg mit Titelstories, Interviews und eigenen Umfragen ein. Ausführlicher griffen die Nachrichtenmagazine Time und Newsweek nicht nur die sexuellen Vergehen auf, sondern behandelten sie in größeren Zusammenhängen die Situation der katholischen Kirche in den USA und in der Weltkirche. Time stellte bereits am 1. April 2002 in einer Titelgeschichte unter der Überschrift "Kann die katholische Kirche sich selbst retten?" die stark autoritäre, hierarchische Struktur der Kirche mit dem Anspruch, alle ihre Angelegenheiten selbst regeln zu wollen, als einen Hauptgrund für die Krise heraus und fragte am 3. Juni angesichts der hohen Entschädigungssummen: "Kann eine Kirche pleite gehen?" Die nationalen Fernseh-Netzwerke von ABC bis CBS richteten eigene Newsblocks über "Die Krise der katholischen Kirche" ein, schön umrahmt mit Kirchenfensterfarben. Dabei ließen sich einige Grundzüge in der Beurteilung der Vorgänge erkennen. Einerseits wurde die katholische Kirche als eine Machtinstitution gesehen, die für das öffentliche Leben in Amerika, und zwar nicht nur für die Katholiken, sondern für die ganze Gesellschaft hohe moralische Werte setzt, jetzt aber weder den eigenen Ansprüchen noch den berechtigten Interessen der Öffentlichkeit entspricht. Andererseits fehlen den meisten säkularen Medien Reporter und Redakteure, die diese Kirche, ihre Struktur und Verfassung, ihre Mission und Verwaltung, ihre Geschichte und ihr Evangelium kennen. Dies ist nicht nur darauf zurückzuführen, dass in den vergangeneo Jahren bei den Fernsehsendern und großen Tageszeitungen die Stellen von "Redakteuren für Religionsfragen" eingespart worden sind, sondern vor allem darauf, dass die Mehrzahl der Reporter und Redakteure - noch stärker als in Deutschland selbst keiner Kirche und meist keinem Glauben anhängen. Während die lokalen Zeitungen und Fernsehsender örtliche Fälle aufgriffen und den Missbrauchten viel Raum für persönliche Anschuldigungen gegen Priester, Bischöfe und Kirche boten, waren die überregionalen Medien in den meisten Fällen bemüht, entsprechend dem amerikanischen Grundsatz der objektiven Tatsachendarstellung durch unkommentierte 0 -Töne bei Verantwortlichen, Sprechern und Vertretern der Kirche Stellungnahmen, Erklärungen, Hintergründe und Meinungen einzuholen. Anders als bei uns erklärten sich auch die meisten Bischöfe zu Auskünften bereit, sohttps://doi.org/10.5771/0010-3497-2002-3-344, am 14.07.2024, 00:36:21 Open Access – - https://www.nomos-elibrary.de/agb 346 DIE US-MEDIEN UND DIE PRIESTERSKANDALE dass etwa Kardinal Avery Dulles, einer der führenden Jesuitentheologen, in der New York Times zu Wort kam, Kardinal Theodore E. McKerrick von Washington USA Today ein Titelseiten-Interview gab, Bischof Gregory, der Vorsitzende der US-Bischofskonferenz, mehrfach im Fernsehen live interviewt wurde, ebenso wie Medienbischof Joseph Galante. Allerdings versuchten die Medien auch kritische Stimmen einzufangen, sowohl von Zusammenschlüssen der Missbrauchsopfer wie SNAP (Survivors Network of those Abused by Priests) als auch bei Theologen, die dafür bekannt sind, dass sie "ungeschminkt" sprechen und oft kontroverse Positionen zur offiziell~m Linie der Kirche vertreten. So wurde z.B. der als progressiv geltende Priester Donald B. Cozzens in seinem freiwilligen Sabbatjahr von einer "endless flood of calls from the media" als Interviewpartner überrollt, wie U. S. Catholic schreibt (und ihn selbst interviewte). Cozzens war Direktor eines Priesterseminars in Cleveland und erregte erst kürzlich mit seinem neuen Buch über "The Changing Face of the Priesthood" öffentliches Aufsehen, in dem er u.a. auf die starke Prägung von Priestern durch ihre enge Mutterbeziehung und ein elitäres Rollenverständnis hinwies. Er gilt als progressiver Denker. Stark konservativ geprägt ist dagegen Richard John Neubaus, Direktor des Katholischen Instituts für Kirche und Kultur in New York und Herausgeber der Monatszeitschrift First Things. Der Konvertit berichtet in seiner Kolumne "The Public Square", dass er "nicht weniger als 30 Stunden in jeder Woche" von Zeitungsreportern oder Rundfunkleuten über die Priesterskandale interviewt worden und des Themas nun überdrüssig sei. Als kompetenter Theologe war und ist gleichfalls der Herausgeber der Jesuitenzeitschrift America, P. Thomas J . Reese gefragt, der seine offene theologische Position sowohl in seiner Monatszeitschrift als etwa auch in der Catholic Theological Society of America (CTSA) vertritt. In einem Interview mit mir unterschied P. Reese im übrigen bei der Medienbehandlung der Skandale zwischen den Printmedien und dem Fernsehen. Die Printmedien seien in der Nachrichtengebung weitgehend um Objektivität bemüht gewesen, während die Uphead- und EditorialWriters, also die Verantwortlichen für die Aufmachung und Kommentierung, oft ins Sensationelle verfallen seien. In den Redaktionen seien viele feministische Journalistinnen tätig, die die Gelegenheit wahrgenommen hätten, den Bischöfen "eins auszuwischen" wegen ihrer strengen Sexualmoral. In den Printmedien handele es sich weniger um anti-katholische Tendenzen als vielmehr um anti-hierarchische Haltungen. Das Fernsehen, so Reese, sei infolge seiner publikumsorientierten Bildwahl oberflächlicher gewesen. Der Jesuit beurteilt die Mediendarstellungen grundsätzlich unter dem Gesichtspunkt, dass die Medien in Amerika ausschließlich profitorientiert seien und ihre Skandalberichterstattung das https://doi.org/10.5771/0010-3497-2002-3-344, am 14.07.2024, 00:36:21 Open Access – - https://www.nomos-elibrary.de/agb 347 DERTEL Ziel habe, durch Auflagensteigerungen und hohe Zuschauerquoten Geld zu verdienen, nicht aber in erster Linie die Kirche zu vernichten. Dem Vorwurf, die Medien seien an den Skandalen Schuld, widersprach schon sehr früh der frühere Sekretär für Public Affairs bei der US-Bischofskonferenz, Russell Shaw. In einem Beitrag für die Wochenzeitung Our Sunday Visitor über "What we've learned from the present debacle" nennt er als erste Erkenntnis: "Geheimhaltung hilft nicht", gefolgt von der zweiten Erkenntnis: "Gebt nicht den Medien die Schuld". Einige befriedigten zwar die Sensationslust und hätten es darauf abgesehen, die Auffassung über den Zölibat und die moralische Autorität der Kirche zu untergraben (um möglicherweise "den Skalp eines Kardinals und den Pulitzer-Preis zu erlangen"); doch es sei Tatsache, dass die Medien das Problem nicht geschaffen hätten. Indem sie die schmerzliche Realität aufdeckten und darüber berichteten, hätten sie "das getan, wozu sie da sind". Wenn die Kirche es versäumt habe, ihre Haus selbst zu reinigen, könne man sich jetzt nicht beschweren, wenn die Medien es täten. Richard John Neuhaus vertritt ebenfalls die Auffassung, dass "natürlich ein Teil der Berichterstattung sensationell aufgeputscht war und es amüsant ist zu beobachten, wie die New York Times jeden Tag auf der Titelseite praktisch dieselbe Geschichte (über die Skandale) veröffentlichte, als ob zu befürchten sei, dass die Leute die Vorgänge vergessen würden". Doch Neuhaus stellt ebenso eindeutig fest: "Die Führer der Kirche, hauptsächlich die Bischöfe, sind verantwortlich für die Krise und die Sensationsberichte." Der frühere Direktor des New Yorker katholischen Medienunternehmens The Christophers, P. John Catoir, verneint noch entschiedener die Frage, ob die Presse antikirchlich sei. "Einige behaupten", so schreibt er in einem syndikatisierten Kommentar für die katholische Presse, "dass die (säkulare) Presse es darauf abgesehen hat, die Kirche zu zerstören. Ich sehe die Presse hingegen als einen notwendigen Dienst im Interesse der Wahrheit an". Es müsse zugegeben werden, dass es Sensationsberichterstattung gegeben habe, um die Verkaufszahlen der Zeitungen und die Einschaltquoten der Sender in die Höhe zu treiben; aber "das Entscheidende ist die Wahrheit", die die Bischöfe durch "den Geist der Geheimhaltung" vertuscht hätten. Catoir zitiert in diesem Zusammenhang einen der bekanntesten amerikanischen Kirchenhistoriker, Msgr. J ohn Tracy Ellis, der an der Catholic University of America in Washington lehrte und in einem Essay schrieb: "Wenn ich gefragt würde, was die größte Schwäche in der römisch-katholischen Kirche sei, würde ich ohne Zögern antworten: der Fehler vieler ihrer Repräsentanten, offen und ehrlich zu sein." Dasselbe Versäumnis bekannte kurz vor der Vollversammlung der UShttps://doi.org/10.5771/0010-3497-2002-3-344, am 14.07.2024, 00:36:21 Open Access – - https://www.nomos-elibrary.de/agb DIE US-MEDIEN UND DIE PRIESTERSKANDALE Bischofskonferenz Mitte Juni in Dallas, wo es um Maßnahmen gegen die pädophilen Verfehlungen von Priestern und Bischöfen ging, schließlich auch Medienbischof Joseph Galante in einem Informationsgespräch vor 300 katholischen Journalisten in Minneapolis: "Es war die Geheimniskrämerei, die uns zu Fall gebracht hat!" Und er forderte: "Wir müssen eine offene und transparente Kirche werden!" Doch gerade dies erwies sich in der Behandlung der Skandale für die Kirchenpresse als problematisch. Der Präsident der American Society of Newspaper Editors, Tim McGire, selbst ein aktiver Katholik, versicherte den katholischen Redakteuren in Minneapolis zwar, dass sie durchaus kirchentreue Katholiken und gleichzeitig "tough journalists" seien können, Journalisten also, die hart an der Wahrheit bleiben. McGire ging allerdings nicht auf das Problem ein, vor das sich katholische Journalisten gestellt sehen, wenn sie in ihren von der Kirche oder einer kirchlichen Institution herausgegebenen Zeitungen an die kirchenoffizielle Position gebunden sind. Welche Folgen eine Missachtung dieser Bindung haben kann, hatte im Fall der Priesterskandale der Chefredakteur der Bostoner Bistumszeitung The Pilot, Msgr. Peter V. Conley, erfahren müssen. Als er in einem Kommentar geschrieben hatte, dass im Zusammenhang mit den sexuellen Verfehlungen so vieler Priester auch die Frage des Zölibats neu durchdacht werden müsste, erklärte das Erzbistum Boston in der folgenden Ausgabe, dass es sich bei dem Kommentar um eine "Privatmeinung" gehandelt habe, denn die Kirche halte am Zölibat weiterhin fest, die pädophilen Vergehen hätten damit nichts zu tun. Der Chefredakteur trat daraufhin zurück. McGire mag indirekt die Grenzsituation kirchlicher Journalisten angesprochen haben, wenn er auf die im amerikanischen Journalismus übliche strikte Trennung von News und Editorial hinwies. Im Nachrichtenbereich haben die katholischen Zeitungen in Amerika immer schon offen über alle Vorgänge und Ereignisse berichten können, auch über Kontroversen und Dissonanzen zwischen Konservativen und Progressiven, Laien und Bischöfen, Bischöfen und Rom. Allerdings beziehen die Kirchenzeitungen diese Nachrichten hauptsächlich durch den Catholic News Service (CNS) in Washington DC, der zwar im Haus der US-Bischofskonferenz sitzt, sich aber unabhängig finanziert und dem Agenturprinzip einer uneingeschränkten faktischen Berichterstattung folgt. Insofern sind die Bistumszeitungen einer eigenen Berichterstattung über die öffentlichen Vorgänge um die Priesterskandale weitgehend enthoben. Wenn es sich um Fälle im eigenen Bistum handelte, druckten sie darüber vorwiegend die offiziellen Erklärungen des Bischofs oder Ordinariates ab. Offene Diskussionen über die mit den pädophilen Priesterskandalen zutage getretenen Themen wie die Zölibatsfrage, die Frage der Priesterweihe von Frauen, aber auch die hierarchische Struktur und den Klerikahttps://doi.org/10.5771/0010-3497-2002-3-344, am 14.07.2024, 00:36:21 Open Access – - https://www.nomos-elibrary.de/agb 349 DERTEL lismus in der amerikanischen Kirche, die autarken personellen, finanziellen und Verwaltungsentscheidungen der kirchlichen Behörden - Diskussionen über diese Themen können höchstens in unabhängigen katholischen Zeitschriften aufgegriffen werden. Im Zusammenhang mit dem Verschweigen der Bischöfe ist z.B. in Erinnerung gerufen worden, dass es die während des Konzils von Laien gegründete unabhängige katholische Wochenzeitung National Catholic Reporter (NCR) war, die bereits vor 15 Jahren kirchliche Interventionen gefordert hatte, als eine erste Enthüllungswelle über Fälle pädophiler Priestervergehen die Öffentlichkeit schockierte. Nachdem sich damals herausgestellt hatte, dass der Erzbischofvon Chicago, Kardinal Bernardin, fälschlicherweise eines Vergehens beschuldigt worden war und die öffentliche Erregung verebbte, waren die Bischöfe weiterhin ihrer Politik des Verschweigens gefolgt. Und in der Bistumspresse war das Thema tabu geblieben. Außer im NCR werden kontroverse Themen vor allen in Magazinen wie Commonwealund America aufgegriffen. "In der Kirche muss alles diskutiert werden können", sagt P. Reese, Chefredakteur von America. Unzufriedenheit herrscht jedoch bei einigen katholischen Medienbeobachtern darüber, dass die Informationsverantwortlichen der Bistümer und insbesondere die PublicRelations-Arbeit der US- Bischofskonferenz während der ganzen Skandalzeit kaum aus einer defensiven Pressepolitik herausgefunden und es bis zuletzt höchstens bei einem pauschalen Schuldeingeständnis sowie Entschuldigungen für ihr Versagen belassen hätten. Wenn der CNS-Korrespondent in Rom, John Thavis, über die Pressekonferenz im Vatikan nach dem Gipfeltreffen zwischen dem Papst und den amerikanischen Kardinälen feststellte, sie sei nach vatikanischem Standard sogar "offen" gewesen, viele der angereisten amerikanischen Reporter von säkularen Medien aber oft einfach die Kirchensprache nicht verstanden hätten, dürfte dies umso mehr für die allgemeine amerikanische nichtkatholische Öffentlichkeit gelten. Hätte in Amerika, so fragen Medienbeobachter, nicht die Chance bestanden für eine offensive Öffentlichkeitsarbeit, die in zeitgemäßer verständlicher Form über das Wesen der Kirche, ihre Sendung, ihre Botschaft von Vergebung und Erlösung informiert, ohne von den Skandalen abzulenken? Sie können deshalb wohl schwerlich der Meinung von Richard J ohn Neuhaus zustimmen, dass "eines Tages die gegenwärtigen Skandale Nachrichten von gestern" sein werden und "die Reporter anderen Skandalen nachjagen" werden. Denn die Kirche wird nur "erneuert bestehen bleiben", wie Neuhaus hofft, wenn sie auch in der Öffentlichkeit besteht. https://doi.org/10.5771/0010-3497-2002-3-344, am 14.07.2024, 00:36:21 - https://www.nomos-elibrary.de/agb 350 Open Access – ERICHT Reinhold Jacobi SIGNIS - Die neue katholische Weltorganisation für Kommunikation Am 26. November 2001 wurde in Rom die Organisation SIGNIS gegründet. Diese wird in Artikel! der Statuten als "The World Catholic Association for Communication" definiert und als professionelle, autonome "Non-Profit"-Organisation deklariert. Der Name ist ein gewissermaßen im Zufallsverfahren zustande gekommenes Mixturn aus signum (lat. Zeichen) und ignis (lat. Feuer). Mit Gründung von SIGNIS ging ein langjähriger Prozess der Selbstreflexion und kritischen Bestandsaufnahme der bis dahin nebeneinander arbeitenden internationalen katholischen Medienorganisationen OCIC (Organisation Catholique International du Cinema de l'Audio-visuel) und UNDA (lat. Welle; Internationale Katholische Organisation für Radio und Fernsehen) zu Ende. Die bis dahin existierenden insgesamt drei internationalen katholischen Medienorganisationen, zu denen als dritte UCIP gehört, die Internationale Katholische Union der Presse, hätten sich insgesamt zu einer einzigen Organisation zusammenfinden können; dezidierte Anhänger einer Auffassung, dass die sehr unterschiedlichen Kräfte katholischer Medienkompetenz auf internationaler Ebene nur durch eine rigide Bündelung zu einer effizienten "Truppe" zusammenzufassen seien, konnten (noch) nicht zum Zuge kommen, weil sich alle drei Unternehmen dadurch unter Umständen von Anfang an übernommen hätten. Zudem folgt UCIP einer anderen Organisationsstruktur als OCIC und UNDA insofern, als sie stärker personenbezogen strukturiert ist und in ihrer Arbeit auch lange deutlicher einem einzigen Medienfeld, dem Prilltbereich, zugeordnet war. Im Falle von OCIC und UNDA war nach einer langen Phase der neuerlichen Konsolidierung der Organisation nach dem zweiten Weltkrieg in den vergangeneu etwa 20 Jahren die Überschneidung der Arbeitsfelder zu deutlich geworden, als dass man ein Nebeneinander noch allzu lange hätte verantworten können. Für OCIC waren nach der mit Gründung (1928) deutlichen Konzentration auf den Kinofilm als das Aufmerksamkeit heischende Massenmedium schlechthin in den letzten Jahrzehnten andere audiovisuelle Medien wie insbesondere die Video-Kassette als zusätzliches Trägermedium für den· Film jenseits des Kinos stark ins Blickfeld geraten. Was wiederum bedeutete, dass sich damit eine Überschneidung mit Inhalten ergab, die mit einem gewissen Automatismus als Fortsetzung des Rundfunks (Fernsehen) mit anderen Mitteln verstanden wurde. Neben diesen medialen Überschneidungen gab es auch personelle, https://doi.org/10.5771/0010-3497-2002-3-344, am 14.07.2024, 00:36:21 Open Access – - https://www.nomos-elibrary.de/agb 1351 JA CO BI nämlich insofern als gerade die Vertreter aus Ländern der Dritten Welt in vielen Fällen die Vertretung ihres Landes respektive ihrer Bischofskonferenz in Sachen Film für OCIC wie auch in Sachen Rundfunk für UNDA in Personalunion wahrnahmen, was den Aufwand getrennter Organisationen nicht mehr rechtfertigte. Diese Konstellation hatte zur Folge, dass die vom 01. bis 09. August 1998 in Montreal versammelten Delegierten der Mitgliederversammlungen von OCIC und UNDA im Kontext ihres gemeinsamen Weltkongresses beschlossen, beide Organisationen zu einerneuen gemeinsamen Organisation zusammenzulegen. Nach gut dreijährigen getrennten und gemeinsamen Beratungen erfolgten die Auflösung von OCIC und UNDA sowie die Gründung einerneuen Organisation SIGNIS im Rahmen des Weltkongresses beider Partner in der Zeit vom 19. bis 27. November 2001. Dass man dazu Rom wählte, machte insofern Sinn, als der gesamte Prozess am Ort zentraler weltkirchlicher Bedeutung den Rang der alten wie der neuen Organisation für die medialen Anliegen der Kirche auch nachdrücklich signalisieren konnte. Die deutliche Präsenz des Präsidenten des Päpstlichen Rates für die Sozialen Kommunikationsmittel, Erzbischof John P. Foley, bei vielen Sitzungen und sonstigen Begegnungen am Rande des römischen Geschehens verlieh der Wahrnehmung auch seitens des Vatikan selbst Ausdruck. Hätte man die bei OCIC und UNDA üblichen, jeweils seit 73 Jahren gültigen Strukturen lediglich übernommen, so hätte man sicherlich kaum eine Verbesserung der Arbeitsmethoden gefunden. Schaffung von Synergien, Bündelung der Kräfte in organisatorischer und auch finanzieller Hinsicht konnten letztlich nur zustande kommen durch maßgebliche Veränderungen der Struktur der Statuten. Die wichtigste Neuerung ist dabei die Umorientierung der früheren Rolle der Mitgliederversammlungen. Auch wenn die Mitglieder von OCIC und UNDA nur alle drei bis vier Jahre aus den verschiedenen Ländern in aller Welt zusammenkamen, so konnten die Versammlungen von 120 bis 130 Mitgliedsländern auch dann kaum etwas bewegen. Dynamik war inzwischen ein Fremdwort, Unbeweglichkeit ein üblicher Standard geworden. Der Generalversammlung von SIGNIS als höchstem legislativem Organ (von insgesamt lediglich drei Organen) sind grundlegende Rechte wie Wahl des Namens, der Zielsetzung und wichtige Strukturänderungen der Satzung vorbehalten. Diese Versammlung wird nur aus Gründen einer maßgeblichen Satzungsänderung zusammengerufen oder wenn ein Fünftel der Mitglieder dieses wünscht. Immerhin betrifft dies den nunmehrigen Mitgliederstand von 140 nationalen und 30 internationalen (Organisations-)Mitgliedern. Wichtiger als die General- bzw. Mitgliederversammlung ist dagegen die Delegiertenversammlung: Sie ist die eigentliche operative und polihttps://doi.org/10.5771/0010-3497-2002-3-344, am 14.07.2024, 00:36:21 - https://www.nomos-elibrary.de/agb 352 Open Access – SIGNIS- KATHOLISCHE WELTORGANISATION FÜR KOMMUNIKATION tisch handelnde Einrichtung. Dies ist der wichtigste strukturelle Zuerwerb der gesamten Reorganisation. Denn die Delegiertenversammlung ist zahlenmäßig überschaubar und daher handlungsfähig. Sie setzt sich aus jeweils sechs Delegierten der (für SIGNIS) sechs Kontinente Afrika, Lateinamerika und Karibik, Nordamerika, Asien, Europa sowie der Pazifischen Region zusammen. Hinzu kommt als gewissermaßen siebter Kontinent die Gruppe der Internationalen Organisationen, die ebenfalls sechs Delegierte entsendet. Diese sieben mal sechs Delegierten sowie der Präsident und seine beiden Stellvertreter, eine Gruppe also von insgesamt 45 Personen, haben die eigentliche legislative Kompetenz für die gesamte Organisation von SIGNIS. Der Delegiertenversammlung stehen zu die Aufnahme neuer (nationaler und internationaler) Mitglieder, die Bildung und Bestätigung regionaler Gruppen, die Bestätigung des J ahreshaushalts, die Wahl und die Entlastung von Präsident und Vizepräsidenten, die Bestätigung der Mitglieder des Board, des Exekutivkomitees. Das Gremium trifft sich - und auch dies ist eine Konsequenz aus der weltweiten Dimension der Organisation und der daraus resultierenden Reisewege - nur alle zwei Jahre zu routinemäßigen Versammlungen. Das Exekutivorgan zur Unterstützung des Präsidenten und seiner Vertreter als das dritte Organ von SIGNIS ist das Board. Bezeichnenderweise ist dem Board rund ein Sechstel des Gesetzestextes der Statuten und damit der maßgebliche Teil gewidmet. Das Board managt die Organisation. Dabei stützt es sich auf je zwei Mitglieder der sechs regionalen und der internationalen Gruppierungen, so dass es insgesamt aus 15 Mitgliedern besteht. Dieses Drittel der insgesamt 45 Mitglieder der Delegiertenversammlung soll auf Weltebene so arbeitsfähig sein wie die je sechs gewählten Delegierten für die Region, sprich den jeweiligen Kontinent. Schon die ersten Erfahrungen mit Board und kontinentaler /regionaler Delegiertengruppe stimmen zuversichtlich. Denn die jeweiligen numerischen Stärken der Gruppierungen sorgen für ein vergleichsweise hohes Maß an Selbstverpflichtung, Solidarität und Kollegialität. Diese markieren die Stoßrichtung der Hauptarbeit: Sie soll auf kontinentaler Ebene liegen, weil der Aufwand von Zusammenkünften und von anderen Vereinbarungen überschaubar, kalkulierbar und für die einzelnen Mitglieder umsetzbar wird. Als Spitze der neuen Organisation SIGNIS wurden in Rom Peter Malone (Australien). bisheriger Präsident von OCIC, Gaye Ortiz (Großbritannien), bisherige Präsidentin von OCIC Europa, und Augustine Loorthusamy (Malaysia). bisher OCIC Asien, gewählt. Auch der Generalsekretär Robert Molhaut (Belgien), kommt von OCIC, wo er für mehr als zwei Jahrzehnte die entsprechende Funktion wahrnahm. Der für das neue Amt ursprünglich vorgesehene bisherige Generalsekretär von UNhttps://doi.org/10.5771/0010-3497-2002-3-344, am 14.07.2024, 00:36:21 Open Access – - https://www.nomos-elibrary.de/agb 1353 JA CO BI DA, PierreBelanger SJ (Kanada) , konnte von seinem Orden nicht länger für diese Medienaufgabe freigestellt werden. Insgesamt ist auch an der Zusammensetzung des Board abzulesen, dass das katholische Medienschaffen immer mehr als eine Aufgabe angesehen wird, die von Laien nicht nur wahrgenommen werden kann, sondern auch wahrgenommen werden soll: Von den 15 Mitgliedern des Board gehört gerade noch ein Drittel dem Klerikerstand an. Aus europäischer Perspektive werden an die neue Organisation SIGNIS insofern hohe Erwartungen geknüpft, als die europäischen Sektionen derbeidenbisherigen Organisationen - Euroeie und UNDA Europa aufgrund der extrem komplizierten nationalen und insbesondere sprachlichen Strukturen auf dem Kontinent kaum arbeitsfähig waren: Allzu heterogen waren die Mitgliedschaften, allzu unterentwickelt das Bewusstsein der nationalen Bischofskonferenzen, den Überschritt über die nationalen Grenzen riskieren zu müssen, um die Evangelisierung auf dem Kontinent insgesamt und damit auch im eigenen Lande medial konditionieren zu können. Letztlich konnten sich gewählte schmale Vorstände der europäischen Organisationen von UNDA und OCIC aus Mangel an personaler Unterstützung, an finanziellen Ressourcen und an operationaler Handlungs- und Verantwortungsstruktur nicht wirklich politisch etablieren. Womit die Organisationen selbst lediglich Signale für ,good will' blieben. Mit der Delegiertenstruktur von sechs Verantwortlichen für den Kontinent Europa wird zumindest der Versuch zu verbinden sein, endlich eine breite, aber wiederum nicht allzu diffus angelegte personelle Verantwortlichkeit zu strukturieren, um dann europäische Fragestellungen (z.B. die Zukunft des öffentlich-rechtlichen Rundfunks; internationaler Fernseh-Kirchenkanal?) und europäische Dauerpräsenz (z.B. Filmfestivals) angemessen zu berücksichtigen. https://doi.org/10.5771/0010-3497-2002-3-344, am 14.07.2024, 00:36:21 Open Access – - https://www.nomos-elibrary.de/agb 354 IERICHT Markus Breuer Wie aus Bedenken Begeisterung wurde Ein Erfahrungsbericht über das Pilot-Projekt .. Der DOM macht Schule" Eigentlich begann das Projekt "Der DOM macht Schule" im Mai 2000 mit der Antrittsrede des neuen Chefredakteurs Gerd Felder. Den Gästen des Erzbistums Faderborn und den Kollegen der deutschen Kirchenpresse erklärte der Nachfolger von Hermann Multhaupt damals, dass er die feste Absicht habe, mit der Kirchenzeitung an die Schulen zu gehen - ganz nach dem Vorbild der meisten deutschen Tageszeitungen, die mit ähnlichen Projekten große Erfolge erzielen. Für den "DOM" wurde die Zusammenarbeit mit den Schulen schließlich Großprojekt Nummer zwei, nachdem sich die Kirchenzeitung für das Erzbistum Faderborn zum Osterfest des Jahres 2001 mit einem völlig neuen Gesicht präsentiert hatte. Wie "Zeitung in der Schule" funktioniert, hatte Felder als Redakteur der Westfälischen Nachrichten in Münster miterlebt, die das Projekt in Zusammenarbeit mit dem Institut zur Objektivierung von Lern- und Prüfungsverfahren (lZOP) mit Sitz in Aachen schon vor Jahren gestartet hatten. Felder verband mit dem Schulprojekt die Hoffnung, sich ein Stück von dem Image zu lösen, dass man eine Zeitung für alte Menschen mache. Die Schülerinnen und Schüler sollten erkennen, dass auch hinter einer Kirchenzeitung professioneller Journalismus stecke, der sich hinter dem von anderen Blättern nicht zu verstecken braucht. Über die Schüler wollte Felder darüber hinaus vor allem junge Eltern erreichen, um langfristig eine neue Leserschaft zu gewinnen, für die es im "DOM" seit Erscheinen der neugestalteten Zeitung ein breites Angebot von Themen gibt, vor allem im Ressort "Lebenshilfe". Aufgrund dieser Überlegungen spielte der Chefredakteur eine Zeit lang mit dem Gedanken, bei dem Projekt mit Grundschulen zusammen zu arbeiten - auch auf Anraten von Peter Brand, Geschäftsführer des Aachener IZOP-Institutes, auf dessen langjährige Erfahrung sich "Der DOM" bei der Planung und Durchführung des Schulprojektes stützen wollte. Das Projekt wurde schließlich aber doch für die Sekundarstufen I und II ausgeschrieben: Der Schritt von der Kirchenzeitung zur Grundschule schien Felder beim Pilot-Projekt zu groß zu sein - trotz der wöchentlichen Kinderseite, die Peter Brand als hervorragenden Anknüpfungspunkt für die Klassen eins bis vier ausgemacht hatte. Mit folgenden Rubriken wollte man die Schülerinnen und Schüler für die Kirchenzeitung begeistern: Zum einen mit dem "Islam-Lexikon", behttps://doi.org/10.5771/0010-3497-2002-3-344, am 14.07.2024, 00:36:21 Open Access – - https://www.nomos-elibrary.de/agb 1355 BREUER stehend aus 20 Folgen, das sich vor allem zur Bearbeitung und Diskussion im Religionsunterricht anbot: Mit dieser Reihe wollte Felder nach den Anschlägen des 11. Septembers zum besseren Verständnis zwischen Christen und Muslimen beitragen. Zum anderen mit dem Internet-Tipp und einem Fragebogen, in dem sich prominente Personen aus allen Bereichen der Gesellschaft zu ihrem Glauben äußern. Mehr als 650 Schülerinnen und Schüler beschäftigten sich von Anfang März bis Mitte Juli 2002 im Unterricht mit der Zeitung und schrieben eigene Artikel. 16 Schulen aus allen Regionen des Erzbistums Paderborn, die sich überwiegend in katholischer Trägerschaft befinden, hatten sich für das PilotProjekt gemeldet. Die Schüler haben größtenteils mit großer Begeisterung für den DOM recherchiert und geschrieben. Mögliche Themen für die Kirchenzeitung waren den Lehrerinnen und Lehrern beim Einführungsseminar in Olpe genannt worden - trotzdem blieb den Schülerinnen und Schüler bewusst viel Platz für die eigene Kreativität. Verwunderlich war es deshalb, dass die ersten eingesandten Artikel sich fast ausschließlich auf rein kirchliehe Themen bezogen. Auf den ersten Schulseiten wurden eine Umfrage zum Thema Fastenzeit und das Portrait eines Paderborner Küsters veröffentlicht. Es war in den ersten Wochen übrigens von großem Vorteil, dass das Bürener Mauritius-Gymnasium vor dem "DOM" schon mit verschiedenen anderen Zeitungen zusammengearbeitet hatte (darunter mit der FAZ) . Die teilnehmende Klasse 10c konnte schnell Beiträge liefern und füllte die Schulseiten der ersten Projekt-Ausgaben fast allein - und auf formal hohem Niveau. Die anderen Schulen benötigten mehr Zeit, um eigene Ideen zu entwickeln und erste Artikel einzusenden. Dabei handelte es sich fast ausschließlich um Reportagen. Gerade am Anfang wurden aber auch viele Beiträge geschrieben, die keiner journalistischen Darstellungsform zuzuordnen waren, vielmehr erinnerten sie an Schulaufsätze. Sehr oft waren die Texte chronologisch aufgebaut und in der Wir-Form geschrieben. Die Verantwortlichen beschäftigte die Frage, ob die Textarbeit beim Einführungsseminar in Olpe zu kurz gekommen sei. Das IZOP-Institut in Aachen, das während des Projektes vor allem für die didaktische Begleitung zuständig war, fasste daraufhin die wichtigsten J;{egeln für das Verfassen von Zeitungsartikeln noch einmal zusammen. Die Regeln und eine gemeinsame Analyse von Zeitungsartikeln im "DOM" trugen dazu bei, dass sich das Niveau der im "DOM" abgedruckten Schülerartikel schließlich immer weniger von Beiträgen in überregionalen Tageszeitungen unterschied. Auffallend war, wie völlig unterschiedlich die Projekt-Lehrerinnen und Lehrer mit den Schülertexten umgingen: Während einige von ihnen die https://doi.org/10.5771/0010-3497-2002-3-344, am 14.07.2024, 00:36:21 Open Access – - https://www.nomos-elibrary.de/agb WIE AUS BEDENKEN BEGEISTERUNG WURDE Beiträge mehrmals vor dem Einsenden korrigiert hatten, kamen andere Schülertexte völlig unbearbeitet in der Redaktion an. Die eingesandten Texte wurden von Projektredakteur Markus Breuer nur so weit redigiert, wie es nötig war. Schließlich sollten ja die Artikel der Schüler in der Zeitung erscheinen. "Durch die Zusammenarbeit mit der Zeitung ließen sich auch Schüler begeistern, die sonst nur selten oder gar nicht am Unterricht beteiligt sind", erklärte Oberstudienrätin Dr. Christa-Maria Schieifer-König beim Abschlussseminar in Paderborn. Erfreulich war auch, dass sich die Schulseiten im Laufe des Projektes durch eine immer größere Vielfalt an Themen auszeichneten. Zwar gab es weiterhin Beiträge zu den klassischen Themen einer Kirchenzeitung zum Beispiel über das Verhältnis von Jugendlichen zur Kirche -, aber die Texte waren so nah an der Lebenswirklichkeit der jungen Menschen, dass jeder Redakteur einen ähnlichen Artikel nur mit größter Mühe und erheblichem Recherche-Aufwand hätte schreiben können. Dass die Schülerbeiträge nicht selten von den üblichen Vorurteilen gegenüber der Institution Kirche geprägt waren, mag man den Jugendlichen verzeihen. Darüber hinaus erreichten die DOM-Redaktion viele Beiträge, die für das Ressort "Lebenshilfe" ideal waren. In diesem viel gelesenen Teil der Zeitung wurde der Platz für Schülertexte aufgestockt (von zwei auf vier Farbseiten), weil sich auf dem Schreibtisch des Projektredakteurs die eingesandten Artikel gegen Mitte des Projektes immer mehr stapelten. So berichteten die Jugendlichen unter anderem über Erfahrungen, die sie während eines Sozialpraktikums in Altenheimen und Krankenhäusern gesammelt hatten. Große Beachtung fand vor allem der Beitrag einer Schülerin, die ein Kind mit Lese- und Schreibschwäche portraitiert hatte. Auch der Artikel über das Kinderhospiz "Balthasar" in Olpe begeisterte die Leser- das Thema war der Süddeutschen Zeitung im Juli 2002 immerhin die SEITE DREI wert. "Der DOM" ging zudem zu einer Zeit in die Schule, in der die Themen Bildung und Erziehung durch die Pisa-Studie und den Amoklauf von Erfurt in eine große öffentliche Diskussion geraten waren. Neben zwei eigenen Reportagen unter den Überschriften "Das Gebet muss unsere Antwort sein" (Ausgabe Nr. 19) und "Schule was nun?" (Ausgabe Nr. 23), für dessen Recherche die bestehenden Kontakte zu den Schulen eine große Hilfe waren, veröffentlichte "DER DOM" den Kommentar eines Lehrers, der an einem Gymnasium in Bielefeld mit der Schulseelsorge betraut war. Die "DOM" -Redaktion hatte ihn über einen Musikkurs kennen gelernt, der ebenfalls an dem Projekt beteiligt war. Die Zusammenarbeit von Schule und Kirchenzeitung zog also immer weitere Kreise. Völlig unterschiedlich liefen die Besuche des Projektredakteurs in den beteiligten Klassen ab. In der Regel waren die Schülerinnen und Schüler https://doi.org/10.5771/0010-3497-2002-3-344, am 14.07.2024, 00:36:21 Open Access – - https://www.nomos-elibrary.de/agb 357 BREUER sehr interessiert an der Arbeit eines Journalisten im Allgemeinen und an der Arbeit für die Kirchenzeitung im Speziellen. "Der DOM" kam bei der Kritik allerdings nicht immer gut weg. Vor allem wurde bemängelt, dass die Kirchenzeitung zu wenig Themen für junge Menschen böte. Oft wus· sten die Schülerinnen und Schüler jedoch selbst nicht, was sie gerne im "DOM" lesen würden. Dass die Kirchenzeitung aber nicht über die "Feten vom Wochenende" berichten konnte (das haben einige Schüler tatsächlich vorgeschlagen) war am Ende einer Doppelstunde meist geklärt. Für zwei Klassen, eine aus Faderborn und eine aus Bielefeld, hatte sich der DOM etwas ganz Besonders einfallen lassen: Die Kirchenzeitung gab den Schülerinnen und Schülern nämlich die Möglichkeit, ihre Innenstadt mit dem Rollstuhl zu erkunden. "DER DOM" stützte sich bei der Vorbereitung dieser Aktion auf die Erfahrung des Paderborner Jugendreiseunternehmens "Youth-Adventure-Tours", das deutschlandweit Ferienfahrten für Menschen mit Behinderungen anbietet. Ein Behinderten· Sportverein, der in der Basketball-Bundesliga spielt, hatte die robusten Rollstühle zur Verfügung gestellt. Die Aktion wurde aus zwei Gründen ein Erfolg: Die Schüler erklärten später bei der Reflexion, dass sie nach der Aktion gut nachfühlen könnten, welche Probleme ein Rollstuhlfahrer im Alltag bewältigen muss. Sie listeten konkret "aus eigener Erfahrung" Behinderungen von Rollstuhlfahrern aufund reichten diese an die Stadtverwaltung weiter. Alle Schüler trugen während ihrer Fahrt durch die Innenstadt einT-Shirtmit dem Logo des Schulprojektes- für den "DOM" hatte die Aktion so zusätzlich einen Werbeeffekt Außerdem berichteten die Tageszeitungen ausführlich darüber, welche Erfahrungen die Schüler mit dem Rollstuhl in den Innenstädten gemacht hatten. Fazit: "Der DOM macht Schule" war ein vielfältiges Projekt, dass mit Sicherheit dazu beigetragen hat, das Image der Kirchenzeitung zu verbessern. Der Großteil der Schüler erklärte in verschiedenen Gesprächen, dass die Kirchenzeitung bei weitem nicht so langweilig sei, wie sie immer gedacht hatten - natürlich würden sie den "DOM" nicht alle abonnieren, aber das war auch nicht das Ziel. Bei Eltern, die den "DOM" meist nur vom Hörensagen kannten, stieß das Projekt auf sehr positive Resonanz. Manche Schülerartikel erreichten über das Leserbrief-Echo hinaus weitere Kreise im lokalen Schulumfeld. Die Lehrerinnen und Lehrer, die am Abschlussseminar in Faderborn teilnahmen, waren mit dem Verlauf des Projektes rundum zufrieden. Die Zusammenarbeit zwischen Kirchenzeitung und Schule trägt jedoch nur dauerhaft Früchte, wenn sie langfristig angelegt ist. Deswegen möchte "DER DOM" das Projekt nach Möglichkeit auch im kommenden Jahr wieder anbieten. https://doi.org/10.5771/0010-3497-2002-3-344, am 14.07.2024, 00:36:21 Open Access – - https://www.nomos-elibrary.de/agb 358 ERSONALIA Michael Schmolke in Salzburg emeritiert Nach Erreichen der Altersgrenze von 68 Jahren ist Prof. Dr. Michael Schmolke am 30. September 2002 an der Universität Salzburg emeritiert worden. Schmolke ist seit der Gründung von Communicatio Socialis eng mit dieser 1968 gegründeten Zeitschrift für Publizistik in Kirche und Welt verbunden und hat ihre Entwicklung als Mitglied des damaligen Giiinderkreises aus dem Seminar des Münsteraner Publizistikwissenschaftlers Henk Prakke entscheidend mitgeprägt und ihr fortlaufendes Erscheinen mitgetragen. Als Mitherausgeber hat er sie 1993 mit dem neuen Untertitel Internationale Zeitschrift für Kommunikation in Religion, Kirche und Gesellschaft auf eine breitere Basis gestellt und ihr Ansehen als wissenschaftliche Fachzeitschrift weit über den deutschsprachigen Raum hinaus gestärkt. Eine Würdigung seiner über 30jährigen wissenschaftlichen und publizistischen Tätigkeit kann an dieser Stelle nicht erfolgen; das umfangreiche Tätigkeitsfeld seiner Lehre und Forschung lässt sich an einer Auswahl seiner Beiträge in der Festschrift zu seinem 65. Geburtstag, "Aufklärung und Aberwissen" (Verlag Reinhard Fischer, München 1999) ablesen, die gegliedert sind in Kapitel mit den Schwerpunkten Theoretische Forschungen, Blick in die Geschichte, Kirche und Kommunikation sowie Kommunikationspolitik und politische Kommunikation. An dieser Stelle sollen anlässtich seiner Emeritierung nur ein paar kurze Daten seiner Biographie stehen. Am 13. Februar 1934 in Gleiwitz (Oberschlesien) geboren, studierte Michael Schmolke Publizistik, Geschichte und Pädagogik in Münster, Göttingen und München. Als wissenschaftlicher Assistent am Institut für Publizistik in Münster wurde er 1965 mit einer Dissertation über "Kolping als Publizist" promoviert und habilitierte sich 1970 mit der Arbeit über "Die schlechte Presse - Das Verhältnis der deutschen Katholiken zur Publizistik zwischen Katholik (1821) und Publik (1968)". Die Arbeit erschien 1971 mit leicht modifiziertem Titel als Buch und gilt als ein Standardwerk über die katholische Presse in Deutschland. 1973 erhielt Schmolke den Ruf als Ordinarius für Publizistik und Kommunikationswissenschaft an das Institut für Publizistik und Kommunikationswissenschaft der Universität Salzburg. In die Zeit seines Wirkens in Salzburg fiel die Entwicklung seiner Disziplin vom früheren Verständnis als Zeitungswissenschaft über die Ausweitung zur Begriffsbestimmung Publizistik bis zum gegenwärtigen Verständnis als Kommunikationswissenschaft, die er weithin mitgestaltete. Neben zahlreichen https://doi.org/10.5771/0010-3497-2002-3-344, am 14.07.2024, 00:36:21 Open Access – - https://www.nomos-elibrary.de/agb 1359 PERSONALIA Forschungsaufträgen hat Schmolke in Büchern als Autor und Herausgeber sowie in vielen Artikeln immer wieder kommunikationsgeschichtliche, -theoretische, -praktische und -politische Themen behandelt, darunter 1974: "Publik - Episode oder Lehrstück?"; 1992: "Wegbereiter der Publizistik in Österreich"; 1992: "Kirchenpresse am Ende des Jahrtausends" sowie Mitarbeit an zahlreichen Lexika und Handbüchern vom "Handbuch der Weltpresse" über "Staatslexikon", "Österreich-Lexikon", "Lexikon für Theologie und Kirche" bis zum "Handbuch der Kirchengeschichte". An der Universität Salzburg hat Schmolke Hunderte von Studenten zum Diplom- und Dissertationsahschluss geführt und das Institut für Publizistik zu einem zeitungswissenschaftliehen Zentrum mit internationalem Ruf ausgebaut. In seiner Abschieds-Vorlesungsreihe im Sommersemester 2002 behandelte er unter dem Titel "Die Medienbiographie des Michael S." die Entwicklung der Medienwelt während seiner Lebenszeit von der Tagespresse über den Rundfunk und das Fernsehen bis zu den neuen Medien mit Veranschaulichung durch zahlreiche Originaldokumente. Er wird auch weiterhin Diplomanden und Dissectanten an der Universität Salzburg zur Verfügung stehen. F. 0. https://doi.org/10.5771/0010-3497-2002-3-344, am 14.07.2024, 00:36:21 Open Access – - https://www.nomos-elibrary.de/agb Chronik ASIEN China Unter dem Titel "The Disciple" erscheint seit über zwei Jahren wöchentlich ein kirchlicher Newsletter in den Pfarreien der Kathedrale und St. Peter in Shanghai. In der 100. Jubiläumsausgabe berichtete das Informationsblatt über eine aktive Teilnahme von jungen und alten Katholiken am kirchlichen Leben. Wegen Missbrauchs religiöser Symbole hat sich eine Bar in Xian offiziell beim Ortsbischof und den Schwestern des hl. Herzens im J esuskonvent entschuldigt. Die Bar hatte in Zeitungsanzeigen mit kirchlichen Symbolen und Kellnerinnen im Schwesterngewand geworben. Nach Protesten der Katholiken musste die Lokalzeitung auf Regierungsanweisung dreimal eine Entschuldigung abdrucken. Ende 2001 war eine ähnliche Kampagne verurteilt worden, bei der Muslime verunglimpft worden waren. Indien Eine Förderung kommunaler Einheit durch die Medien wurde auf zwei Tagungen in den Mittelpunkt gerückt: auf einem nationalen Seminar, das SIGNIS-Indien veranstaltete, und einem Treffen katholischer Redakteure in Kerala. In zwei Workshops vermittelte das Jesuiteninstitut zur Förderung von Ethik und Werten am Pastoralzentrum von Nadiad 36 Redakteuren und Kolumnisten eine weiterführende Ausbildung in professioneller Berichterstattung. Japan Die Bewegung "Light of the Heart", die in Japan über Radio, Fernsehen und Internet Evangelisierungssend ungen verbreitet, konnte in der Kathedrale von Tokio ihr Goldenes Jubiläum feiern. Die Bewegung führt auch Korrespondenzkurse in Religion und Englischkurse durch und veranstaltet Pilgerreisen. Der japanische Film "GO" ist von SIGNIS-Japan als bester Film des Jahres ausgezeichnet worden. Der Film behandelt einen Fall der Rassendiskriminierung und leitet zu Reflexionen über menschliches Zusammenleben an. Philippinen Pressefreiheit in der Berichterstattung über Terrorismus forderten über 100 ausländische und inländisehe Journalisten auf einer Konferenz in Manila, die von der UNESCO veranstaltet wurde. Sie beklagten die Einschränkung der Pressefreiheit in vielen Ländern Asiens und Afrikas nach dem 11. September 2001 und die Nichtanerkennung von Journalisten als https://doi.org/10.5771/0010-3497-2002-3-344, am 14.07.2024, 00:36:21 Open Access – - https://www.nomos-elibrary.de/agb 361 CHRONIK Zivilpersonen durch die Militärs und Regierenden. Einen "IT-Injonnationstechnik· Rat" hat die Bischofskonferenz der Philippinen gegründet, der kirchliche Initiativen auf diesem Gebiet fördern soll. Der Rat soll als "Denkfabrik" der Konferenz entsprechende Projekte planen. Am Welttag der Medien setzte die Bischofskonferenz der Philippinen CBCPWorld in Kraft, die den bisherigen Internet Provider CBCPNet ersetzt. Er filtert glaubensschädigendes und pornografisches Material aus dem Internet und bietet Erziehungsinformationen für die Armen und Ausgegrenzten. Ihr fünfjähriges Bestehen im Lokalrundfunkbereich feierten das Catholic Media Network (CMN) von 34 AM- und 14 FM-Sendern sowie eine Gruppe kleiner KabelTV-Sender in 11 Regionen und 35 Provinzen. Auf der Generalversammlung in Manila nannte CMN-Präsident P. Francis Lucas die Strategie dieser Sender "die einzige Alternative zur Globalisierung" . In ihrem Medienapostolat ermutigte der Vorsitzende der Medienkommission, Bischof Cabrera, die 100 Teilnehmer an der ersten Generalversammlung von SIGNISPhilippinen in Manila. Über die Möglichkeiten der Evangelisierung der Jugend durch die Medien informierte der Salesianerpater Ely Cruz die Teilnehmer. Eine dokumentarische Buchreihe startete das Medienbüro der Fernöstlichen Bischofskonferenzen mit Band 1 "Church and Social Communication in Asia Documents, Analysis, Experiences". Er enthält Dokumente über die Jahrestreffen seit 1996, die Auswirkungen der Asien-Synode 1998 auf das Medienapostolat in Asien und die Mediennutzung in Asien. Der 2. Band "Social Communication Formation for Priestly Ministry" dokumentiert das dritte BISCOM 2001, während Band 3 das "Social Communication Handbook Asia" ist. Die Bände erscheinen beim Logos Verlag der Steyler Missionsgesellschaft. Thailand In einem dreijährigen Pilotprogramm beteiligen sich asiatische kirchliche Institutionen an dem Online-Glaubensinformationsprogramm VLCFF, das vom Pastoralinstitut an der amerikanischen Dayton-Universität in Ohio entwickelt wurde. Das beschlossen katholische Erziehungs- und Medienvertreter bei einem Treffen mit Repräsentanten der US- Universität in Bangkok. Das amerikanische Programm wird auf die asiatischen Bedürfnisse abgestellt. Den Ausbau eines interreligiösen Netzwerkes im Kampf gegen den Terrorismus haben die Mitglieder der Vereinigung der katholischen Presse Südostasiens (SEACPA) auf ihrer Generalversammlung in Bangkok beschlossen. Die Jour- https://doi.org/10.5771/0010-3497-2002-3-344, am 14.07.2024, 00:36:21 Open Access – - https://www.nomos-elibrary.de/agb CHRONIK nalisten verpflichteten sich gerade angesichts der ethnischen und religiösen Verschiedenheit zu professioneller Fairness in der Berichterstattung. Taiwan Die Kooperation katholischer Buchverleger im Dienst der Evangelisierung wurde vom Generalsekretär der regionalen Bischofskonferenz in Taiwan, P. Wu Chung-Yuan, begrüßt. An der 10. Internationalen Buchmesse in Taipei hatten acht Verlage erstmals einen eigenen Stand. EUROPA Deutschland Den Katholischen Journalistenpreis 2002 erhielt Asmus Heß, freier Journalist, für seine in der Zeitschrift "Brigitte" erschienene Ruanda-Reportage "Und trotzdem: Gott war da". Den Nachwuchspreis erhielt Stefan Leifert, Assistent an der Bayerischen Akademie für Fernsehen, für sein Krankenhaus-Hörfunkfeature "Wenn Philosophie konkret wird: Ethik im Stationsalltag". Der von der Deutschen Bischofskonferenz gestiftete Journalistenpreis, der am 18. September 2002 vom Katholischen Medienverband (KMV) und der Gesellschaft Katholischer Publizisten (GKP) in Würzburg verliehen wurde, ist mit insgesamt 10.000 Euro dotiert. Delegationen der katholischen und der evangelischen Kirche trafen sich im Mai 2002 in Berlin zu einem Spitzengespräch mit Vertretern des Bundesverbandes Deutscher Zeitungsverleger (BDZV) und des Verbandes Deutscher Zeitschriftenverleger (VDZ). Weihbischof Friedrich Ostermann, Vorsitzender der Publizistischen Kommission der Deutschen Bischofskonferenz, und Dr. Eckart von Vietinghoff, Präsident des Landeskirchenamtes Hannover, unterstrichen die Bedeutung der Zeitungen für die Kommunikationsarbeit der Kirche. Dabei wiesen sie auf die Schwierigkeiten hin, vor allem jüngere Menschen mit der traditionellen Kirchenpresse überhaupt noch zu erreichen. BDZV-Hauptgeschäftsführer Dr. Volker Schulze vertrat die Auffassung, dass die Möglichkeiten der Kooperation zwischen den Tageszeitungen und den Kirchen "noch längst nicht" ausgeschöpft seien. Katholische Verlage wollen das im Herbst 2001 probeweise gestartete Buchversand-Projekt "inspiration" fortführen. Mehrere Testläufe mit einem Versandkatalog für religiöse Bücher hätten positive Ergebnisse gebracht, teilte der Katholische Medienverband (KMV) in Bonn mit. Angestrebt wird eine Mitarbeit evangelischer Medienhäuser. An dem Versandunternehmen sind bislang der Bergmoser + Höller Verlag, die Bonifatius Gesellschaft, der Pattloch-Verlag, der Sankt Michaelsbund, die Verlagsgruppe "engage- https://doi.org/10.5771/0010-3497-2002-3-344, am 14.07.2024, 00:36:21 Open Access – - https://www.nomos-elibrary.de/agb 363 CHRONIK ment" und der Weltbild Verlag beteiligt. Der Bundesverband des Katholischen Deutschen Frauenbundes (KDFB) und dessen bayerischer Landesverband wollen künftig eine gemeinsame Mitgliederzeitschrift herausgeben. Die Kooperation ist in Augsburg mit einem Vertrag besiegelt worden, wie der KDFB mitteilte. Bisher erscheinen in Bayern und der Pfalz die Mitgliederzeitschrift "KDFB Engagiert" und im übrigen Deutschland die "Christliche Frau". Das neue Produkt soll ab dem kommenden Jahr unter dem Titel "KDFB Engagiert - Die christliche Frau" herauskommen. Redaktionssitze bleiben München und Köln. Der Fensehsender RTL unterstützte die Image-Kampagne der Evangelischen Kirche Deutschlands (EKD) mit drei Werbespots. So waren parallel zur bundesweiten Plakat- und Anzeigenaktion der EKD ab April Spots zu folgenden Fragen zu sehen: "Ist der Mensch nur so viel wert, wie er verdient?", "Was ist Glück?" und "Wohin wollen Sie eigentlich?". Der bisherige Geschäftsführer der in München ansässigen katholischen Film-Produktionsgesellschaft Tellux, Bernd Grote, ist neuer Aufsichtsratsvorsitzender des Unternehmens. Er tritt die Nachfolge von Leopold Schu1er an, der in den Ruhestand verabschiedet wurde. Italien Der deutschsprachige Medienverbund GIG- "Centrum Informationis Catholicum" -hat in Rom sein 40jähriges Bestehen begangen. Seit Beginn des II. Vatikanischen Konzils 1962 unterhalten die katholischen Nachrichtenagenturen von Deutschland, Österreich und der Schweiz ein gemeinsames Korrespondentenbüro, das täglich über den Papst und den Vatikan informiert. Es ist der einzige in Rom tätige übernationale katholisehe Medienverband. Neben der KNA mit Sitz in Bonn, der kathpress aus Wien und der KIP A aus Freiburg/Schweiz gehörte auch die inzwischen aufgelöste niederländische KNP zu den Gründungsmitgliedern des CIC. Ein zweiteiliger Spielfilm über das Leben von Papst Johannes XXIII. hat in Italien rekordverdächtige Zuschauerzahlen erzielt. 11,7 Millionen Menschen sahen den zweiten Teil des Spielfilms "Papa Giovanni" im staatlichen Fensehen RAI Uno. Damit erzielte der von Regisseur Giogi Capitani gedrehte Historienfilm eine Quote von 43,5 Prozent. Die RAI plant derzeit ähnliche Filme über weitere Persönlichkeiten des 20. Jahrhunderts, darunter auch Mutter Theresa von Kalkutta. Frankreich Frankreichs nationale Pressevereinigung und die J ournalistenorganisation "Reporter ohne Grenzen" haben sich besorgt über die https://doi.org/10.5771/0010-3497-2002-3-344, am 14.07.2024, 00:36:21 Open Access – - https://www.nomos-elibrary.de/agb CHRONIK Lage der Pressefreiheit in ihrem Land geäußert. In den vergangenen Jahren seien mehrfach für die Meinungsfreiheit ungünstige Gerichtsurteile gefällt und die Rechte der Journalisten gesetzlich eingeschränkt worden. Diese Tendenz habe sich 2001 verstärkt. Im vergangeneu Jahr seien rund ein Dutzend Journalisten wegen ihrer Berichterstattung über Affären juristisch verfolgt worden. Die französische Tageszeitung "Le Monde" hat vor dem Europäischen Menschenrechtsgerichtshof einen Rechtsstreit gegen Frankreich gewonnen. Die französischen Behörden hätten gegen das Grundrecht auf Meinungsfreiheit der Europäischen Menschenrechtskonvention verstoßen, indem sie 1995 die Zeitung wegen eines kritischen Artikels über den damaligen marrokanischen König Rassan II. wegen Beleidigung verurteilt hätten, urteilte das Straßburger Gericht. Nach Ansicht der Richter wurde mit dem Urteil die Einschränkung der Pressefreiheit unverhältnismäßig weit getrieben. In dem Fall ging es um einen 1995 in "Le Monde" erschienenen Artikel über einen vertraulichen Bericht über Drogenexporte aus Marokko. Darin wurden Vorwürfe auch gegen die Umgebung des Königs erhoben. Der Film "Der Mann ohne Vergangenheit" des finnischen Regisseurs Aki Kaurismäki ist mit dem diesjährigen Preis der Ökume- nischen Jmy in Cannes ausgezeichnet worden. Der von Zärtlichkeit und Humor geprägte Film sei eine Parabel über die Wiedergeburt eines Menschen und die Geburt einer Gemeinschaft, begründete die J my ihre Preisvergabe. Kaurismäkis Film hatte auch von der offiziellen Cannes-Jmy mehrere Preise bekommen. Lobend erwähnt wurden von der Ökumenischen Jmy der Film "Le Fils" der belgiseben Regisseure J ean-Pierre und Luc Dardenne sowie "L'ora di religione" des Italieners Marco Belloccio. Der simbabwische Journalist Geoffrey Nyarota erhält den diesjährigen Preis für Pressefreiheit der Weltkulturorganisation UNESCO. Der Chefredakteur und Gründer der einzigen unabhängigen Tageszeitung in Simbabwe habe sich für die Aufrechterhaltung einer kritischen und unabhängigen Presse in dem afrikanischen Land eingesetzt, erklärte die UNESCO in Paris. "The Daily News" sei zur auflagenstärksten Zeitung in Simbabwe geworden. Trotz zahlreicher Zerstörungen der Büros und einer Explosion in der Druckerei habe die Zeitung seit ihrer Gründung im Jahr 1999 nie auf eine Ausgabe verzichtet. Nyarota hat nach UNSECO-Angaben bereits Todesdrohungen erhalten und wurde etliche Male von der Polizei verhaftet und angeklagt. Der UNESCO-Preis für Pressefreiheit "Guillermo Canno" wird seit 1997 verliehen. https://doi.org/10.5771/0010-3497-2002-3-344, am 14.07.2024, 00:36:21 Open Access – - https://www.nomos-elibrary.de/agb 365 CHRONIK Niederlande Neun protestantische Kirchen in den Niederlanden wollen ihre sonntäglichen Fernsehgottesdienste einstellen. Ab Ende Oktober 2002 soll eine neue Sendung mit einer mehr dem Medium Fernsehen angepassten Form ausgestrahlt werden. Nach Angaben des IKON -Sprechers werden die Fernsehgottesdienste am Sonntag vor einem "mehr und mehr wegzappenden Publikum" ausgestrahlt. Deswegen wird kaum noch ein Gottesdienst von Anfang bis zum Ende am Bildschirm verfolgt. Der Dachverband IKON ist einer von drei Zuarbeitern für die protestantischen Gottesdienstübertragungen am Sonntagvormittag. Daneben werden jeden Sonntag im Sender Nederland 1 auch katholische Fernsehgottesdienste übertragen. der "qualitativen Neuausrichtung des Blattes und der angestrebten Schärfung seines Profils auch optisch Rechnung tragen", teilten beide in Wien mit. "Die Furche" ist die älteste Wochenzeitung des Landes und besteht seit 1945. Mit 80 Jahren ist Felix Gamillscheg als langjähriger Mitherausgeber der Wochenzeitung "Die Furche" ausgeschieden. Seine journalistische Laufbahn begann er 1951 bei der Wiener Tageszeitung "Die Presse", später war er Chefredakteur der katholischen Nachrichtenagentur "kathpress". 1976 übernahm der Journalist die Chefredaktion der "Furche", deren Mitherausgeber er 1978 wurde. Vatikan J ohannes Paul II. hat von der internationalen Radio- und Fernsehvereinigung "AIR-IAB" einen Österreichs Wochenzeitung "Die Furche" erscheint in einem neuen Preis für die Verteidigung der Meinungsfreiheit, der Menschenwürde und der Demokratie erhalten. Eine Layout. Gerda Schaffelhofer, seit Januar 2002 zuständige Geschäftsführerin, und der im Oktober letzten Jahres neu ernannte Chefredakteur Rudolf Mitlöhner wollen durch die Umgestaltung entsprechende Urkunde bändigte der Generaldirektor der weltweiten Vereinigung privater Radio- und Fernsehstationen dem Papst nach einer Generalaudienz aus. Österreich https://doi.org/10.5771/0010-3497-2002-3-344, am 14.07.2024, 00:36:21 Open Access – - https://www.nomos-elibrary.de/agb
https://openalex.org/W2607749638	https://europepmc.org/articles/pmc5431018?pdf=render	English	null	The mechanism of iron binding processes in erionite fibres	Scientific reports	2,017	cc-by	8,711	Alessandro Pacella1, Carlo Cremisini2, Elisa Nardi2, Maria Rita Montereali2, Ida Pettiti3 & Paolo Ballirano 1,4 Received: 10 January 2017 Accepted: 29 March 2017 Published: xx xx xxxx Received: 10 January 2017 Accepted: 29 March 2017 Published: xx xx xxxx Fibrous erionite-Na from Rome (Oregon, USA) was K-exchanged and characterized from the structural point of view. In addition, the modifications experienced after contact with a Fe(II) source were investigated for evaluating if the large potassium ions, blocking off nearly all the erionite cavity openings, might prevent the Fe(II) binding process, which is currently assumed to be one of the reasons of the toxicity of erionite. The K-exchanged sample had a 95% reduction of the BET surface area indicating that it behaves as a mesoporous material. Exchanged K is segregated at K2 and at OW sites commonly occupied by H2O. The latter K cations provide a relevant contribution to the reduction of the surface area. Surprisingly, despite the collapse of its surface area the sample preserves the tendency to bind Fe(II). Therefore, yet in the case of a peculiar and potentially hostile structural environment the Fe(II) ion-exchange process has essentially the same kinetics observed in a typical erionite sample. This is a clear evidence of the very limited effect of the chemical composition of erionite on the Fe(II) binding process and reasonably it does not play a significant role in its toxicity. Erionite is a fairly common zeolite often occurring with fibrous habit in several geological environments1. In the recent years, a relevant number of papers has been published aimed at investigating the chemical and structural properties of this zeolite owing to its well-known link, upon inhalation, with malignant mesothelioma2. As a result, erionite has been recently classified as a Group 1 Human-Carcinogen by the International Agency for Research on Cancer (IARC)3, 4. Obviously, a crucial point to be addressed is the definition of the mechanism/s inducing carcinogenicity to devise an inactivation route of the fibres. A common view attributes genotoxicity of erionite to the production of reactive oxygen species (ROS) by Fenton reaction5, 6. Moreover, it has been shown that erionite is able to induce transformation of human mesothelial cells (MET5-A), but on the other hand, asbes- tos is unable to cause such transformation7. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Received: 10 January 2017 Accepted: 29 March 2017 Published: xx xx xxxx Scientific Reports \| 7: 1319 \| DOI:10.1038/s41598-017-01477-x The mechanism of iron binding processes in erionite fibres Alessandro Pacella1, Carlo Cremisini2, Elisa Nardi2, Maria Rita Montereali2, Ida Pettiti3 & Paolo Ballirano 1,4 Alessandro Pacella1, Carlo Cremisini2, Elisa Nardi2, Maria Rita Montereali2, Ida Pettiti3 & Paolo Ballirano 1,4 Alessandro Pacella1, Carlo Cremisini2, Elisa Nardi2, Maria Rita Montereali2, Ida Pettiti3 & Paolo Ballirano 1,4 Pristine (erionite-Na) KCl treated (erionite-K) Mg 5 (0) 2 (0) Ca 14 (1) 1 (0) Na 473 (2) 5 (0) K 42 (1) 445 (33) Table 2. ICP-OES analyses of the supernatant for cation release (calculated as nmol/mg sample). Data of pristine sample taken from ref. 16. Data report the net cation release obtained by subtracting the cation release obtained at the same pH (ca. 5) in H2O. Pristine (erionite-Na) KCl treated (erionite-K) Mg 5 (0) 2 (0) Ca 14 (1) 1 (0) Na 473 (2) 5 (0) K 42 (1) 445 (33) Table 2. ICP-OES analyses of the supernatant for cation release (calculated as nmol/mg sample). Data of pristine sample taken from ref. 16. Data report the net cation release obtained by subtracting the cation release obtained at the same pH (ca. 5) in H2O. Table 2. ICP-OES analyses of the supernatant for cation release (calculated as nmol/mg sample). Data of pristine sample taken from ref. 16. Data report the net cation release obtained by subtracting the cation release obtained at the same pH (ca. 5) in H2O. Table 2. ICP-OES analyses of the supernatant for cation release (calculated as nmol/mg sample). Data of pristine sample taken from ref. 16. Data report the net cation release obtained by subtracting the cation release obtained at the same pH (ca. 5) in H2O. and Cametti21: erionite-K 3.50–5.05 Å; Ballirano and Pacella22: erionite-Na 3.45–5.05 Å). The K-exchanged form experienced a dramatic reduction of 95% of the surface area resulting in a complete removal of its adsorption properties, apart from a residual adsorptive capacity for H2O20. Moreover, it has been reported that zeolites having a large K content show reduced catalytic properties23. On this basis, the present work reports the results of a detailed crystal chemical and structural characterization of K-exchanged erionite. In addition, the modifications experienced by the K-exchanged fibres whenever in con- tact with a ferrous chloride solution were investigated to evaluate if the large potassium ions, blocking off nearly all the adsorption cavities, may prevent the Fe(II) binding process. Alessandro Pacella1, Carlo Cremisini2, Elisa Nardi2, Maria Rita Montereali2, Ida Pettiti3 & Paolo Ballirano 1,4 Other factors may be significant in the development of malignant mesothelioma by erionite, such as levels of exposure that are indeed dangerous for the population8 as well as genetic predisposition9.i g p p Relevantly, erionite is a nominally Fe-free phase but it has been supposed that it is able to fix iron, provided by protein injury, owing to its cation-exchange properties10. Recently, it has been shown that erionite can act as a vehiculating media in the human body for iron as several Fe-bearing mineral species (nano-oxydes, sulphates, phyllosilicates) were found to adhere to its surface11–14. Therefore, the behaviour of erionite fibres kept in contact with iron sources has been thoroughly investigated to define the fate of both Fe(II) and Fe(III) and in particular the tendency to bind to the surface or to be fixed as extra-framework (EF) cations15–17. Results unequivocally showed that Fe(III) is mainly fixed to the surface at relatively high concentration, whereas at low concentrations typical of body fluids it is prevalently segregated at the Ca3 site within the erionite cavity17. Differently, Fe(II) is segregated at Ca3 at all concentrations16. Unfortunately, this picture indicates that erionite under typical phys- iological conditions may act as a very efficient ROS generator because iron is located at well-defined crystallo- graphic sites and it possesses a very low nuclearity18, 19. Therefore, it appears to be of great interest to investigate different physico-chemical procedures to prevent the access of iron species to the erionite cavity as future roads of (partial) inactivation of such fibres.i i Under such aspect, it is worth noting that Eberly20 first reported the adsorption properties of erionite and its cation-exchanged forms. He measured a surface area of 203 m2 g−1 and an effective pore diameter comprised between 4.5 and 5.4 Å, in substantial agreement with the minor and major axes of the largest opening (Ballirano 1Department of Earth Sciences, Sapienza Università di Roma, Piazzale Aldo Moro 5, I-00185, Roma, Italy. 2ENEA, C. R. Casaccia, Via Anguillarese 301, I-00123, Roma, Italy. 3Department of Chemistry, Sapienza Università di Roma, Piazzale Aldo Moro 5, I-00185, Roma, Italy. 4Rectorial Laboratory Fibres and Inorganic Particulate, Sapienza Università di Roma, Piazzale Aldo Moro 5, I-00185, Roma, Italy. Correspondence and requests for materials should be addressed to P.B. Alessandro Pacella1, Carlo Cremisini2, Elisa Nardi2, Maria Rita Montereali2, Ida Pettiti3 & Paolo Ballirano 1,4 (email: paolo.ballirano@uniroma1.it) Scientific Reports \| 7: 1319 \| DOI:10.1038/s41598-017-01477-x 1 www.nature.com/scientificreports/ Oxides (wt.%) Pristine KCl treated KCl treated FeCl2 SiO2 60.06 (47) 57.35 (14) 58.58 (60) Al2O3 12.81 (19) 12.78 (8) 12.82 (17) Na2O 4.03 (27) 1.46 (11) 0.67 (27) K2O 4.01 (42) 9.32 (13) 6.74 (67) MgO 0.59 (24) 0.59 (9) 0.75 (22) CaO 0 0 0 FeO 0 0 1.94 (38) H2O* 18.50 18.50 18.50 Total 100.00 100.00 100.00 Si 28.76 (11) 28.51 (5) 28.62 (10) Al 7.24 (11) 7.49 (5) 7.38 (10) Na 3.75 (27) 1.41 (10) 0.63 (29) K 2.45 (27) 5.91 (9) 4.20 (45) Mg 0.42 (17) 0.44 (7) 0.55 (15) Ca 0 0 0 Fe 0 0 0.79 (15) O 72.10 (14) 72.35 (4) 72.07 (17) H2O 29.60 (17) 30.73 (4) 30.20 (25) R 0.799 (3) 0.792 (1) 0.795 (3) M/(M + D) 0.936 (27) 0.944 (9) 0.896 (46) E% 3.1 −8.6 −1.7 Tot. s.s. EF cat. (e−) 92.8 133.0 114.0 Table 1. Chemical analyses, by SEM-EDS, of both pristine16 and KCl treated fibres (3 cycles). Standard deviations are reported in parenthesis. Estimated. Oxides (wt.%) Pristine KCl treated KCl treated FeCl2 SiO2 60.06 (47) 57.35 (14) 58.58 (60) Al2O3 12.81 (19) 12.78 (8) 12.82 (17) Na2O 4.03 (27) 1.46 (11) 0.67 (27) K2O 4.01 (42) 9.32 (13) 6.74 (67) MgO 0.59 (24) 0.59 (9) 0.75 (22) CaO 0 0 0 FeO 0 0 1.94 (38) H2O 18.50 18.50 18.50 Total 100.00 100.00 100.00 Si 28.76 (11) 28.51 (5) 28.62 (10) Al 7.24 (11) 7.49 (5) 7.38 (10) Na 3.75 (27) 1.41 (10) 0.63 (29) K 2.45 (27) 5.91 (9) 4.20 (45) Mg 0.42 (17) 0.44 (7) 0.55 (15) Ca 0 0 0 Fe 0 0 0.79 (15) O 72.10 (14) 72.35 (4) 72.07 (17) H2O 29.60 (17) 30.73 (4) 30.20 (25) R 0.799 (3) 0.792 (1) 0.795 (3) M/(M + D) 0.936 (27) 0.944 (9) 0.896 (46) E% 3.1 −8.6 −1.7 Tot. s.s. EF cat. (e−) 92.8 133.0 114.0 Table 1. Chemical analyses, by SEM-EDS, of both pristine16 and KCl treated fibres (3 cycles). Standard deviations are reported in parenthesis. Estimated. Table 1. Chemical analyses, by SEM-EDS, of both pristine16 and KCl treated fibres (3 cycles). Standard deviations are reported in parenthesis. Estimated. Table 1. Chemical analyses, by SEM-EDS, of both pristine16 and KCl treated fibres (3 cycles). Standard deviations are reported in parenthesis. Estimated. Results ft Note: Sample pre-treated at 65 °C for 15 hour and then at 200 °C for 2 hours. Figure 1. Drawing of (left) the framework of the K-exchanged form of erionite and of (right) the EF cations and water molecules location within the erionite cage. H2O residing at OW sites may be partly replaced by K cations. Figure 1. Drawing of (left) the framework of the K-exchanged form of erionite and of (right) the EF cations and water molecules location within the erionite cage. H2O residing at OW sites may be partly replaced by K cations. of traces of admixed impurities (see methods), being Ca absent in the pristine sample as indicated by SEM-EDS analysis (Table 1). The calculated charge balance evidences that the number of Fe acquired charges is comparable with that of released charges (Supplementary Table S1), highlighting that Fe(II) was mainly acquired by erionite through ion-exchange with the EF cations, as reported by Ballirano et al.16 (Supplementary Table S1). However, in the case of erionite-K, fibres fix Fe(II) within the structure through an ion-exchange process, mainly involving K, while for erionite-Na the process mainly affected Na (Supplementary Table 2). SEM-EDS analysis of the Fe(II) loaded sample shows a significant reduction of both K and Na content [from 5.91 (9) to 4.2 (4) apfu and from 1.41 (10) to 0.6 (3) apfu, respectively], counterbalanced by an upload of 0.79 (15) apfu Fe(II). However, ICP data indicated only a very marginal Na release (Supplementary Table 2). The observed discrepancy between SEM-EDS and ICP data may be attributed to the relevant Na migration occurring in the external layer of fibres during micro-analysis17, 24. Summarizing, the following crystal chemical formula (Mg0.6Fe0.7Na1.5K4.2)[Al7.5Si28.5O72]∙ 30.5H2O may be retrieved for Fe(II) erionite.i y Pacella et al.17 indicated for erionite-Na (pristine material) a BET specific surface area, measured in the con- ventional p/p0 0.05–0.3 range, of 252 (5) m2 g−1 and of 325 (5) m2 g−1 in the p/p0 0–0.1 range, typical of micropo- rous materials (Table 3). The BET specific surface area of the K-exchanged samples collapses to about 12 m2 g−1. It is worth noting that erionite-Na (pristine material) possesses an external surface area of 10.1 (5) m2 g−1, i.e. a value very close to the BET surface area of K-exchanged samples (erionite-K), indicating that upon treatment the sample behaves exclusively as a mesoporous material. Results ft After an enrichment procedure, erionite was K-exchanged by immersion in a concentrated KCl solution (see methods). ICP data indicate that erionite fibres released significant amounts of Na (581 nmol/mg) at the end of the treatment with the KCl solution. SEM-EDS data of the erionite treated with the KCl solution provide clear evidence that the K binding was successful, since the relevant increase of the K content from 2.5 (3) to 5.91 (9) atoms per formula unit (apfu) in the fibres (Table 1). Coherently with ICP data, a marked reduction of the Na con- tent from 3.8 (3) to 1.41 (10) apfu was evidenced. The final average crystal-chemical formula of (Na1.41K5.91Mg0.44) [Al7.49Si28.51O72.35] · 30.73H2O allows a sample classification as erionite-K.i i Fe(II)-loading of the K-exchanged fibres was performed by suspending erionite in a 500 µM FeCl2 solution. ICP data reveal that erionite fibres bind 290 (13) nmol/mg of Fe. Notably, the Fe amount acquired by erionite-K is perfectly comparable with that observed by Ballirano et al.16 for erionite-Na after suspension in the 500 µM FeCl2 solution [291 (1) nmol/mg]. ICP analysis of supernatants for EF cation release evidences a significant net release of K [445 (33) nmol/mg], coupled with a very minor release of Na, Ca, and Mg during the suspension of the fibres in the FeCl2 solution (Table 2). It should be pointed out that the low Ca release detected is due to the presence Scientific Reports \| 7: 1319 \| DOI:10.1038/s41598-017-01477-x 2 www.nature.com/scientificreports/ Sample BET surface p/p0 0.05–0.3 (m2 g−1) BET surface p/p0 0.0–0.1 (m2 g−1) Pore volume overall (cm3 g−1) Pristine* 252 (5) 325 (5) 0.151 (0.130) KCl1 12.0 (5) 0.0213 KCl3 11.0 (5) 0.0195 Fe-exchanged 73.7 (17) 0.0495 Fe-exchanged* 308 (7) 393 (4) 0.182 (0.151) Table 3. Results of N2 adsorption analyses. In italics, in parenthesis, volume of micropores. Data of the pristine sample were taken from ref. 23. KCl1 and KCl3 refer to fibres subjected to, respectively, 1 and 3 KCl treatments (see Materials and Methods for details). Note: *Sample pre-treated at 65 °C for 15 hour and then at 200 °C for 2 hours. Table 3. Results of N2 adsorption analyses. In italics, in parenthesis, volume of micropores. Data of the pristine sample were taken from ref. 23. KCl1 and KCl3 refer to fibres subjected to, respectively, 1 and 3 KCl treatments (see Materials and Methods for details). Results ft Comparison between the site scattering (s.s.) of extra-framework (EF) cation sites calculated from SEM-EDS chemical data (s.s. from partition) and Rietveld refinement (s.s. from Rietveld refinement). In italics, in parenthesis, the same calculation for an estimated content of Na equal to that of the KCl treated sample, due to the very small release of Na detected by ICP-OES. The low Na content observed by SEM-EDS is due to the renewed Na (and possibly K) cation mobility under the electron beam arising from the leaching of the K cations located at the OW sites. Table 4. Comparison between the site scattering (s.s.) of extra-framework (EF) cation sites calculated from SEM-EDS chemical data (s.s. from partition) and Rietveld refinement (s.s. from Rietveld refinement). In italics, in parenthesis, the same calculation for an estimated content of Na equal to that of the KCl treated sample, due to the very small release of Na detected by ICP-OES. The low Na content observed by SEM-EDS is due to the renewed Na (and possibly K) cation mobility under the electron beam arising from the leaching of the K cations located at the OW sites. and water molecule sites is referred to (Supplementary Table S4). In the case of the K-exchanged sample we observe a complete depletion of the Ca1 site, which is preferentially occupied by Mg and Na in the pristine sample (erionite-Na) and a very strong reduction of the s.s. at Ca3 (occupied by Na). Conversely, the K2 site (occupied by K) significantly increases its s.s. (Fig. 1). It is worth mentioning that the total EF s.s. drops from ca. 113 e− of the pristine sample to ca. 94 e− of the KCl treated sample apparently indicating a significant cationic discharge during the K binding process. The Fe(II) loading process induces a small refilling of the Ca1 site (ca. 4 e−), a significant increase of s.s., from ca. 5 to ca. 20 e− at Ca3, whereas K2 remains substantially unchanged. Concerning the OW sites occupied by H2O, the most relevant differences between pristine and KCl treated sample are related to a sig- nificant s.s. increase at OW7, OW10 and OW12 and a marked reduction at OW11 in the case of the KCl treated sample (Fig. 1). As a net result, the total OW sites s.s. rises from 245 (11) e− for the pristine to 282 (7) e− for the KCl treated sample. Discussion Textural analysis of the KCl treated sample indicates values of the BET surface that are ca. 5% of the pristine material. The corresponding values of ca. 10 m2 g−1 compare favourably with those observed in both amphibole and chrysotile asbestos (3–20 m2 g−1) and are consistent with the 7–9 m2 g−1 reported by Eberly20 for erionite-K from Rome, Oregon, USA. At the structural level, the abrupt abatement of the BET surface implies the occlusion of all microporosity. Erionite possesses two apertures that have been considered to act as possible ways for cation mobility, the so-called 8-member (8MR) and 6-member (6MR) rings. The first one, which is the largest, repre- sents the preferential way for any exchange process, whereas the second one, that is shared between the cancrinite and erionite cages, albeit of small dimensions, has been claimed to act as a revolving door for the so-called “inter- nal ionic exchange” mechanism occurring at high temperature21, 22, 26. It is worth noting that the K2 site is located approximately at the centre of 8MR. Therefore, the reported increased population at this site implies a more extended occlusion of 8MR leading to a reduction of the BET surface. Despite an exchange process involving the replacement by K (19 electrons) of (mainly) Na (11 electrons) and Mg (12 electrons), the total EF s.s (i.e. the sum of the s.s. of Ca1, Ca2, Ca3, K1, and K2 sites) decreases (Supplementary Table S4). This is a clear indication that a fraction of the EF cations was re-located at sites previously occupied by H2O (Fig. 1). This is confirmed by the simultaneous increase of the total OW sites s.s. from 245 (11) e− of the pristine sample to 282 (7) e− for the KCl treated sample mainly justified by a significant s.s. increase at OW7, OW10 and OW12 (Supplementary Table S4). Recent investigations of the thermal behaviour of erionite21, 22 have detected relevant s.s. at both OW7 and OW12 at temperatures well exceeding that of dehydration (280–330 °C thermal range). This fact has been attributed to the migration of EF cations toward those sites to obtain a favourable coordination with oxygen atoms of the framework. We may hypothesize the onset of a similar mechanism in the case of the K-binding process. Results ft In the case of the Fe(II) loaded sample the s.s. of OW7, OW10 and OW12 reduces back and the total water molecules s.s. decreases to 268 (9) e−. Results ft The total volume of pores decreases from 0.151 cm3 g−1 of the pristine sample (of which 0.130 cm3 g−1 attributable to micropores) to ca. 0.02 cm3 g−1. The Fe(II) loaded sample exhibited an increase of the BET specific surface area to 73.7 (17) m2 g−1 significantly smaller than that of the pristine sample. In addition, the total volume of pores slightly increases with respect to the K-exchanged samples to 0.0495 cm3 g−1. g XRPD data reveal that the cell volume expansion of erionite-K is caused by the c-axis expansion only partly counterbalanced by the a-axis contraction (Supplementary Table S2). Iron binding produces a further minor contraction of both a- and c-axis leading to a cell volume smaller than that of erionite-Na (pristine material). The c/a ratio passes from ca. 1.138 to ca. 1.140 of the two treated samples (Supplementary Table S2). As expected no significant modifications of the framework (Fig. 1) were detected with the variation of <T-O> bond distances (Supplementary Table S3) and T-O-T bond angles (not shown) being confined within a few standard deviations. The slightly higher R values as calculated from Jones’ determinative curves25 with respect to those from SEM-EDS might be related to the effect of protonation at some oxygen sites of the framework progressively occurring dur- ing treatments17. On the contrary, relevant differences are observed as far as the site scattering (s.s.) of both EF Scientific Reports \| 7: 1319 \| DOI:10.1038/s41598-017-01477-x 3 www.nature.com/scientificreports/ Site KCl exchanged Fe-exchanged EF cation s.s from partition (e−) s.s. from Rietveld refinement (e−) EF cation s.s from partition (e−) s.s. from Rietveld refinement (e−) Ca1 Mg0.6 7.2 4.1 (7) Ca2 Na1.5K0.6 27.9 26.1 (8) Na0.6(1.5) 6.6 (16.5) 24.3 (6) Ca3 Mg0.3 3.6 5.3 (5) Fe0.8 20.8 20.2 (9) K1 K2 38 38 K2 38 38 K2 K1.3 24.7 24.7 (7) K1.1 20.9 23.1 (6) OW7–12 K2 38 37 K1.1 20.9 23 total 132.2 131 (2) total 124.3 (134.2) 133 (3) Table 4. Comparison between the site scattering (s.s.) of extra-framework (EF) cation sites calculated from SEM-EDS chemical data (s.s. from partition) and Rietveld refinement (s.s. from Rietveld refinement). In italics, in parenthesis, the same calculation for an estimated content of Na equal to that of the KCl treated sample, due to the very small release of Na detected by ICP-OES. Results ft The low Na content observed by SEM-EDS is due to the renewed Na (and possibly K) cation mobility under the electron beam arising from the leaching of the K cations located at the OW sites. Site KCl exchanged Fe-exchanged EF cation s.s from partition (e−) s.s. from Rietveld refinement (e−) EF cation s.s from partition (e−) s.s. from Rietveld refinement (e−) Ca1 Mg0.6 7.2 4.1 (7) Ca2 Na1.5K0.6 27.9 26.1 (8) Na0.6(1.5) 6.6 (16.5) 24.3 (6) Ca3 Mg0.3 3.6 5.3 (5) Fe0.8 20.8 20.2 (9) K1 K2 38 38 K2 38 38 K2 K1.3 24.7 24.7 (7) K1.1 20.9 23.1 (6) OW7–12 K2 38 37 K1.1 20.9 23 total 132.2 131 (2) total 124.3 (134.2) 133 (3) Table 4. Comparison between the site scattering (s.s.) of extra-framework (EF) cation sites calculated from SEM-EDS chemical data (s.s. from partition) and Rietveld refinement (s.s. from Rietveld refinement). In italics, in parenthesis, the same calculation for an estimated content of Na equal to that of the KCl treated sample, due to the very small release of Na detected by ICP-OES. The low Na content observed by SEM-EDS is due to the renewed Na (and possibly K) cation mobility under the electron beam arising from the leaching of the K cations located at the OW sites. Site KCl exchanged Fe-exchanged EF cation s.s from partition (e−) s.s. from Rietveld refinement (e−) EF cation s.s from partition (e−) s.s. from Rietveld refinement (e−) Ca1 Mg0.6 7.2 4.1 (7) Ca2 Na1.5K0.6 27.9 26.1 (8) Na0.6(1.5) 6.6 (16.5) 24.3 (6) Ca3 Mg0.3 3.6 5.3 (5) Fe0.8 20.8 20.2 (9) K1 K2 38 38 K2 38 38 K2 K1.3 24.7 24.7 (7) K1.1 20.9 23.1 (6) OW7–12 K2 38 37 K1.1 20.9 23 total 132.2 131 (2) total 124.3 (134.2) 133 (3) Table 4. Comparison between the site scattering (s.s.) of extra-framework (EF) cation sites calculated from SEM-EDS chemical data (s.s. from partition) and Rietveld refinement (s.s. from Rietveld refinement). In italics, in parenthesis, the same calculation for an estimated content of Na equal to that of the KCl treated sample, due to the very small release of Na detected by ICP-OES. The low Na content observed by SEM-EDS is due to the renewed Na (and possibly K) cation mobility under the electron beam arising from the leaching of the K cations located at the OW sites. Table 4. Discussion Therefore, the extra 37 e− detected at the water molecule sites of the KCl treated sample with respect to the pristine one may be attributed to EF cations, preferentially K. Summing this extra s.s. to that arising from the EF cations sites a total of 131 e− is obtained in perfect agreement with that determined by SEM-EDS (Table 1). A close inspection of the spatial distribution of the OW sites indicates a few possible coordination types for EF cations residing at (near) the OW7, OW10 and OW12 sites. However, it is expected that the reported fractional coordinates of H2O (+cations) sites represent the average position of very close neighbouring sites. Analysis of the s.s. at the various EF cations sites (Table 4) seems to suggest, except for Na, an unusual occurrence of K at Ca2. This hypothesis is supported by the presence of several potential ligands in the 3.1–3.3 Å range (OW12 × 3, OW9 × 3, O5 × 3). Both s.s. and coordination are consistent with the presence of Mg at Ca3 (Table 4). It must be noted that, considering the s.s. from SEM-EDS data and those from Rietveld refinement, there is a remarkably, albeit uncommon, good agreement, the possible mismatch being attributed to alkali-metal migration23. In the present case, we may hypothesize that the almost complete pore occlusion caused by the additional K cations prevent alkalies from migration/volatilization. Scientific Reports \| 7: 1319 \| DOI:10.1038/s41598-017-01477-x 4 om/scientificreports/ Figure 2. BET surface area as a function of the K content in the OW sites. www.nature.com/scientificreports/ tificreports/ Figure 2. BET surface area as a function of the K content in the OW sites. Figure 2. BET surface area as a function of the K content in the OW sites. It must be pointed out that, despite the collapse of the surface area and the occlusion of all microporosity, the K-exchanged form unfortunately preserved its tendency to bind Fe whenever in contact with a source of Fe(II). Regarding the Fe(II) loaded sample, a significant increase of s.s. at Ca3 may be attributed to the segregation of Fe(II) at this site (Table 4), similarly to the experiments by Ballirano et al.16. In addition, Ca1 site, previously completely depleted during the K-exchange process, upon iron-loading acquires a s.s. that reasonably matches that of Mg as determined by SEM-EDS. Discussion Mobilization of Mg from Ca3 to Ca1 is favoured by a relatively small, non-occurring, distance between the two sites of 1.3 (2) Å. Therefore, we may hypothesize that Mg occupies Ca1 whereas Na is preferentially allocated at Ca2. However, the calculated s.s. of Ca2 is significantly smaller than that retrieved from the Rietveld refinement (Table 4). We may suggest that the Na content has been underestimated in the SEM-EDS analysis due to the well-known, above mentioned, analytical effects. The absence of a coupled significant reduction of s.s. at K2 indicates that the Fe(II) → K exchange process, devised by ICP analysis, preva- lently involves K ions located at the OW sites as well as those at Ca2. This hypothesis is confirmed by the reported decrease of the total s.s. of the OW sites from 282 (7) to 268 (9) e− (Supplementary Table S4). Accordingly, the renewed Na (and possibly K) cation mobility under the electron beam can be justified by the leaching of the K cations located at the OW sites. On this basis, hypothesizing the same Na content as in the case of the KCl treated sample (1.5 apfu instead of 0.6), the overall s.s. balance improves significantly. As above mentioned, the BET spe- cific surface area fairly increases with respect to the KCl treated sample passing from ca. 10 to ca. 75 m2 g−1. Our results showed that this is prevalently due to the release of K ions allocated in the OW sites, that therefore play a primary role in governing the specific surface area of the fibres (Fig. 2).i p y g g pii g Notably, the BET specific of the Fe(II) loaded sample measured in the p/p0 0.05–0.3 range [and in the 0–0.1 range], significantly increases from 73.7 (17) m2 g−1 [and from 75.6 (17) m2] to 308 (7) m2 g−1 if the sample is fur- ther pre-treated under vacuum at 200 °C for 1.6 hours (Table 3). The sample heating at this temperature promotes its dehydration and consequently the migration of K hosted from the water molecular OW sites. Accordingly, the sample reacquires the peculiarity of microporous material with the total volume of the sample increasing to 0.182 cm3 g−1 (of which 0.151 cm3 g−1 attributable to micropores), value comparable with that measured for the pristine sample (0.151 cm3 g−1, of which 0.130 cm3 g−1 attributable to micropores). Materials and Methods Then, solid and solution were separated following the same procedure described for KCl1 sample and stored for BET (KCl2 sample) and ICP-OES analysis, respectively. In the third experiment, the procedure of KCl treatment was repeated three times (KCl3 sample). A blank proce- dure, suspending the fibres in deionized water, was also performed. Notably, BET results (Table 3) showed no sig- nificant differences in the surface area between samples undertaking one (KCl1) or three KCl treatments (KCl3). Accordingly, only one KCl treatment was performed on the enriched material for the K-binding experiment. Fe(II) binding to erionite experiment. Fe(II) binding was performed using the enriched erionite sample previously treated with the KCl solution and following the procedure described in Ballirano et al.19. In detail, 25 mg of erionite fibres were suspended in 25 mL of a 500 µM FeCl2 solution, in a 50 mL FalconTM polypropylene tube. All sample suspensions were prepared in a nitrogen-filled glove-bag to prevent iron oxidation. Later, all the polypropylene tubes, closed and sealed with Parafilm®, were removed from the glove-bag and instantly placed in an oscillating bath at 37 °C for 1 hour. After 1 h the tubes were placed again in the oxygen-free glove bag and the supernatant solution was sampled and filtered, using a 0.22 µm nitrocellulose membrane filter, for ICP-OES analysis. The fibres were recovered from the tube on filter, rinsed with ultrapure deionized water to remove any residues of iron not bound, dried and stored under nitrogen prior to the SEM-EDS and XRPD investigations. Blank samples were prepared by suspending fibres in deionized water adjusted to the appropriate pH (ca. 5: i.e. the same pH of the 500 μM FeCl2 solution) by adding HCl (Ultrex II, ultrapure reagent, JT Baker). Experiments were done in triplicate. ICP-OES analysis. To measure the concentration of released cations from the fibres suspended in both the KCl and FeCl2 solutions, the filtered solutions were properly diluted with 1% nitric acid solution and analysed by ICP-OES. All measurements were performed using an Optima 2000 DV ICP-OES spectrometer (Perkin-Elmer, Waltham, MA, USA) equipped with a cross flow nebulizer placed inside a Scott spray chamber. ICP Aristar (BDH) standard solutions in nitric acid for the investigated elements were used to prepare the calibrating solu- tions for ICP-OES analyses. To ensure adequate quality assurance, the measures of the standard solutions were regularly repeated after the analysis of a defined number of samples. Materials and Methods In the second experiment, the fibres after having been subjected to the first treatment were added again in a fresh KCl solution and stirred for other two hours. Then, solid and solution were separated following the same procedure described for KCl1 sample and stored for BET (KCl2 sample) and ICP-OES analysis, respectively. In the third experiment, the procedure of KCl treatment was repeated three times (KCl3 sample). A blank proce- dure, suspending the fibres in deionized water, was also performed. Notably, BET results (Table 3) showed no sig- nificant differences in the surface area between samples undertaking one (KCl1) or three KCl treatments (KCl3). Accordingly, only one KCl treatment was performed on the enriched material for the K-binding experiment. K binding to erionite experiment. K binding was performed following the indications of Eberly20 except for rescaling of the quantity of the materials. The KCl solution was obtained by dissolving 75 g of potassium chloride (KCl anhydrous ACS ≥99%, Sigma Aldrich) in 250 mL of deionized water. About 500 mg of erionite were placed in a 50 mL FalconTM polypropylene tube with 2 mL of KCl solution at 65 °C in an oscillating bath. A non-enriched material was used for a trial to define the best operating conditions for the K-binding procedure. In detail, three different treatments of the fibres with KCl solution were done. In the first one, fibres were stirred with KCl solution for 2 hours. Then, the fibres were separated from the solution by centrifugation at 4000 rpm for 10 min. The solution was filtered with a 0.2 µm membrane filter and stored for ICP-OES analysis. The fibres were recovered on filter and repeatedly rinsed with ultrapure deionized water to remove any residues of Cl ions and K not bound. The washing procedure was repeated until the absence of Cl ions was evidenced by the absence of opalescence in the washing solution after the addition of few drops of a solution of AgNO3 (0.1N). At the end of these washing cycles the fibres were dried and stored prior to the BET surface area measurement (KCl1 sample).iti g yi p ( p ) In the second experiment, the fibres after having been subjected to the first treatment were added again in a fresh KCl solution and stirred for other two hours. Conclusions Erionite-Na experienced a dramatic reduction of the BET surface area of ca. 95% upon K-loading indicating that the sample behaves exclusively as a mesoporous material, coherently with literature data. Although the large potassium ions blocked off nearly all the adsorption cavities, erionite-K preserves its capacity of binding Fe(II) in the erionite cavity by ionic exchange, with efficiency comparable to that observed for the pristine sample (erionite-Na). Rietveld refinement highlighted that the K ions residing at the OW sites provide a significant con- tribution to the reduction of the BET surface area. Coherently, the Fe(II) loaded sample showed a BET specific surface reduced of ca. 25% with respect to that of the pristine sample (erionite-Na). Notably, since the well-known relationship between surface properties of fibres and their interacting ability with biological medium27–31, this abrupt reduction supposedly plays a fundamental role in governing the corresponding chemical reactivity of erionite. It is worth noting that our experiments testify the very strong tendency of Fe(II) to bind to the erionite structure owing to its relatively high ionic potential exceeding that of the common EF cations. In fact, it is able to enter even into a very crowded erionite cage hosting additional large K cations at sites previously occupied by water molecules. Therefore, yet in the case of a very peculiar and potentially hostile crystal chemical and struc- tural environment for a Fe(II) ion-exchange process we found essentially the same kinetics observed in a typical erionite sample16. This is a clear evidence of the very limited effect of the chemical composition of fibres on the Fe(II) bind process and, accordingly, we may hypothesize that their chemical composition does not play a signifi- cant role in inducing toxicity. Present results are of interest in future attempts to engineer alternative roads toward the (partial) inactivation of those fibres. Scientific Reports \| 7: 1319 \| DOI:10.1038/s41598-017-01477-x 5 www.nature.com/scientificreports/ Materials and Methods Materials and Methods Materials. We used erionite-Na from Rome, Oregon, USA that has been previously characterized from the chemical and structural point of view by Ballirano et al.15. It has a crystal chemical formula (Na3.75K2.45Mg0.42) [Al7.24Si28.76O72.10] · 29.60H2O. About 5 g of the raw sample were enriched following the protocol devised by Ballirano and Cametti32. SEM-EDS analysis. Chemical analyses were performed using a Quanta 400 SEM equipped with an EDAX Genesis EDS system (FEI, Hillsboro, OR, USA) and operating at 15 kV accelerating voltage, 11 mm working distance and 0° tilt angle. Chemical data were collected at least at 8 analytical points and the crystal chemical for- mulae were calculated, after renormalization of the chemical analyses hypothesizing a water content of 18.5 wt.% (corresponding to ca. 30 apfu), on the basis of 36 (Si + Al) apfu. The balance error formula E%33 and the K content test34 were evaluated for selecting the reliable analyses. K binding to erionite experiment. K binding was performed following the indications of Eberly20 except for rescaling of the quantity of the materials. The KCl solution was obtained by dissolving 75 g of potassium chloride (KCl anhydrous ACS ≥99%, Sigma Aldrich) in 250 mL of deionized water. About 500 mg of erionite were placed in a 50 mL FalconTM polypropylene tube with 2 mL of KCl solution at 65 °C in an oscillating bath. A non-enriched material was used for a trial to define the best operating conditions for the K-binding procedure. In detail, three different treatments of the fibres with KCl solution were done. In the first one, fibres were stirred with KCl solution for 2 hours. Then, the fibres were separated from the solution by centrifugation at 4000 rpm for 10 min. The solution was filtered with a 0.2 µm membrane filter and stored for ICP-OES analysis. The fibres were recovered on filter and repeatedly rinsed with ultrapure deionized water to remove any residues of Cl ions and K not bound. The washing procedure was repeated until the absence of Cl ions was evidenced by the absence of opalescence in the washing solution after the addition of few drops of a solution of AgNO3 (0.1N). At the end of these washing cycles the fibres were dried and stored prior to the BET surface area measurement (KCl1 sample). References Crystal structure and iron topochemistry of erionite-K from Rome, Oregon, USA. Am. Mineral. 94, 1262–1270, doi:10.2138/am.2009.3163 (2009). g 2. Matassa, R., Famigliari, G., Relucenti, M., Battaglione, E., Downing, C., Pacella, A., Cametti, G. & Ballirano, P. A Deep Look Into Erionite Fibres: an Electron Microscopy Investigation of their Self Assembly Sci Rep 5 16757 doi:10 1038/srep16757 (2015) 2. Matassa, R., Famigliari, G., Relucenti, M., Battaglione, E., Downing, C., Pacella, A., Cametti, G. & Ballirano, P. A Deep Look Into Erionite Fibres: an Electron Microscopy Investigation of their Self-Assembly. Sci. Rep. 5, 16757, doi:10.1038/srep16757 (2015). 3 C A All i M Ri d C G di G Y H & C b M N I Ri h P ti l Li th S f 13. Croce, A., Allegrina, M., Rinaudo, C., Gaudino, G., Yang, H. & Carbone, M. Numerous Iron-Rich Particles Lie on the Surface of Erionite Fibers from Rome (Oregon, USA) and Karlik (Cappadocia, Turkey). Microsc. Microanal. 21, 1341–1347, doi:10.1017/ S1431927615014762 (2015).i 14. Gualtieri, A. F., Bursi Gandolfi, N., Pollastri, S., Pollok, K. & Langenhorst, F. Where is iron in erionite? A multidisciplinary study of fibrous erionite-Na from Jersey (Nevada, USA). Sci. Rep. 6, 37981, doi:10.1038/srep37981 (2016).f i 15. Eborn, S. K. & Aust, A. E. Effect of iron acquisition on induction of DNA single-strand breaks by erionite, a carcinogenic mineral fiber. Arch. Biochem. Biophys. 316, 507–514, doi:10.1006/abbi.1995.1067 (1995).iii f Biochem. Biophys. 316, 507–514, doi:10.1006/abbi.1995.1067 (1995 i p y 6. Ballirano, P. et al. Fe(II) segregation at a specific crystallographic site of fibrous erionite: A first step toward the understanding of the mechanisms inducing its carcinogenicity. Micropor. Mesopor. Mat. 211, 48–63, doi:10.1016/j.micromeso.2015.02.046 (2015). g g y p p j 17. Pacella, A., Fantauzzi, M., Atzei, D., Cremisini, C., Nardi, E., Montereali, M. R., Rossi, A. & Ballirano, P. Iron within the erionite cavity and its potential role in inducing its toxicity: Evidence of Fe(III) segregation as extra-framework cation. Microp. Mesop. Mat. 237, 168–179, doi:10.1016/j.micromeso.2016.09.021 (2017). j 18. Schwidder, M., Santhosh Kumar, M., Klementiev, K. V., Pohl, M. M., Brückner, A. & Grünert, W. Selective reduction of NO with Fe-ZSM-5 catalysts of low Fe content I. Relations between active site structure and catalytic performance. J. Catal. 231, 314–330, doi:10.1016/j.jcat.2005.01.031 (2005). j j ( ) 19. Zecchina, A., Rivallan, M., Berlier, G., Lamberti, C. & Ricchiardi, G. Materials and Methods Concerning the Fe(II) binding to erionite experiments, the amount of Fe bound to the fibres (nmol/mg) was calculated by difference between the Fe con- centration measured in the initial solution and that measured in the solution after 1 h of sample incubation. The net cation release was calculated by difference between the values measured after suspension in the FeCl2 solution and those measured after suspension of fibres in water at the appropriate pH (ca. 5). In order to verify if Fe(II) was acquired by erionite through an ion exchange mechanism, a charge balance was calculated using the same approach of Eborn and Aust15. The number of charges of each element was determined multiplying the nanomoles by the valence of the corresponding element and therefore, the net charges released into solution were compared to the total charges acquired in form of Fe. Surface area measurements. Surface area, Brunauer–Emmett–Teller (BET) multipoint method35, and textural analysis were obtained by N2 adsorption/desorption measurements at the liquid nitrogen temperature (−196 °C), using a 3-Flex analyser (Micromeritics, Norcross, GA, USA). Before analysis, samples were pre-treated under vacuum at 65 °C for 15 hours. The pores distribution was determined from the adsorption curve by the Barret–Joyner–Halenda (BJH) method36 and from the analysis of the micropore isotherm by the t-test37 taking the curve of Harkins and Jura38. The total pore volume was determined by the rule of Gurvitsch39. Scientific Reports \| 7: 1319 \| DOI:10.1038/s41598-017-01477-x 6 www.nature.com/scientificreports/ X-ray powder diffraction (XRPD). XRPD data were collected in transmission mode, using SiO2-glass cap- illaries, on a D8 Advance (Bruker AXS, Karlsruhe, Germany) equipped with incident-beam focussing Göbel mirrors and a PSD VÅntec-1. 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Author Contributions A.P. performed SEM-EDS, analysed the results, wrote and revised the manuscript; C.C., E.N. and M.R.M. engineered and performed binding experiments, carried out ICP-OES measurements, analysed the results, wrote and revised the manuscript; I.P. performed surface area measurements, analysed the results, wrote and revised the manuscript; P.B. conceived the research and conducted XRPD experiments, performed structure refinements, analysed the results, wrote and revised the manuscript. Scientific Reports \| 7: 1319 \| DOI:10.1038/s41598-017-01477-x References Dehydrated natural erionite with stacking faults of the offretite type. Acta Crystallogr. B33 3265 3268 doi:10 1107/S0567740877010784 (1977) 6. Schlenker, J. L., Pluth, J. J. & Smith, J. V. Dehydrated natural erionite with stacking faults of the offretite type. Acta Crystallogr. B33 3265–3268, doi:10.1107/S0567740877010784 (1977).hi 27. Fubini, B. 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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: 1319 \| DOI:10.1038/s41598-017-01477-x 8
https://openalex.org/W2166189895	https://refubium.fu-berlin.de/bitstream/fub188/14928/1/Molecules18-13p06679.pdf	English	null	Isotope Effects in ESR Spectroscopy	Molecules/Molecules online/Molecules annual	2,013	cc-by	21,935	Molecules 2013, 18, 6679-6722; doi:10.3390/molecules18066679 Molecules 2013, 18, 6679-6722; doi:10.3390/molecules18066679 Keywords: effects of nuclear spin; mass dependency; magnetic moment; local symmetry; time dependency; vibronics and tunnelling in ESR 1. Introduction The physical explanation of the phenomenon and the usage of isotopes for industrial or medical purposes are highly developed technical and scientific areas [1,2]. This is also true for the method of electron spin resonance (ESR) [3,4]. ESR supplies a well-developed instrumental as well as a theoretical basis. The ESR method is suitable to examine systems with unpaired spins [3,5], which interact with each other, as well as with stationary or time-dependent magnetic and electric fields, coherent electromagnetic radiation (in general microwaves), and the magnetic moments of neighbouring nuclei. On this the microscopic degrees of freedom of the system under examination act mediating and modulating in addition to the local structure constraints. Based on the experimental findings and the respective theoretical interpretation many basic phenomena of quantum mechanics can be understood. Moreover, important contributions to the solution of basic problems of physics, chemistry, biology, medicine, geology, and environmental science could be delivered. The method is indispensable on examining the effect of ionizing radiation of foodstuff and other objects. This requires the cooperation of scientists from different fields of work. The difficulty of the method is and will remain the complex interpretation of the experimental findings, even on considering the technical progress in the field. Essential problems of ESR can be solved by the use of isotopes. Last but not least the application of the isotope methods in ESR requires good skills in the art of chemical preparation and a certain “chemical feeling” for discussing the results. Moreover, using isotopes in spectroscopy has been done for a long time already, and is highly developed nowadays [1]. A large number of publications speak for that fact. Finally, it is the task of spectroscopy to deliver information about the system under investigation. Applications in mass spectrometry, infrared spectroscopy, and nuclear magnetic resonance (NMR) prove that statement. Altogether, the thermal, mechanical, electrical, optical, magnetical, and chemical properties of the samples, including their enclosed atoms, are specifically changed on using isotopes. The usage of isotopes in ESR spectroscopy supports the signal assignment and, thus, to obtain the desired physical, chemical, biological, and geological information about the examined systems out of the recorded spectra. Reinhard Stößer 1 and Werner Herrmann 2,* 1 Institute of Chemistry, Humboldt University of Berlin, Brook-Taylor-Str. 2, Berlin 12489, Germany; E-Mail: rs1939@gmx.net 1 Institute of Chemistry, Humboldt University of Berlin, Brook-Taylor-Str. 2, Berlin 12489, Germany; E-Mail: rs1939@gmx.net 2 Institute of Pharmacy, Free University Berlin, Kelchstr. 31, Berlin 12169, German * Author to whom correspondence should be addressed; E-Mail: wfh@zedat.fu-berlin.de Tel.: +49-30-8385-0695; Fax: +49-30-8385-0685. * Author to whom correspondence should be addressed; E-Mail: wfh@zedat.fu-berlin.de; Tel.: +49-30-8385-0695; Fax: +49-30-8385-0685. Received: 26 March 2013; in revised form: 14 May 2013 / Accepted: 29 May 2013 / Published: 7 June 2013 Received: 26 March 2013; in revised form: 14 May 2013 / Accepted: 29 May 2013 / Published: 7 June 2013 Abstract: In order to present the relationship between ESR spectroscopy and isotope effects three levels are considered: (i) ESR spectroscopy is described on a general level up to the models for interpretation of the experimental spectra, which go beyond the usually used time and mass independent spin-Hamilton operator, (ii) the main characteristics of the generalized isotope effects are worked out, and finally (iii) the basic, mainly quantum mechanical effects are used to describe the coupling of electron spins with the degrees of freedom, which are accessible under the selected conditions, of the respective paramagnetic object under investigation. The ESR parameters and the respective models are formalized so far, that they include the time and mass depending influences and reflect the specific isotope effects. Relations will be established between the effects in ESR spectra to spin relaxation, to spin exchange, to the magnetic isotope effect, to the Jahn-Teller effects, as well as to the influence of zero-point vibrations. Examples will be presented which demonstrate the influence of isotopes as well as the kind of accessible information. It will be differentiated with respect to isotope effects in paramagnetic centres itself and in the respective matrices up to the technique of ESR imaging. It is shown that the use of isotope effects is indispensable in ESR spectroscopy. Keywords: effects of nuclear spin; mass dependency; magnetic moment; local symmetry; time dependency; vibronics and tunnelling in ESR Keywords: effects of nuclear spin; mass dependency; magnetic moment; local symmetry; time dependency; vibronics and tunnelling in ESR Molecules 2013, 18 Molecules 2013, 18 6680 1. Introduction Basically there are three fields of application on using isotopes: (i) Proof and application of quantum effects by means of ESR, (ii) isotopes as means for signal assignment in case of complex ESR spectra, and (iii) detection and interpretation of kinetic and isotope effects under participation of the electron spin. Concerning the topic “isotopes as a means for the identification in the case of complex ESR spectra” one can find a large number of papers, i.e., the respective methodology has been developed farthest, while publications in the other two fields are relatively rare due to their higher theoretical standard. The usage of isotopes may significantly contribute to simplify complex spectra, to explain the origin of specific atoms in chemical products, or to allow the observation of structural and micro-dynamic processes, respectively. This is based on the characteristic differences in the properties of isotopes such as mass, nuclear volume, nuclear spin, and nuclear magnetic moment. Using these differences in properties requires good skills in chemical synthesis for the defined placement of isotopes in the molecular structure or in the lattice of a solid in case there is no possibility to use the natural abundance. Molecules 2013, 18 6681 All this is connected to the underlying theoretical models such as Jahn-Teller effects, tunnel effects, vibronic couplings, zero-point oscillations, spin exchange, spin-phonon couplings, and magnetic isotope effects. All this is connected to the underlying theoretical models such as Jahn-Teller effects, tunnel effects, vibronic couplings, zero-point oscillations, spin exchange, spin-phonon couplings, and magnetic isotope effects. The detection and identification of paramagnetic species in ESR is still a problem, e.g., in order to prevent fast chemical reactions many radical systems have been made observable at low temperatures or in a chemical way by introducing steric hindrances into their structures (spin probes, spin traps [6,7]). Simultaneously there has been a fast technical progress in the ESR equipment particularly with respect to sensitivity and spectral resolution [3,8–13]. Nevertheless the isotopic marking is of great importance for the decryption of the hyperfine structure patterns. Thus, it is an aim of the present paper to present the ‘state of the art’ of ESR (somehow deviating from the existing monographs [3,14–16]) in such a way that the relations between the ESR parameters and the characteristic properties of isotopes become evident. 1. Introduction These relations will be discussed by means of selected properties and suitable examples starting with the schematic representation of the interactions of a paramagnetic particle (see Figure 1). 2.1. Basic Principle of ESR Schematic representation of the realization of an ESR spectrum (blue) starting from the different properties of a paramagnetic particle including different internal and external interactions as well as specific influences of the matrix. The green marking shows those parameters including the resulting effects which are not part of the common Spin-Hamiltonian. The possible isotope effects are marked red. Using the given models for interpretation detailed information about structure and dynamics of the examined paramagnetic systems will be accessible. Figure 1. Schematic representation of the realization of an ESR spectrum (blue) starting from the different properties of a paramagnetic particle including different internal and external interactions as well as specific influences of the matrix. The green marking shows those parameters including the resulting effects which are not part of the common Spin-Hamiltonian. The possible isotope effects are marked red. Using the given models for interpretation detailed information about structure and dynamics of the examined paramagnetic systems will be accessible. ill be accessible. 2.1. Basic Principle of ESR ESR is the most important analytical instrument for the examination of paramagnetic compounds. At first this is related to the structure of molecules, distributions of spin densities [17,18] and the dynamics of molecules. There are many applications of the aspects, which will be discussed in greater detail further on, in the fields of biology, medicine, the chemistry of radicals [6,7], metal-organic compounds, solid state physics and chemistry, food chemistry, pharmacy, in geology, and archaeology. ESR differs from nuclear magnetic resonance (NMR) only quantitatively [19] It belongs to those ESR is the most important analytical instrument for the examination of paramagnetic compounds. At first this is related to the structure of molecules, distributions of spin densities [17,18] and the dynamics of molecules. There are many applications of the aspects, which will be discussed in greater detail further on, in the fields of biology, medicine, the chemistry of radicals [6,7], metal-organic compounds, solid state physics and chemistry, food chemistry, pharmacy, in geology, and archaeology. ESR differs from nuclear magnetic resonance (NMR) only quantitatively [19]. It belongs to those spectroscopic methods which apply magnetic fields and coherent electromagnetic radiation. Usually the high-frequency susceptibility χ”(ω) of a sample is measured in a device in which the high-frequency magnetic induction 1 B  is perpendicular to the static field 0 B  . According to the resonance condition: compounds, solid state physics and chemistry, food chemistry, pharmacy, in geology, and archaeology. ESR differs from nuclear magnetic resonance (NMR) only quantitatively [19]. It belongs to those spectroscopic methods which apply magnetic fields and coherent electromagnetic radiation. Usually the high-frequency susceptibility χ”(ω) of a sample is measured in a device in which the high-frequency magnetic induction 1 B  is perpendicular to the static field 0 B  . According to the resonance condition: h ν = g β B  or ω = γ B  (1) (1) where γ: gyromagnetic ratio, the internal magnetic moments (of the unpaired electrons) will flip. Thus, the ESR experiment consists of microwave radiation entering a cavity (with a sample) which is critically coupled to the microwave generator and absorbs the microwave completely. On passing the resonance, e.g., by variation of the static field 0 B  , a mismatching of the resonator and hence, and a part of the microwave radiation will be registered at the microwave diode. Molecules 2013, 18 6682 Figure 1. Molecules 2013, 18 6683 Figure 2(a) shows a simplified scheme of an ESR apparatus. The sample is located between the poles on an electromagnet and will be exposed to microwave irradiation of frequency ν. Plotting the microwave diode current iD versus the magnetic induction B0 gives a horizontal line with a dip in the case of resonance (h ν = g β B0), which is an expression of the microwave absorption by the electron spins of the sample at Bres. Figure 2. Strongly simplified scheme of an ESR spectrometer (a) and splitting of the nuclear spin states with I = ½ in the zero field by the electron spin S = ½ (↓) (b). From a theoretical point view this means that if the energy levels and the wave function of a paramagnetic system are known, the disturbance of the system by a magnetic field (Zeeman effect, [3,20,21]) and transition between the Zeeman levels are the precondition for the ESR. Then that and the resonance absorption of electromagnetic radiation in the microwave or radio frequency (RF) range together with spin relaxation and other temperature and pressure depending effects (see Figure 1) determine the resulting ESR spectra [3,19]. Generally many ESR spectra can be described by a simple Spin-Hamilton operator (see [3,22]). Its matrix elements determine position and intensity of the absorption lines in the microwave or RF range of the electromagnetic radiation. But explicit mass and time dependent effects are ignored and only a part of isotope effects (magnetic moments N μ and nuclear spins I  ) can be taken into account. To grasp the full spectrum of isotope effects further models (see Figure 1 and Section 2.9.) are necessary. The electron and nuclear spin (S  and I  ) as well as the magnetic induction 0 B  (and their combinations; see Figure 1) are represented by operators connected by spin coupling coefficients [17,18], which depend on structure and properties of the examined system (see e.g., [23,24]). Favourably the arrangement of the Spin-Hamilton terms will be selected in such a way that they transform as the irreducible presentation of the point groups of the system [25]. Next to the experimental possibility to determine the components of the Spin-Hamiltonian (see e.g., [3,17,18,22]), they can be calculated by quantum mechanical means on using electronic state (or wave) functions. 2.1. Basic Principle of ESR interaction with 0 B  (Zeeman effect) interaction with 1 B  h ν = γ β B properties of paramagnetic particles (spin, orbits which can be described by parameters independent of time properties of paramagnetic particles depending on m, T, p, and t paramagnetic particles S ≠ 0 (I ≠ 0) ESR experiment ESR spectrum models isotope effects f(I, γN, μN, nuclear volume) spin-spin interaction static Jahn-Teller effect Spin-Hamiltonian ⋅ zero point energy ⋅ phonon effects ⋅ dynamic Jahn-Teller effect ⋅ orbital-lattice interaction ⋅ thermal modulation of the crystal field and the zfs ⋅ molecular reorientation ⋅ spin relaxation ⋅ tunnel effect; spin exchange ⋅ chemical reaction isotope effects f(m, (T, t) …) equations of motion [ ] B dt d H t i     ∧ = Ψ =      Ψ μ γ μ ϑ ϑ , Matrix (all states of aggregation) Characterization of the paramagnetic system with respect to dynamics, to the mechanisms of its chemical reactions, to the spin density distributions in ground and excited states, to local symmetry and to the mechanisms of couplings of the electron spin with the degrees of freedom of the matrix information properties of paramagnetic particles depending on m, T, p, and t properties of paramagnetic particles (spin, orbits which can be described by parameters independent of time Matrix (all states of aggregation) paramagnetic particles S ≠ 0 (I ≠ 0) spin-spin interaction static Jahn-Teller effect interaction with 0 B  (Zeeman effect) interaction with 1 B  h ν = γ β B Spin-Hamiltonian models information Characterization of the paramagnetic system with respect to dynamics, to the mechanisms of its chemical reactions, to the spin density distributions in ground and excited states, to local symmetry and to the mechanisms of couplings of the electron spin with the degrees of freedom of the matrix Molecules 2013, 18 Which effects or processes explicitly involve spins or which observables are produced by the spin? There are two more items to be named: (i) Taking into account the Pauli principle in form of quantum numbers inclusive the spin conservation, e.g., in chemical reactions, during Pauli repulsion (see Section 3.8.11.), and finally in anti-symmetry of the electronic wave function. Due to the vector character of the spin the couplings with the vector components of electric and magnetic fields are to represent by coupling matrices with tensor character. If these matrices are determined (gˆ , Aˆ ,Tˆ , …, see [3,27]), important information about structure and dynamics of the paramagnetic species can be gained on using further models (see Sections 2.8. and 2.9. and Figure 1). Simplified the spin can be considered to be a further indicator of the total state (wave) function [17,18]. Contrary to the indistinguishable electrons having only one spin quantum number S = ± ½ (see also [17,28]), the nuclei including the isotopes are described by a considerable number of nuclear spins and the corresponding magnetic dipole and electric quadrupole moments. Together with this a large number of applications exist which are based on nuclear moments. To name only the most important one: nuclear magnetic resonance (NMR). Which effects or processes explicitly involve spins or which observables are produced by the spin? There are two more items to be named: (ii) The electron-spin interaction leads to a splitting of the originally degenerated energy levels and the transitions between them represent the fine structure (see also Section 2.3.). (ii) The electron-spin interaction leads to a splitting of the originally degenerated energy levels and the transitions between them represent the fine structure (see also Section 2.3.). (iii) It is not always necessary to solve the complete many-electrons equation of motion (e.g., the complete Schrödinger equation) in order to achieve an acceptable adaption to the experimental fine structure. In most of theses cases the effects of spins are essential and a Spin-Hamilton operator can be established which is (only) a function of the spin operators [17,18]. Thus, the Spin-Hamilton operator connects theory and many of the spin depending experimental results. By means of ESR one could contribute to answering basic questions in physics, chemistry, geology, archaeology, biology, and medicine [3,29]. Molecules 2013, 18 As mentioned above the Spin-Hamiltonian described in such a way does not depend on m, T, p, n (n: Amount of substance), …, phonons, etc. It is assumed, that on applying that spin-Hamilton model the atomic nuclei are “fixed”. On doing so, vibronic effects cannot be considered explicitly, despite the fact that they may change experimental spectra considerably. This is true, e.g., for the Jahn-Teller effect [25], which in ESR spectroscopy is manifested in vibronic reduction, tunnel splitting, random- strain, and relaxation effects, and causes e.g., a distinctive dependency of fine and hyperfine structure on the temperature. These phenomena get eye-catching e.g., on observing the un-common paramagnetic resonance properties of Cu(II) compounds [25,26]. A qualitatively correct interpretation Molecules 2013, 18 Molecules 2013, 18 6684 of the respective spectra can be achieved as a function of the Jahn-Teller dynamics of the nuclear configuration (and therefore the nuclear masses, too [25]). Therefore there is a necessity for a considerable extension of the original Spin-Hamilton operator for the interpretation of real ESR spectra, including the consideration of isotope effects. Before that a deeper understanding about the phenomena and properties of electrons and nuclear spins should be developed. Under aspects of applications, electron and nuclear spins have to be considered as follows: (i) Taking into account the Pauli principle in form of quantum numbers inclusive the spin conservation, e.g., in chemical reactions, during Pauli repulsion (see Section 3.8.11.), and finally in anti-symmetry of the electronic wave function. Due to the vector character of the spin the couplings with the vector components of electric and magnetic fields are to represent by coupling matrices with tensor character. If these matrices are determined (gˆ , Aˆ ,Tˆ , …, see [3,27]), important information about structure and dynamics of the paramagnetic species can be gained on using further models (see Sections 2.8. and 2.9. and Figure 1). Simplified the spin can be considered to be a further indicator of the total state (wave) function [17,18]. Contrary to the indistinguishable electrons having only one spin quantum number S = ± ½ (see also [17,28]), the nuclei including the isotopes are described by a considerable number of nuclear spins and the corresponding magnetic dipole and electric quadrupole moments. Together with this a large number of applications exist which are based on nuclear moments. To name only the most important one: nuclear magnetic resonance (NMR). Molecules 2013, 18 That means in particular, the detection and characterization of paramagnetic states including their surrounding in all states of aggregation, mainly in fluid systems, crystalline and amorphous insulators and semiconductors. The majority of the up to now presented papers is related ground state phenomena, but important and in part unique information about optically excited states (e.g., triplet states [3,27]) can be obtained, too. The application of spin probes allows the characterization of chemically and biologically important systems concerning their structures, dynamics, and function [6,7]. It has to be emphasized, that investigations of the mechanisms of photosynthesis, other biochemical, as well as chemical reactions have been carried out on using the techniques of spin trapping and spin polarization [6,7,30]. Unique contributions to the consequences of the Jahn-Teller [25,31] and tunnel effects [32,33] could be provided by ESR spectroscopy. The phenomenon of spin exchange [34,35] is an important field of ESR because it not only allows the characterization of the matrix but also the differentiation between chemically reactive and non-reactive collision of particles. Molecules 2013, 18 Molecules 2013, 18 6685 Last but not least, ESR delivered proofs for basic theories in physics and chemistry, as well as for the existence of quantum effects (see e.g., Pauli principle, Hund’s rule, e.g., the quartet ground state of N, exchange interactions [36]). For that the usage of isotopes plays an important role which should not be underestimated (see Sections 3.3.2. and 3.8.1.1.). The technical progress contributed much to raise the information content of ESR spectra mainly through higher sensitivity, the usage of non-stationary methods (e.g., pulse methods [14,15]), multiple frequencies, multi-dimensional procedures, imaging, and finally the complete field of chemical means for spectra interpretation (use of isotopes, introducing steric hindrances, trapping techniques). Nonetheless, the usage of isotopes remains of great importance for improving the spectral assignments despite the latest technical progress in electronic equipment and the development of (new and improved mathematical) models, and there is no sign for a replacement of isotope usage. This will be clear on looking e.g., at the important element oxygen. The direct ESR detection of the paramagnetic molecule is still difficult [3]. In compounds it can be observed only in case of its isotope 17O (I = 5/2, 0.0373% natural abundance) directly. Other spectroscopic methods (NMR, MS, IR, etc.) have to use isotopic patterns for a doubtless interpretation of their results, too. It should be mentioned here, that the usage of isotopes as replacement of the naturally occurring atoms is expensive. Moreover, the synthesis of isotopically labelled compounds requires high technical skills. 2.2. Parameters of the Spin-Hamiltonian 2.2.2. The Hyperfine Interaction 2.2.2. The Hyperfine Interaction 2.2.2. The Hyperfine Interaction 2.2.2.1. Nuclear Spin, Nuclear Magnetic Moment, Hyperfine Structure, and General Relations to Isotope Effects 2.2.2.1. Nuclear Spin, Nuclear Magnetic Moment, Hyperfine Structure, and General Relations to Isotope Effects Due to the fact that the nucleus can be considered to be an electromechanic system it should have electromagnetic moments. The first hypotheses concerning a nuclear spin and the nuclear moments have been made by Allen in 1915, Oxlay in 1921, Pauli in 1924, and others, while Pauli considered the nuclear spin to be reason for the hyperfine structure of optical spectra already [40]. First experimental results came from Back and Goudsmit [41] based in particular on their observations of the bismuth nucleus (see also [28]). A first value for the nuclear magnetic moment (7Li) was delivered by Breit and Doerman [42] and Jackson [43] tried to measure the nuclear moment of 133Cs directly by means of optical spectroscopy (it is interesting that starting from 1963 the transition between two hyperfine levels of that isotope serves as the unit of time; see also [44]). In general, by improving the resolution of the atomic spectra it could be shown, that the fine structure again splits (e.g., the further splitting of the doublet of the yellow sodium line by the nuclear spin of sodium) and the originally four quantum numbers of the electronic states (n, l, m, s [45–47]) are not sufficient to explain that phenomenon. A nuclear spin had to be assigned to the respective nuclei, too. It turned out that in case of isotope effects the charge distribution in the nucleus is no longer spherically symmetric; the nucleus-energy level is different for different isotopes. Moreover, another isotope effect comes along; the mass dependence of the Rydberg constant [46,47]. That dependency results from a small ‘joint movement of the nucleus’ due to the movement of the surrounding electrons. That effect is largest with the lightweight elements and it was used by Urey [48] for discovering 2H (D). Comparably accurate (and unambiguous) values for the nuclear moments were given by Rabi [49] by means of atomic beam method. Today the magnetic nuclear moments of all known isotopes have been determined with adequate accuracy [1,50]. 2.2. Parameters of the Spin-Hamiltonian 2.2.1. The gˆ Tensor, the Interaction of Spin and Orbital Moments with the External Magnetic Induction 0 B  2.2.1. The gˆ Tensor, the Interaction of Spin and Orbital Moments with the External Magnetic Induction 0 B  The electronic gˆ tensor represents the coupling of the components magnetic induction B  and electron spin S  [see Equation (2)]. With respect to its transformation behaviour that coupling element (coupling matrix) is a tensor object. It mainly determines the Zeeman term: HZeeman = S gˆ B   β (2) (2) Under a physical aspect, gˆ is determined by the orbital momentum L  (see quenching of orbital moments in molecular systems [3,17,18,23,24]) as well as the kind and energy levels of the excited electronic terms. The frequently determined “mean value” of gˆ mainly inform about the nature of the participating heavy atoms in the paramagnetic centre. For free electrons a value of ge = 2.0023 was determined, while nitroxyl spin probes have value around 2.05 [6,7,37], and for heavy ion like Cu2+ it will be around 2.2 [38]. In comparison to NMR the large difference in mass between the electron and the nucleus is obvious and results in chemical shifts of some ppm in NMR, whereas it might be in the percentage range in ESR. The difference between the variation range of the g-factor and the NMR chemical shift is based on different facts: The magnetic moment is indirect proportional to the mass whereas the chemical shift is the result of the interactions with the electron shell. This interaction is dominated by electron states with non-zero orbital moments in the case of the g-factor and is much smaller for the shielding Molecules 2013, 18 6686 effect of the nuclei because the effects of higher electronic states reach the nuclei only by spin polarization effects. But it should be mentioned that the isotopic influence on the gˆ tensor is relatively small (see Section 3.2.). The nuclear gˆ factor is determined by the respective nuclear properties and is specific for any nuclear paramagnetic isotope [1,39]. 2.2.2. The Hyperfine Interaction In general the nuclear moment is a function of the spin moment and the orbital moment of the nucleus, whereas the nuclear spin and the integral orbital angular momentum contribute to the total angular momentum of the nucleus. The magnetic dipoles of the nuclei contribute to the following phenomena [50]: (i) hyperfine structure and magnetic splitting of the hyperfine structure in atomic and ESR spectra [3]; (i) hyperfine structure and magnetic splitting of the hyperfine structure in atomic and ESR spectra [3]; (ii) band spectra of homonuclear molecules [1]; (ii) band spectra of homonuclear molecules [1]; (iii) magnetic deflection and resonance with atomic and molecular beams [51]; (iv) resonance and relaxation effects of the nuclear paramagnetism [22]; (v) hyperfine structure of the microwave spectroscopy [52]. Molecules 2013, 18 Molecules 2013, 18 6687 rfine structure is caused by two phenomena: (i) interaction of the electrons with nuclear moments (magnetic dipole and electric quadrupole moments), (ii) isotopic shifts in the atomic spectra, i.e., a splitting in isotopic mixture occur; there are no effects with individual atoms. (ii) isotopic shifts in the atomic spectra, i.e., a splitting in isotopic mixture occur; there are no effects with individual atoms. The hyperfine structure is usually around 103 smaller than the fine structure (see Sections 2.3.and 3.4.). Different values of the energy of magnetic interaction correspond to the values of the different mutual orientations of electron and nuclear spin. The direct determination of the nuclear moments of atoms with unpaired electrons is challenging due to the strong magnetic field at the nucleus caused by these electrons. On the other hand this is advantageous, e.g., to obtain sensitively detailed information about the paramagnetism of the respective sample by means of the magnetic resonance in the GHz range [50,53–57]. Nevertheless it has to be considered that exchange interactions [36] average out the hyperfine structure on magnetically undiluted samples [51]. If the nucleus of an atom in a paramagnetic system has a magnetic moment a magnetic field of some mT will result that acts upon the electrons. The magnitude of the field depends on the orientation of the nucleus, which is usually fixed during electronic transitions. Therefore any transition (or the respective spectral line) will split in 2 I + 1 components. 2.2.2. The Hyperfine Interaction It has to be mentioned that the field strength of ESR at about 10 GHz is about 0.3 T and does not have a strong influence on the orientation of the nuclear moments. Thus the nuclear magnetic moment or the nuclear spin of the respective nucleus can be identified. Gorter [58] developed the hypothesis that beside 0 B  the nuclear magnetic moment plays a role on splitting the ground state of paramagnetic systems. At the nucleus that field should have a magnitude of some 10,000 mT and should cause a hyperfine splitting larger than 0.01 cm−1. Penrose [59] delivered the first successful experimental proof of the ESR hyperfine structure. The coupling constant of the hyperfine structure of paramagnetic systems is one of the most important parameters in ESR and from the very beginning it is connected to the different action of isotopes (e.g., 63Cu and 65Cu) [59–62]. The hyperfine structure of paramagnetic systems allows the determination of the nuclear spin simply and directly, as well as the relation of the magnetic moments of two isotopes of the same atom. After Penrose’s discovery the nuclear magnetic moments could be determined by means of ESR as there are 53Cr [63], 143Nd and 145Nd [64], 159Tb [65], 165Ho [66], 56Co and 57Co [67], 235U [68], 63Cu and 65Cu [62], and 60Co [69] (see also [70,71]). After Penrose’s discovery the nuclear magnetic moments could be determined by means of ESR as there are 53Cr [63], 143Nd and 145Nd [64], 159Tb [65], 165Ho [66], 56Co and 57Co [67], 235U [68], 63Cu and 65Cu [62], and 60Co [69] (see also [70,71]). Another milestone in the sequence of discoveries derived from the hyperfine structure had been the proof of hyperfine interactions in the system Na2[Ir(IV)Pt]Cl6 H2O [72,73]. The careful analysis of the ESR spectra not only revealed the hyperfine contribution which results from the interaction of the unpaired d-electrons and the nuclear moments of the iridium isotopes 191Ir (I = 3/2) and 193Ir (I = 3/2), but the interaction with the ligand atoms 35Cl (I = 3/2) and 37Cl (I = 3/2), too. That had been the proof that unpaired electrons are not only located at the central atom. In the following the models of chemical bonding had been refined by assigning covalent contributions [55] (see also [74]). 2.2.2. The Hyperfine Interaction In general the analysis of hyperfine structures, in particular those of organic radicals and radical ions [75], significantly contributed to the understanding of the phenomena of delocalization of electron and spin densities in molecules and their (quantum mechanical) interpretation [3,18,19]. Spin density Molecules 2013, 18 Molecules 2013, 18 6688 as the difference of electron densities with α and β spin [18] can also have negative values contrary to electron densities. Further on, by means of the hyperfine findings the spin and exchange polarizing action of unpaired valence electrons or outer shell d and f states (i.e., electrons whose magnetization disappears formally near the nucleus) onto the 1s states and, thus, the hyperfine fields at the nucleus could be discussed [18,19,76]. Actual ideas concerning the chemical bond are given in [74]. Real atomic nuclei have a finite mass, a charge, which is distributed over a finite volume, and (possibly) a nuclear spin I different from zero. Due to that magnetic dipole moments and electric quadrupole moments result, which couple to an overall moment. That property influences the atomic energy level slightly (about 10−5 Rydberg [45–47]) but measurably. The finite mass leads to a “mass shift” and the finite volume for the charge to a “volume shift” (some ppm) of the spectral lines, which at best can be observed due to their isotopic shifts. With that elements with atomic numbers <40 deliver a positive shift and elements >60 negative shifts. The mass shifts give positive and the shifts in nuclear volume negative contributions [32,33,76]. On the other hand the magnetic dipole and the electric quadrupole moments lead to hyperfine structure, which is based on the respective splitting of the energy levels. Today the importance of the coupling of magnetic moments and the surrounding unpaired electrons goes beyond the determination of nuclear moments or structural and dynamic details of atomic und molecular states, respectively. Due to the influence of the hyperfine structure onto the optical selection rules and the transfer of moments, e.g., from light to atoms, laser experiments allow the manipulation of atoms by means of light. The hyperfine structure is of greatest importance in the case of free and bound atoms which have unpaired electrons. Particularly via these interactions several isotope effects can be detected or used, respectively. where m: mass of the electron; M: mass of the nucleus where m: mass of the electron; M: mass of the nucleus Particularly for spectra of systems resembling that of hydrogen, the mass dependent isotope effect may be interpreted considering the reduced mass K e K e m m m m + ⋅ = μ instead of the real mass (9.109 × 10−31 kg). In a descriptive way this can be explained by a joint movement of the nucleus (proton mass: 1.672·× 10−27 kg, muonium mass: 9.106·× 10−31 kg. (9.109 × 10−31 kg). In a descriptive way this can be explained by a joint movement of the nucleus (proton mass: 1.672·× 10−27 kg, muonium mass: 9.106·× 10−31 kg. The influence of the nuclear volume on the hyperfine interaction will be explained in the following sub-chapter (under “static (stationary) hfs”) and [77]. The influence of the nuclear volume on the hyperfine interaction will be explained in the following sub-chapter (under “static (stationary) hfs”) and [77]. 2.2.2. The Hyperfine Interaction According to the formula for the Bohr energy levels the energy increases in the state n with increasing mass:    − = M m 1 E E 0 n n (3) (3) Molecules 2013, 18 Molecules 2013, 18 6689 This interaction involves electronic as well as nuclear properties. In the simplest case the splitting a depends on the nuclear parameters gN (nuclear g factor), βN (nuclear magneton), and the spin density \|Ψ(0)\|2 at the nucleus: This interaction involves electronic as well as nuclear properties. In the simplest case the splitting a depends on the nuclear parameters gN (nuclear g factor), βN (nuclear magneton), and the spin density \|Ψ(0)\|2 at the nucleus: 2 N N 0 ) 0 ( g 3 2 a Ψ β μ = (4) (4) where μ0: magnetic field constant. By means of a well-known pair of isotopomers, hydrogen 1H and deuterium D or 2H, the differences in hyperfine coupling (hfc) based on the nuclear constants (Table 1) shall be shown. Table 1. Selected physical constants of 1H and 2H. 1H 2H nuclear magnetic moment μH = gN βN I = 1.4106 × 10−26 J T−1 μD = gN βN I = 1.4106·× 10−26 J T−1 nuclear spin I = ½ I = 1 nuclear g factor gH = 5.5856 gD = 0.8574 gyromagnetic ratio γH = μH/I  = 2.6752 × 108 s−1 T−1 γD = μD/I  = 4.1067 × 107 s−1 T−1 Table 1. Selected physical constants of 1H and 2H. In a good approximation the ratio γi/γj for two isotopomers i and j can be obtained via the ratio of the experimental hyperfine coupling constants ai and aj. E.g., from the experimental ratio aH/aD = 50.685/7.780 = 6.514 follows γH/γD = 6.514. In a good approximation the ratio γi/γj for two isotopomers i and j can be obtained via the ratio of the experimental hyperfine coupling constants ai and aj. E.g., from the experimental ratio aH/aD = 50.685/7.780 = 6.514 follows γH/γD = 6.514. Generally that ratio may be larger/smaller than 1 depending on the degrees of freedom of the respective paramagnetic molecule. In particular the zero-point vibration delivers about details of the structure, e.g., the distribution of the spin density. 2.2.2.1.2. Static and Dynamic Aspects of the Hyperfine Interaction 2.2.2.1.1. Phenomena of Magnetic Hyperfine Structure Exemplified on the Pair of Isotopomers 1H and 2H 2.2.2.1.1. Phenomena of Magnetic Hyperfine Structure Exemplified on the Pair of Isotopomers 1H and 2H 2.2.2.1.1. Phenomena of Magnetic Hyperfine Structure Exemplified on the Pair of Isotopomers 1H and 2H As mentioned above the hyperfine structure (hfs) [78] is one of the most important parameters in ESR spectroscopy. Due to the interactions of electron spin S  and nuclear spin I  energy levels of the electron split. 2.3. The Zero-Field Splitting By means of ESR the phenomenon zero-field splitting (zfs) [3,27] can be observed with systems with S≥1; i.e., there will be a splitting of the originally degenerated spin states (in spherical symmetry) already in the absence of an outer magnetic field (B0 = 0) due to internal magnetic (dipole and spin orbit) interactions. These splittings, giving the ESR fine structure, react sensitively on very small changes of the symmetry of the electric field (e.g., of a coordination polyhedron) in which the unpaired spins are located (see also [82]): ) O b O b ( 60 1 O b 3 1 S gˆ B H 3 4 3 4 0 4 0 4 0 2 0 2 B + + + μ =   (5) (5) The corresponding spin-Hamilton operator i j S TS  , (collecting magnetic dipolar and exchange spin-spin interactions) which is usually used for the parametrization of the ESR fine structure [Equation (5)], does not contain a mass depending term. Thus, for the description of the respective influence of isotopes the interaction with phonons have to be considered, e.g., in form of the spin-phonon operators [29,83] as it was shown by Shrivastava [84] for the system 52Cr, 53Cr in the system MgO:Cr3+. (ii) Dynamic hfs effects: These effects can be recognized easily by means of the (mostly temperature and concentration depending) changes of the line width of the cw spectra. More detailed information can be obtained by non-stationary ESR techniques (see e.g., [14]). These effects are caused by e.g., spin-rotation interactions, molecular reorientation diffusion [6,7], dynamic polarization of S  and I  [30,34,35], spin exchange [35], dynamic Jahn-Teller effect [25], tunnel effect [33,80], vibronic coupling, and others. Under a dynamic aspect the isotope effects act upon the zero-point energies by vibrational averaging with amplitudes depending on isotopes. Morton and Preston used this effect for investigating the hyperfine structure of F2NO [81]. (i) Static (stationary) hfs: Within the respective ESR spectrum the following hyperfine phenomena can be observed: isotropic and anisotropic (Fermi contact and dipolar) contributions [17,18], number of lines and ratio of intensities (as function of the number of involved atomic nuclei and their nuclear spins), and values (and sign, respectively) of the hyperfine coupling constants. The stationary hfs is an important mean to identify the kind of paramagnetic species. This is mainly related to stable (or stabilized) radicals in solutions and the solid state, paramagnetic defects in the solid state, in transition metal ions in chemical and biochemical processes, as well as in optically excited electronic states. Then the influence of the isotopes is here mainly related to differences in the nuclear moment μN, the nuclear spin I, and the nuclear volume. The importance of the nuclear volume for the hyperfine interaction results from the following considerations: The electrostatic potential inside a nucleus is reduced and does not behave like Ze/r. If the valence electrons penetrate into this region, especially the s electrons, then their energy will become larger because of the reduced potential. In general the charge distribution is spread out if neutrons are added to a nucleus because the energy is raised further. Therefore a reduction of the binding energy results and the transition energy is decreased. It means a negative mass shift can be observed if the s electrons represent the lower energy state at the transition [77]. The resulting volume correction is given by Sobel’man et al. [79]. Moreover, for Molecules 2013, 18 Molecules 2013, 18 6690 reducing the line width, caused by magnetic dipole interactions with atoms of the matrix, the isotopic substitution may be favourable, too (e.g., exchange of hydrogen by deuterium in K2Cu(SO4)2·H2O). 2.4. Spin Relaxation Spin relaxation processes generally belong to the family of processes in which a retarded reaction of the system proceeds after the end of an outer action. That means for spin systems, a process in an (originally) magnetically ordered system returns in a state of equilibrium. E.g., experiments on adiabatic magnetic cooling have shown that a sudden process of demagnetization in an ensemble of electrons (S = 1/2) in a crystal lattice leads to an increase of the entropy ΔS. In other words: that ΔS takes up a certain part of the thermal energy of the lattice vibrations produced via interactions with the spins. In this connection, it is of interest to know how much time it takes for that exchange of thermal energy [4] after the thermal contact of the ensemble of “electronic magnets” to the degrees of freedom of the vibrations of the recent surrounding. That process will be named the spin-lattice relaxation, while the phonon system (elementary excitation of lattices oscillations) plays an important role [29,83,84]. Under a mechanistic aspect the interaction proceeds via modulation of the electric field due to the Molecules 2013, 18 Molecules 2013, 18 6691 vibration of the neighbours with the consequence of the modulation of “orbital movement”. These processes depend on the mass of the involved atoms, and therefore give rise to isotope effects in ESR. If one considers the spin-lattice relaxation with a time constant T1 (or other models [3,4]) two important mechanisms have to be kept in mind; (i) direct and (ii) indirect (Raman) processes (Figure 3). In process (i) a spin of mS = +1/2 and the energy E = ½ gβB0 changes into the state mS = −1/2 and the energy E = −½ gβB0 under emission of a phonon of the frequency ν = h β gB0. Process (ii) is characterized by the action of a phonon of the energy hν’ on a spin with mS = +1/2 and E = ½ gβB0 and in a non-elastic scattering process the spin state changes to mS = −1/2 and energy E = −½ gβB0 under emission of a phonon with the energy hν” The following energy results: hν’ + gβB0 = hν” [4] If one considers the spin-lattice relaxation with a time constant T1 (or other models [3,4]) two important mechanisms have to be kept in mind; (i) direct and (ii) indirect (Raman) processes (Figure 3). 2.4. Spin Relaxation In process (i) a spin of mS = +1/2 and the energy E = ½ gβB0 changes into the state mS = −1/2 and the energy E = −½ gβB0 under emission of a phonon of the frequency ν = h β gB0. Process (ii) is characterized by the action of a phonon of the energy hν’ on a spin with mS = +1/2 and E = ½ gβB0 and in a non-elastic scattering process the spin state changes to mS = −1/2 and energy E = −½ gβB0 under emission of a phonon with the energy hν”. The following energy results: hν’ + gβB0 = hν” [4]. Figure 3. Mechanisms of spin-lattice relaxation. 2.5. Spin Exchange On colliding of atoms or molecules with incompletely occupied electronic states (unpaired electrons), respectively, the process of spin exchange may occur [35]: On colliding of atoms or molecules with incompletely occupied electronic states (unpaired electrons), respectively, the process of spin exchange may occur [35]: A(↑) + B(↓) Ý A(↓) + B(↑) (6) (6) In doing so, the hyperfine state of the colliding particles will change [32]. Spin exchange can be considered as an example of tunnelling exchange of electrons (see 2.6.). It is a process where electrons of two atoms (or molecules) are exchanged via simultaneous tunnelling in opposite directions. E.g., if one has a look onto the spin exchange of the collision of two particles with 1s electrons (e.g., hydrogen atoms), the wave function of the particles with opposite spins (until collision) can be written as follows: ψ (t→−∞) = (α1 β2 ϕa (1) ϕb (2) − α2 β1 ϕb (1) ϕa (2))/ 2 . (7) (7) On this ϕa and ϕb stand for the atomic orbitals of the two colliding atoms and α and β for the spin functions. On this ϕa and ϕb stand for the atomic orbitals of the two colliding atoms and α and β for the spin functions. The corresponding time-dependent Schrödinger equation supplies the following equation: The corresponding time-dependent Schrödinger equation supplies the following equation: ψ± (t) = ((ψ+ exp (−i  ∞ − + t U (τ) dτ)) − ( ψ− exp (−i  ∞ − − t U (τ) dτ))) / 2 (8) (8) where U+, U−: energy of the symmetric and antisymmetric states and ψ±: symmetric and antisymmetric wave function, which in case of large distances can be written as: ψ± = ((α1 β2  α2 β1) ( ϕa (1) ϕb (2) ± ϕa (2) ϕb (1))/ 2 (9) (9) Formally the spin exchange and resonance charge transfer coincide [32]. Formally the spin exchange and resonance charge transfer coincide [32]. Figure 3. Mechanisms of spin-lattice relaxation. Figure 3. Mechanisms of spin-lattice relaxation. Figure 3. Mechanisms of spin-lattice relaxation. The paramagnetic relaxation is mainly determined by the spin-phonon interactions in paramagnetic crystals [85,86]. The oscillations of the neighbouring atoms and atomic groups, respectively, lead to the modulation of the orbital moments of the unpaired electrons and, thus, to the induction of spin transitions via spin-orbit couplings. The respective theoretical basic ideas have been developed by Waller in 1932 [21] already. An example for the influence of isotope marking, i.e., for the shift of the fine structure parameters, has been given by Shrivastava [84] by means of 52Cr and 53Cr doted MgO. Based on an effective spin-phonon operator he was successful in a correct interpretation of observed isotope effects. Within the Bloch model the second kind of spin relaxation is named transversal relax Differently to the longitudinal relaxation, which is characterized by releasing energy to the surrounding matrix (e.g., crystal lattice), spin-spin relaxation means the exchange of energy by spins of similar type. This causes the homogenization of the temperature of a spin system within a characteristic period of time T2. E.g., a nucleus which returns from the excited spin state releases energy to a neighbouring nucleus which gets excited then. With this only the phase relation of the participating spins changes (for applications see Section 3.8.2.1). Molecules 2013, 18 Molecules 2013, 18 6692 2.6. The Magnetic Isotope Effect Isotope effects, as they are usually observed on chemically kinetic examinations, are linked to changes in the nuclear mass (and, thus, to the molecular mass), to changes of the moments of inertias, and on shifts of the oscillation frequency. The theory of kinetic isotope effects [1] yields in some cases quantitative descriptions of averaged energies of oscillation of molecules and activated complexes. In contrast to this the magnetic isotope effect is based on differences in the magnetic properties of isotopes which cause the change of the singlet-triplet evolution in radical pairs. Usually the hyperfine energies of different isotope nuclei differ (see Section 2.2.1.1.) and, thus, there are different efficiencies for recombinations of radical pairs, which contain these isotopes. E.g., the hyperfine coupling constants of 1H and 2H differ by factor 6.514 (see [3]). The following will be favourable combinations for isotope pairs for the observation of this isotope effect: 1H – 2H, 12C – 13C, 16O − 17O. Moreover, in the cases of 12C and 16O it is advantageous that these isotopes do not have nuclear magnetic moments and hence the paramagnetic resonance spectra do not superimpose. According to [30] there are four remarkable findings, which make the difference between the magnetic isotope effect and the common kinetic isotope effect, which results due to differences in the mass of the isotopes [1]: Molecules 2013, 18 6693 (i) Contrary to the classical isotope effect the magnetic isotope effect only occurs in reactions which involve the formation or recombination of radical pairs. (i) Contrary to the classical isotope effect the magnetic isotope effect only occurs in reactions which involve the formation or recombination of radical pairs. (i) Contrary to the classical isotope effect the magnetic isotope effect only occurs in reactions which involve the formation or recombination of radical pairs. (i) Contrary to the classical isotope effect the magnetic isotope effect only occurs in reactions which involve the formation or recombination of radical pairs. (ii) The magnetic isotope effect may achieve the dimension of some per cent, even for the heavy nuclei 13C, 15N, 17C, etc., while the classical kinetic effect is usually smaller. (ii) The magnetic isotope effect may achieve the dimension of some per cent, even for the heavy nuclei 13C, 15N, 17C, etc., while the classical kinetic effect is usually smaller. 2.6. The Magnetic Isotope Effect (iii) The classical effect does not depend on the outer magnetic field, while the magnetic effect shows a clear dependency on B  . It decreases with increasing 0 B  . In this case the triplet states [3,27] T+ and T− of the singulet-triplet evolution are separated and only the S-T0 channel is active. At lower fields all three channels contribute. (iv) The magnetic isotope effect depends on the hyperfine energy, the spin, the magnetic moments of the nuclei, and parameters which determine the molecular radical pair dynamics, such as the solvent viscosity, radical diffusion coefficients, etc. Beside the named kinetic-mechanistic examinations it will be possible to separate the isotopes and to determine the magnetic moments of nuclei in excited states. 2.7. Tunnel Effect Phenomenon and Its Relation to ESR and to Isotope Substitution The passage of a particle through a potential barrier can occur even if the kinetic energy of the particle is smaller than the height of the barrier. The possibility of a tunnel event decreases rapidly with increasing width of the barrier and decreasing energy of the particle. It is a typical wave phenomenon [32,33,45], whose explanation is delivered by quantum mechanics: The particle (•) “tunnels” the barrier (Figure 4). ransfer of an electron can be described as follows [32]: The tunnel transfer of an electron can be described as follows [32]: A− + B ↔ A + B− (10) (10) where A: Donator, B: Acceptor, and its respective state (wave) function ψ is: eptor, and its respective state (wave) function ψ is: where A: Donator, B: Acceptor, and its respective state (wave) function ψ is: ψ ∝ exp (−γ r) (11) (11) where γ: coefficient of the decay of ψ with distance r where γ: coefficient of the decay of ψ with distance r The probability W of the tunnel effect in a unit of time is proportional to ψ2 at the donator near the acceptor: W(r) = ν exp (−γ r) (12) (12) ν: frequency factor The tunnel effect is not only of importance on the decay of radioactive atoms, but for the kinetics of low-temperature reactions with the transfer of atoms [88], e.g., of H+, H•, and H−, respectively, for biologically relevant processes [1], for the field of spectroscopy, for processes at electrodes, solid state phenomena, for the visualization of small and smallest particles (e.g., tunnel microscopy). The temperature dependence is another characteristic of the tunnel effect. While at elevated temperatures nearly Arrhenius behaviour results, at lower temperatures quantum effects dominate the movement of the nuclei and the temperature dependency decreases (double minima potentials). The NH3 molecule is an example for this. The double minimum potential is indicated by two equilibrium configurations. And the effect of tunnelling is manifested by doubling every energy level into two levels with a difference according to the tunnel frequency. Some the empiric criteria for the ‘proof’ of the tunnel effect should be named. According to Bell [33] and LeRoy [89] the following could be stated: Some the empiric criteria for the ‘proof’ of the tunnel effect should be named. According to Bell [33] and LeRoy [89] the following could be stated: (i) The velocity of a reaction (process) is (mostly) larger than it is predicted by a semi-classical kinetic treatment. (i) The velocity of a reaction (process) is (mostly) larger than it is predicted by a semi-classical kinetic treatment. (ii) Deviations from the validity of the simple Arrhenius equation are detected, in which the energy of activation decreases with decreasing temperature and at sufficiently low temperature it becomes independent from temperature (e.g., giving strongly bent curves in the Arrhenius plot). Even at smaller effects the Arrhenius parameters will be smaller as predicted by the semi-classical methods. (iii) Isotope effects can be anomalously large as well as comparably small [90–92]. E.g., the activation energy ΔEa for the hydrogen abstraction by •CH3 in CH3CN after γ irradiation at 77 K will be 1.4 kcal, while it is 10.1 kcal in the gas phase at 300 K. The rate for the deuterium abstraction is extremely low for low temperature. The ratio kH/kD at 77 K is about 20 times larger than in the absence of the tunnel effect. ESR is one of the most favourite techniques for the detection of tunnel effects [32,33]. E.g., isomeric radicals show clearly different ESR spectra even if they have ‘identical’ energies. ν: frequency factor ν: frequency factor It is interesting that in general the frequency factor ν does not depend on the temperature and has a dimension of the movement of the electron of about 1016 s−1 [32]. The parameter γ is associated to the ionization potential Ip of the donator: γ2 = 2 mIp/ 2  (13) γ2 = 2 mIp/ 2  (13) m: mass 6694 Molecules 2013, 18 Figure 4. Allegory of the tunnel effect (according to an idea by J. H. Van Vleck cited in [38]) with a classical barrier in Gauss form (particle (•) does not have sufficient energy to get over the barrier) and the probability for tunnelling the barrier (at E’ the probability is 0.5; the dotted line represents the classical course; see also [33]). an electron transfer is considered, it proceeds from one discrete state of the donator to a d of the acceptor. This requires changes in the coordinates of the nuclei, which again determi ies of the discrete states of the electrons. i.e., the frequency factor ν does not characteri If an electron transfer is considered, it proceeds from one discrete state of the donator to a discrete state of the acceptor. This requires changes in the coordinates of the nuclei, which again determine the energies of the discrete states of the electrons. i.e., the frequency factor ν does not characterize the If an electron transfer is considered, it proceeds from one discrete state of the donator to a discrete state of the acceptor. This requires changes in the coordinates of the nuclei, which again determine the energies of the discrete states of the electrons. i.e., the frequency factor ν does not characterize the Molecules 2013, 18 6695 movements of the electrons but those of the nuclei. It is a function of characteristic properties of donator and acceptor, and the medium (matrix) as well. The tunnel effect is not limited to changes of the electron state. It appears at the substitution of isotopes, too. The largest effect will occur in the sequence Mu → 1H, 1H → 2H, etc. (Mu: {μ+e−}, the “light hydrogen atom” (see [8,86,87]). As expected the influence of the isotope substitution decreases with increasing atomic number. 2.8. The Jahn-Teller Effect (JTE) Jahn and Teller [25,31] stated on the basis of group-theoretical considerations in 1937 that a non- linear molecule with a degenerated electronic ground state is instable. It changes its configuration in such a way that the state of degeneration will be eliminated (Exceptions: Linear molecules and molecules with Kramers’ degeneracy; see ions in crystals). Therefore the lifting the degeneracy electron states of a paramagnetic centre in a matrix by distortion of the surrounding lattice is called JTE. At sufficiently strong interactions between the centre and the lattice the surrounding lattice atoms can change their states of equilibrium in such way, that a decrease of the local symmetry results, causing the lifting of the state of degeneration (static JTE). The energy content of the system is lower than in the undistorted state. At weaker interactions the gain in energy by distortion is comparable to the zero-point energy of the lattice vibrations and therefore no stable static distortion next to the centre will be formed. Then the state of degeneration will be terminated by the interaction with phonons (dynamic JTE). Due to that, e.g., interesting averaging effects of ESR parameters as a function of the temperature of the solid occur. 2.9. Zero-Point Vibrations and Isotope Effects The zero-point energy of a system is the energy at absolute zero, i.e., the ground state of a system. With respect to the Heisenberg uncertainty principle [45] the kinetic energy of a system does not disappear at absolute zero. Already the (quantum mechanical) harmonic oscillator (e.g., one dimensional) supplies a frequency spectrum with the energy. En = h ν (n + 1/2) (14) (14) Molecules 2013, 18 Molecules 2013, 18 6696 ν: frequency factor The requirement, that the characteristic time t of the technique for measurement must be small compared to 1/νt (νt: tunnelling frequency), can be fulfilled by the relative wide time window of ESR [3,14,15]. Via a tunnel correction Q value the contribution for the tunnel effect can be quantified [33]. For 1 < Q < 1.1 it can be neglected. For the range 1.1 < Q < 4 a correction is typical for chemical reactions at room temperature. There are quantitative changes only, no new phenomena. There are nearly no chemical reactions at room temperature with large tunnel effects. At low temperature Q might have values over several orders of magnitude and the examined system might behave completely different (see also [93]). n: 0, 1, 2 …. Parameters Determining the Habit of the ESR Spectra (ESR Response) (i) parameters which influence the shape of experimental ESR spectra or which influence the ESR response S: (i) parameters which influence the shape of experimental ESR spectra or which influence the ESR response S: (i) parameters which influence the shape of experimental ESR spectra or which influence the ESR response S: p instrumental parameters with direct relation to the resonance effect: with direct relation to the resonance effect: S = S (νMW, νRF, PM, PRF, 0 B  , ti, Grad Bi, E  , …) (16) S = S (νMW, νRF, PM, PRF, 0 B  , ti, Grad Bi, E  , …) (16) (16) where νMW: microwave frequency (⊥ or \|\| 0 B  ); νRF: radio frequency; PM, PRF: microwave or radio frequency power; 0 B  : outer ‘static’ magnetic induction; ti: different time functions for the variation of the other parameters (cw and impulse techniques); Grad Bi: gradient of one component of 0 B  , e.g., for recording spatially dissolved spectra (ESR tomography); E  : electric field e.g., for interaction with quadrupole moments. where νMW: microwave frequency (⊥ or \|\| 0 B  ); νRF: radio frequency; PM, PRF: microwave or radio frequency power; 0 B  : outer ‘static’ magnetic induction; ti: different time functions for the variation of the other parameters (cw and impulse techniques); Grad Bi: gradient of one component of 0 B  , e.g., for recording spatially dissolved spectra (ESR tomography); E  : electric field e.g., for interaction with quadrupole moments. (ii) parameters which comprise the changes of the structure of paramagnetic species including the interaction within the paramagnetic unit: anisotropies of the spin coupling matrices, exchange interactions (particularly spin exchange parameters as well as exchange interactions within a species at “fixed” geometry, i.e., mainly the parameters of the spin-Hamilton operator (see [18,22])) (remark: the commonly used spin-Hamilton operator is independent of time and mass; thus isotope effects mainly result in the number of lines and intensity of the hfs as well as on the value of the hyperfine coupling). n: 0, 1, 2 …. With this the ground state has the energy: With this the ground state has the energy: E0 = h ν/2 (15) (15) E0 = h ν/2 At T = 0 K the movement of the particles cannot “freeze completely”, due to the fact that the location would be fixed then (Δq = 0) and the impulse p would be zero (Δp = 0; Δq Δp ≥ /2). At T = 0 K the movement of the particles cannot “freeze completely”, due to the fact that the location would be fixed then (Δq = 0) and the impulse p would be zero (Δp = 0; Δq Δp ≥ /2). Zero-point energy effects are responsible for finite rates of reaction at temperatures close to absolute zero. This is true e.g., for hydrogen abstraction and others, whereas there are predictions for large ratios of kH/kD (see e.g., [93]. The phenomenon of zero-point energy and its effect on processes of dissociation in chemistry can be demonstrated easily by means of the dissociation of X-H and X-D fragments. The force constants kdo not differ significantly for both isotopes ( 2 1 2 1 m m m m ; / k 2 + ⋅ = μ μ = πν ), but the reduced mass is smaller for the X-H than for the X-D fragment and consequently the oscillation frequencies νH > νD (in reality the ratio νH/νD is about 0.7) and the zero-point energies behave as E0 H > E0 D [33]. Thus, the dissociation energies are EH > ED (see Figure 5) and the dissociation of the hydrogen component will be faster (there is a smaller activation energy). This is, among others, manifested in the higher pH value of D2O in comparison to H2O at the same temperature. 6697 Molecules 2013, 18 Figure 5. Influence of the zero-point energy on the dissociation of R-H and R-D fragments (E0 H and E0 D: zero-point energies of hydrogen and deuterium; adapted from [33]). Figure 5. Influence of the zero-point energy on the dissociation of R-H and R-D fragments (E0 H and E0 D: zero-point energies of hydrogen and deuterium; adapted from [33]). 2.10. Parameters Determining the Habit of the ESR Spectra (ESR Response) 2.10. Molecules 2013, 18 Molecules 2013, 18 Molecules 2013, 18 6698 (b) preparation of the microscopic state of the sample by changing the electron configuration of the atoms or molecules, respectively, by redox reactions, optical excitation or ionizing radiation (among others there may be internal changes in concentration; it cannot be assumed that the nuclei are “fixed” in all cases, but the respective movements have not to be considered in any case), respectively. (c) dynamics of paramagnetic centres: parameters of the dynamics of the complete paramagnetic centre, i.e., diffusion, reorientation, rotation of molecular fragments, collisions, and spin exchange, isotope effects in the matrix (isotopic dilution, zero-point energy effects, etc.; even here the atomic nuclei can be considered as mainly “fixed” in the centres) (c) dynamics of paramagnetic centres: parameters of the dynamics of the complete paramagnetic centre, i.e., diffusion, reorientation, rotation of molecular fragments, collisions, and spin exchange, isotope effects in the matrix (isotopic dilution, zero-point energy effects, etc.; even here the atomic nuclei can be considered as mainly “fixed” in the centres) (d) parameters of the phenomena which are caused or modified by the movement of nuclei in the paramagnetic species: vibronic couplings, zero-point vibrations, dynamic Jahn-Teller effects, (tunnel effects) … and the respective isotope effects via the mass of the nuclei or anharmonic contributions to the interaction potentials. Table 2. Matrix arrangement of the spin (S  , I  ), orbital ( L  ), and quadrupole moments Q ††, as well as electric (E  ), and magnetic fields ( B  ) ‡‡ for symbolizing the spin couplings (with “fixed” atomic nuclei §§). Molecules 2013, 18 B  S  I  L  Q  E  B  I II II IV V • S  VI VII VIII • • I  IX X • • L  • • • Q  • XI E  * †† nuclear quadrupole moments which interact with electric and magnetic fields; ‡‡ in form of the magnetic induction; §§ • stands for small terms (see [87,94] within the total Hamilton operator, which are time and mass independent and do not contribute much to the ESR response; I: diamagnetism 1st and 2nd order of perturbation theory; II: Zeeman term HZ with coupling parameter e gˆ ; III: nuclear Zeeman term HN with coupling parameter N gˆ ; IV: Zeeman term HL with coupling parameter L gˆ ; V: nuclear quadrupole interaction with B  ; VI: spin-spin interaction HSS with coupling parameter Tˆ (ESR fine structure) or J, respectively; VII: ESR hyperfine interactions, Fermi-contact interactions, dipolar hyperfine splitting; VIII: spin-orbit coupling with parameter λ; IX: nuclear spin-nuclear spin interaction Jij; X: nuclear spin-orbit interaction; XI: nuclear quadrupole-E  interaction; * these interactions will not be considered and explained. n: 0, 1, 2 …. (ii) parameters which comprise the changes of the structure of paramagnetic species including the interaction within the paramagnetic unit: anisotropies of the spin coupling matrices, exchange interactions (particularly spin exchange parameters as well as exchange interactions within a species at “fixed” geometry, i.e., mainly the parameters of the spin-Hamilton operator (see [18,22])) (remark: the commonly used spin-Hamilton operator is independent of time and mass; thus isotope effects mainly result in the number of lines and intensity of the hfs as well as on the value of the hyperfine coupling). (iii) parameters which determine the change of the macroscopic state and the structure of the complete sample. In principle there many different kinds of influences which have to considered and which act upon the thermal, mechanical, electrical, optical, magnetic, and chemical properties of substances and their coupling with the spin properties (i.e., it is related to the coupling of the electron spins with all accessible degrees of freedom of the recent sample). (a) preparation of the macroscopic state of the sample by variation of T, p, v, E  , H  , ci, … (ci: concentration of components which, e.g., determine the pH value or the concentration of educts or products of chemical reactions); E  is responsible for polarizations or chemical reactions (e.g., electrode reactions; see below); the state of magnetization of the sample may by varied by H  of different origins. (a) preparation of the macroscopic state of the sample by variation of T, p, v, E  , H  , ci, … (ci: concentration of components which, e.g., determine the pH value or the concentration of educts or products of chemical reactions); E  is responsible for polarizations or chemical reactions (e.g., electrode reactions; see below); the state of magnetization of the sample may by varied by H  of different origins. 2.11. Models for the Parameterization of ESR Spectra; Phenomenological ESR Parameters Phenomenology of the ESR parameters (i) “nearly model free” parameters (i.e., general spectrometric parameters) such as position of lines (often given in effective g factors h g' B ν = β or g' const. B ν = ), line form (Gauss, Lorentz, and Voigt profiles) as well as line widths. (i) “nearly model free” parameters (i.e., general spectrometric parameters) such as position of lines (often given in effective g factors h g' B ν = β or g' const. B ν = ), line form (Gauss, Lorentz, and Voigt profiles) as well as line widths. profiles) as well as line widths. (ii) parameters of spin depending but time and mass independent interactions. Similarly, the concept of the spin-Hamilton operator spin Hˆ will be in the central position [3,22,82] including the 6699 Molecules 2013, 18 introduction of statistical distributions of the coupling parameters [89,90]. The spin-Hamiltonian spin Hˆ represent the disturbance energy of a system as a function of the spin operators. Individual terms of Hˆ are shown in Table 2 and e.g., in [3,22]. A more general representation of the relations between the properties of isotopes and the ESR response is given in Figure 1. (iii) models for determining time and mass, as well as spin depending effects such as: dynamic models or equations of motions, respectively, for describing the molecular reorientation [10,95], diffusion, spin exchange [35,96], tunnel effects [32,33], vibronic couplings, the dynamic Jahn-Teller effect [25], chemical reactions, spin relaxation [97] with the parameters T1 and T2 [3,98], and spin-phonon interactions [4,83,85,99]. (iv) quantum mechanical models for the calculation of energies, state functions, electron and spin densities [100–102], calculations of the spin-Hamilton operator under consideration of perturbation terms, solution of the time-independent Schrödinger equation considering isotope effects. 3.1. Determination of Concentrations of Paramagnetic Particles in a Sample by Means o On finding parameters for experimental ESR spectra the first question which arises is that for the number of spins and their relevance in the system under investigation (similarly to all other fields of spectroscopy). That question can be answered nearly without using theoretical models [16,75,103–106]. In other words, ESR can be used for the purpose of quantification. Nevertheless the determination of concentrations is challenging. In ESR spectroscopy generally the first derivative of the signal is registered and the signal area is proportional the number of spins. Thus, the double integration of the spectrum is needed for the determination which easily gives large errors [16,75,103,105,106]. Considering the most important sources of errors, one can determine the concentration of paramagnetic species e.g., as a function of time or other suitable spectral parameters. For this it is important that the spin properties of the particles itself are used. In this way it will be possible to observe kinetic isotope effects. As an example the examinations of the reaction of H or D, respectively, with CH4 by Kurylo et al. [107] shall be used. For the reactions: H• + CH4 Ý H2 + •CH3 (17) (17) 3.2. Influence of the H-D Substitution on the g Factor Coupling of the electron spins with the surrounding magnetic fields will be realized by the gˆ tensor (g coupling matrix). Next to the outer magnetic induction 0 B  the contributions of the orbital momentum has to be considered. The influence of the nuclear masses of the involved atoms is very small, thus there are only very few investigations with respect to the influence of isotopes in that field. Walther et al. [110] was successful in measuring the ratio g(H)/g(D) = G for the 1s states with an accuracy of 3 10−11 by means of hydrogen maser. With it the ratio (G – 1) could be determined to be 7.221 10−9 (see also [111]). For the g factor of 1H• a value of 2.0022838 is given by Weil and Bolton [3] (see also [112,113]). The vibronic reduction factor should be applicable for all physical ground state properties of electronic origin and thus have an influence on the orbital contribution of g, i.e., gL (see [25]). 3.3. Selected Hyperfine Effects 3.3.1. ESR Detection of Effect of Isotopes in Natural Abundance 3.3.1. ESR Detection of Effect of Isotopes in Natural Abundance An impressive demonstration of the ability of the simple cw ESR is supplied by Okazaki [114], with respect to the detection of isotopes in natural abundance (see also [20,115,116]). Solutions of K4[(SO3)2NO]2 (PADS) have been measured in a system of reversed micelles [117] in order to increase the sensitivity and the spectral resolution. With that it was possible to detect beside the well-known 14N hfs triplet (see Figure 6) the hfs of 17O (in SO3; 3.7 × 10−2% natural occurrence; I = 5/2; 6 lines with 0.537 ± 0.1 G hfs splitting), of 33S (0.74%; I = 3/2; 4 lines with 1.282 ± 0.1 G hfs splitting), and of 15N (0.365%; I = 1/2; 2 lines with 18.35 ± 0.056 G hfs splitting) could be observed (the 17O splitting in the NO fragment was 20.55 ± 0.26 G [116]). Altogether the authors were successful in assigning the spectral contributions to the overall spectrum. Moreover they succeeded to determine the different spin densities at 17O in the NO and SO3 fragments. The sign of the 17O hyperfine splitting in Fremy’s salt was determined by Luz et al. [11]. Figure 6. and: and: D• + CH4 Ý HD + •CH3 (18) (18) the constants k1 and k2 have been determined to be k1 = 6.25 1013 exp [−11600 ± 150/RT] and k2 = 4.5 1013 exp [−11100 ± 150/RT], i.e., in the expected relation for the isotopes. Using the much larger isotope effect of muonium [8,108] Roduner could obtain further information [109]: The competing effects zero-point energy and tunnel effect could be separated from the kinetic isotope effect as follows: if kMu/kH < 1 the zero-point energy dominates in the transition state, and kMu/kH > 1 for the domination of the tunnel effect. Molecules 2013, 18 6700 3.2. Influence of the H-D Substitution on the g Factor 3.2. Influence of the H-D Substitution on the g Factor ESR hyperfine structure of K4[(SO3)2NO]2 in a system of reversed micelles including the respective isotope splittings (inset) using the natural abundance of the isotopes (adapted from [38]). Molecules 2013, 18 Molecules 2013, 18 6701 A specific aspect of the hyperfine interaction shall be mentioned here with respect to the application of the isotope 99Tc in radio-pharmacy. ESR supplied much information for refining the structure of 99Tc coordination compounds, as well as the chemical knowledge about that isotope [12,13]. 3.3.2. Evidence for Trapped Muonium, Hydrogen, and Deuterium Atoms at Ambient Temperature Compounds with the structural unit Si8O12 (see Figures 7 and 8) have been proven to be useful for trapping reactive species like 1H•, 2H•, and Mu (μ+e− [86,87,118,119]) and to stabilize them for longer periods of time. These systems are not only interesting objects for investigations of the properties of the named paramagnetic species but they are useful as probes in their surrounding (e.g., for the detection and quantification of 3O2). Due to steric reasons of the cage there will be no real chemical reaction between H• and 3O2 but via the spin exchange the concentration of 3O2 can be determined as well as the frequency of non-reactive collisions in solution and solids. Figure 7. Model of a Si8O12R8 cage with incorporated H• (van der Waals radius). Figure 8. Photograph of a Si8O12R8:1H model. Figures 9 and 10 show the atomic hydrogen trapped in the Si8O12 cage which is symbolized by its van der Waals radius. The respective ESR spectrum for the solid compound is displayed in Figure 9. Figure 7. Model of a Si8O12R8 cage with incorporated H• (van der Waals radius). Figure 7. Model of a Si8O12R8 cage with incorporated H• (van der Waals radius). Figure 8. Photograph of a Si8O12R8:1H model. Figure 8. Photograph of a Si8O12R8:1H model. Figure 8. Photograph of a Si8O12R8:1H model. Figures 9 and 10 show the atomic hydrogen trapped in the Si8O12 cage which is symbolized by its van der Waals radius. The respective ESR spectrum for the solid compound is displayed in Figure 9. Figures 9 and 10 show the atomic hydrogen trapped in the Si8O12 cage which is symbolized by its van der Waals radius. The respective ESR spectrum for the solid compound is displayed in Figure 9. Figures 9 and 10 show the atomic hydrogen trapped in the Si8O12 cage which is symbolized by its van der Waals radius. The respective ESR spectrum for the solid compound is displayed in Figure 9. Molecules 2013, 18 Molecules 2013, 18 6703 Figure 11 reports about the origin of the 1H• and 2H•, respectively. EtT8 was irradiated at 300 K dissolved in C6D12. About 82% of the ESR signal area originate from 2H•, while about 18% are related to 1H•. That means, intermolecular processes take part in the trapping. Only about 18% of the 1H• can have their origin from the CH groups of the cage itself. Figure 11. ESR spectrum of a γ irradiated Si8O12R8 cage dissolved in C6D12 at 300 K (in the centre the line triplet of the D• atoms; at the outside the doublet of H•). The cages proved to be suitable not only for the trapping of 1H and 2H. The light hydrogen isotope muonium could also be trapped inside these cages at room temperature [86]. The muonium is the ultra-light isotope of hydrogen [87,109]. Under physical aspects μ+ is considered to be a heavy electron and in chemistry a light proton. Its mass is only 1/9 of that of hydrogen and it can be incorporated in different systems. Except the mass effect the μ behaves like a hydrogen isotope. It is the most sensitive probe for all kinds of mass dependencies, like effects of zero-point energy and tunnel effects. E.g., in EtT8 a hyperfine coupling constant of muonium of 4405.9 MHz compared to 1415.58 MHz for EtT8:1H, and 217.314 MHz for EtT8:D can be observed. Altogether, the absolute values of the hyperfine coupling constants compared to the values of the isotopes in vacuum clearly show the interaction of these atoms with their surrounding; they act as spin probes (see [123–125]). The cages proved to be suitable not only for the trapping of 1H and 2H. The light hydrogen isotope muonium could also be trapped inside these cages at room temperature [86]. The muonium is the ultra-light isotope of hydrogen [87,109]. Under physical aspects μ+ is considered to be a heavy electron and in chemistry a light proton. Its mass is only 1/9 of that of hydrogen and it can be incorporated in different systems. Except the mass effect the μ behaves like a hydrogen isotope. It is the most sensitive probe for all kinds of mass dependencies, like effects of zero-point energy and tunnel effects. E.g., in EtT8 a hyperfine coupling constant of muonium of 4405.9 MHz compared to 1415.58 MHz for EtT8:1H, and 217.314 MHz for EtT8:D can be observed. Molecules 2013, 18 Altogether, the absolute values of the hyperfine coupling constants compared to the values of the isotopes in vacuum clearly show the interaction of these atoms with their surrounding; they act as spin probes (see [123–125]). Molecules 2013, 18 6702 Molecules 2013, 18 As expected the trapped 2H• gives a 3-line spectrum with the reduced hyperfine coupling constant (a = 7.8) due the smaller gyromagnetic ratio γH/γD = 6.51 and the nuclear spin I(2H) = 1. The proof for the location of the hydrogen atom in the cage can be seen in Figure 10. The cages Q8M8 and EtT8 [120–122] had been irradiated with γ-rays. The line widths in the degassed solution are in the range of 2 – 9 μT. The symbol * indicates the signals of EtT8:1H and ** stands for Q8M8:1H. The satellites to the 1H doublet are a result of the interactions with the 29Si atoms of the cages. It has to be considered that the number of 29Si (I = 1/2) atoms per cage is determined by the natural abundance of 29Si (4.7%) and the probabilities of the occurrence in the cages (1 times 29Si, 2 times 29Si, etc.). The calculated probabilities and, thus, the percentage part of the ESR intensity correspond well with the experimental findings. Altogether this is the proof for the trapping of the hydrogen atom inside the cage. Figure 9. ESR spectra of H• (I = 1/2) and D• (I = 1) in solid Si8O12R8 matrices. Figure 9. ESR spectra of H• (I = 1/2) and D• (I = 1) in solid Si8O12R8 matrices. Figure 9. ESR spectra of H• (I = 1/2) and D• (I = 1) in solid Si8O12R8 matrices. Figure 10. ESR spectra of degassed solutions of γ irradiated Si8O12R8 cages in C2Cl4 at 293 K. : H• in the cage with R = propyl : H• in the cage with R = methyl. Figure 10. ESR spectra of degassed solutions of γ irradiated Si8O12R8 cages in C2Cl4 at 293 K. : H• in the cage with R = propyl *: H• in the cage with R = methyl. Figure 10. ESR spectra of degassed solutions of γ irradiated Si8O12R8 cages in C2Cl4 at 293 K. : H• in the cage with R = propyl **: H• in the cage with R = methyl. 3.4. Selected zfs Effects Isotope effects in the ESR fine structure of Mn2+ in Ca(OH)2 and Ca(OD)2 have been examined by Holuj and Kwan [126]. At low temperatures there are relatively large differences with respect to the zfs parameter 0 2 b . 0 2 b symbolizes a spin coupling parameter of second order in the spin-Hamilton operator [see [22,82] and Equation (5)] and it can be interpreted as a measure for stretching or compressing of a Mn2+ coordination polyhedron. The analysis of the ESR parameters concerning the lattices vibrations revealed, that the hyperfine coupling constant of 55Mn changes only slightly over a large temperature range (about 4.3%), but the fine structure parameter 0 2 b changes even its sign. Next to the thermal expansion of the sample with increasing temperature a modulation of the crystal electric field by the lattice vibrations occurs. The development of that field in terms of displacement of the Molecules 2013, 18 Molecules 2013, 18 6704 participating atoms including orbital lattice interactions showed, that the contributions of the involved acoustic modes to the modulation process are reciprocally proportional to the masses of 1H and D [126]. For these findings it is interesting to watch the subtle changes of the fine structure by isotopic marking, but that the changes appear via changes in the vibrational modes of the matrix. The influence of isotopes on the fine structure of ESR spectra is described in [127–130]. The role of the spin-phonon coupling was investigated by Böttger [99] on examining the line splitting of the ESR spectrum of Ni2+ in MgO, while Tomioka et al. [131] studied steric and isotope effects in triplet carbene systems. 3.5. Examples for the Jahn-Teller Effect and Vibronic Couplings The ESR detection of the static Jahn-Teller distortion in cationic radicals with originally degenerated occupied molecular states has been done already with several examples such as SF6 + [132], benzene+ [133,134], etc. At higher temperatures the hyperfine structure for the benzene cation results in six equivalent protons due to the fast exchange of the three equivalent distorted structures [135]. This phenomenon is called pseudo-rotation. The influence of the isotopic substitution on the Jahn-Teller distortion was demonstrated by means of cations of methane, cyclopropane, and cyclohexane [134]. The respective theoretical background can found in [136]. Partially deuterated benzene cation radicals are the objects in the examinations of Toriyama et al. [134] while Iwasaki et al. [137] examined ethane radicals in SF6. It was found that the kind of distortion of the cations is unique with respect to the sites of the D substitution in all examined matrices. For the ground states D2h symmetry results and the remaining C-H bonds are localized in the distorted structure at sites with higher spin density. In the simplest case the zero-point energy for the stretching vibrations of C-H is proportional to (k/mH)1/2 (k: force constant). On exchanging H and D the complete zero-point energy depends on the position in the cation. The criterion for that consists in the strongest decrease of the zero-point energy in case the D atom occupies a position with a larger force constant. That can be realized with a shorter bonding and smaller spin density. The addition of muonium to benzene gave an interesting effect as described by Eckert et al. [87]. This reaction was about twenty times faster than that of 1H and the activation energy was about two third smaller [138]. That finding will be caused by a dominance of the tunnel effect which will superimpose that of the zero-point energy in the transition state. 3.6. Examples for the Tunnel Effect For the first time Zamaraev and Khairutdinov [139] could provide the experimental proof that the reactants have an unusual large distance from each other, i.e., about 1.4 Å, in the moment of reaction. This was found for the reaction: etr − + O− → O2− (16) (16) Both reactants are ESR active and can be observed separately because of the different spin-orbit coupling contributions to the gˆ tensor. Moreover, from the line widths of the signals the distance between the paramagnetic species can be determined. Further on there is a possibility to determine the actual concentration of the paramagnetic reactants and products, respectively. It turned out that in the Molecules 2013, 18 Molecules 2013, 18 6705 range of 4.2–93 K the main reaction channel appears to be a non-activated tunnel process for the electron transfer, which changed to an activated one at higher temperatures. range of 4.2–93 K the main reaction channel appears to be a non-activated tunnel process for the electron transfer, which changed to an activated one at higher temperatures. Due to the fact that ESR spectra sensitively reflect changes in structure and the local environment of the paramagnetic species, isomerizations, interconversions of radical pairs (see [30]) and isolated radicals, respectively, as well as transformations of diradical pairs in crystals can be observed. Examples for this are described by Bell [33] and Williams et al. [140–143]. E.g., : CH3 + CH3X Ý CH4 + •CH2X (X = CN, NC, OH) (19) CH3 + CH3OD Ý CH4 + •CH2OD (20) (19) (20) Due to the usage of CH3OD the line widths was reduced caused by the smaller magnetic moments of the D nuclei. Due to the usage of CH3OD the line widths was reduced caused by the smaller magnetic moments of the D nuclei. Another example for the influence of isotopes on the tunnel effect will be the isomerisation of 2,4,6-tributylphenol into 3,5-di-t-butylneophyl [144] which was carried out in comparison to the completely deuterated species. In the range from 28–200 K a strong change in activation energy took place indicating a distinctive isotope effect. For that the ratios of the rates kH/kD have been determined which particularly at low temperatures are much above the predicted one for the model of the absolute reaction rate. Thus, one can state that the complete process is dominated by the tunnel effect. 3.6. Examples for the Tunnel Effect The tunnel effect of H atoms could observed on changing radical pairs in γ irradiated crystalline dimethylglyoxime, too. The vanishing activation energy in the range of 4.2–50 K is also a sign for a tunnel process [32,145,146]. The respective 1H/2H isotope effects have been examined by Jakimtschenko and Lebedev [145]. It could be shown that the conversion of radical pairs in the temperature range 77–310 K occurs via intra and intermolecular migration of bonds. The isotope effects are pronounced particularly at low temperatures. The basic mechanisms are mainly determined by hydrogen tunnelling. Another interesting isotope effect at the para-elastic relaxation has been shown by Pfister and Känzig [147] by means of ESR. The paramagnetic species 16O2 − and 18O2 − have been examined in KCl, KBr, and KJ at temperatures of 1.3–4.5 K under uniaxial pressure. The observation of the elastic relaxation of the systems after pressure release was done via recording the ESR intensity as a function of the time. Under a molecular aspect the relaxation was expressed by the turning of a molecule from one equilibrium orientation to another one due to undoing the distortion after the pressure release. Despite the ‘jump rate’ of the paramagnetic molecules had been very small at the selected temperatures the sensitivity of ESR allowed the observation of the time-depending course via changes of the line intensity of the ESR signals. At T < 4 K the rate of reorientation was directly proportional to the temperature. On turning the molecules the present potential barriers have been tunnelled successfully. On that a relatively large isotope effect of the rates of reorientation for 16O2 − and 18O2 − could be detected. It is interesting to state that the differentiation between the species 16O2 − and 18O2 − (both have I = 0) by classical ESR parameters is not possible. 3.8. Isotope Effects of the Matrix 3.8. Isotope Effects of the Matrix 3.7. Influence of the Zero-Point Vibrations The influence of the zero-point vibrations on the superhyperfine interactions of 1H• and 2H• in KCl could be shown by Spaeth [148] by means of ESR. The isotropic superhyperfine splitting constants Molecules 2013, 18 Molecules 2013, 18 6706 had been smaller in case of 2H•. The ratio of the couplings with the potassium nuclei (I = 3/2 for 39K) had been aH(K)/aD(K) = 1.165 and with chlorine (I = 3/2) 1.026. About 30% of aH(K) and about 10% of aH(Cl) could ascribed to zero-point vibrations. The lighter 1H• causes a larger energy and amplitude of the zero-point vibration and a stronger influence on the hyperfine interaction. Finally the average effect of the vibrations on the superhyperfine structure was observed. This means an admixture of different core-orbital contributions to the spin density of the ESR active centres takes place. Further results for H and D atoms in alkali halide crystals are given in [149]. The examinations of Morton and Preston [81] supplied an example for the influence of isotopes on the isotropic hyperfine interactions using the F2NO radical. The 19F coupling increases with the mass of oxygen and nitrogen (15N and 17O substitution). As expected the original nitrogen coupling changes from a triplet to the 15N doublet with a larger splitting. Hints to quantum-mechanical tunnelling processes of methyl protons at 20 K could be obtained from the analysis of the ESR spectra of the partially deuterated tetramethysilanes [Si(CH3)4 + and others] [150]. By means of the 1H-2H substitution in radical cations of CH3OCH2F and CH3CCl3 and the complete analysis of the ESR spectra, accompanied by ab initio DFT-MO calculations, the conformation of the cations could be determined. The influence of the 2H substitution onto the conformation and rotation of the CH3 groups in the dimethylether cation could be shown by Itagaki et al. [101]. The 1H hyperfine constant showed up to 100 K nearly no temperature dependency different to the 2H system because of the difference in the zero-point vibrations of the different rotational conformers. The proof for intermolecular hydrogen bridges in the hydroxysemiquinone provided Gendell et al. [151] by means of 1H-2H isotope effects on the hyperfine structure. Obviously the vibrational effects in a hydrogen bridge play a decisive role. The ratio of the coupling constants a(1H)/a(2H) was in the range of 7.2−7.8 larger than the expected value of 6.51 out magnetic data. 3.7. Influence of the Zero-Point Vibrations The reduced mobility of 2H has an effect on the values of the hyperfine splitting in isotopically substituted 1-hydroxypyridil radicals [152]. Papers concerning the simulation of ESR spectra considering isotopic substitutions on the hyperfine structure including the respective computer programmes are widespread in the literature (see e.g., [101,153,154]). 3.8.1. Stationary Effects This concerns the detection of the properties of the surrounding of paramagnetic species. In this connection the term matrix should be understood to be the lattice of a solid, as well as solvents, or other fluid systems. Thus the isotopic substitution might be done in: (i) the spin probes as well as in (ii) the units of the matrices. Further differentiation in: (iii) static and (iv) dynamic effects seems to be useful. Molecules 2013, 18 Molecules 2013, 18 6707 3.8.1.1. H-D Exchange and Fermi Contact 3.8.1.1. H-D Exchange and Fermi Contact The values for the Fermi contact interaction [3] of the hydrogen isotopes in liquid and solid H2O are below the vacuum values. They depend on the mass and give a negative temperature coefficient in the liquid phase. It means there is some spin delocalization to solvent molecules. Moreover, there is a strong isotope effect in mixtures of H2O and D2O [124]. Furthermore there is an indication for the existence of clathrate-like cages in water. Other chemically interesting effects of H-D exchange were observed by Magin and Morse [155] and Pfirrmann et al. [156]. 3.8.1.2. Spin Probes and Polarity of the Matrix Under practical chemical considerations the term polarity is used in chemical synthesis, e.g., to describe the influence on the course of chemical reactions by selecting and applying suitable solvents. The relative dielectric constant εr is a possible means to indicate the polarity of a solvent [157]. The hyperfine coupling constants of spin probes, dissolved in the respective solvent are related to polarity [131]. The reason for that is the fact that in polar media the accommodation of the polar constituents of the structure of the spin probes is favoured and the spin density at the nitrogen atom will increase (see Figure 12). With it the hyperfine coupling increases, too. As shown in Figure 12 fluid media can be formally subdivided into polar and non-polar (εr < 25). In that way the selection of a suitable solvent for specific reaction can be selected by their hyperfine coupling. For the determination of the polarity the usage of 15N spin probes is useful, because the larger hyperfine coupling responds more sensitively on changes of εr. Distance measurements in complex systems have been performed by Jeschke et al. [158] using marked nitroxyl spin probes. Figure 12. Formal differentiation of the polarity of solvents by means of the values of the hyperfine coupling constants in relation to the respective dielectricity constants εR. The ordinate shows the differences ΔAiso with respect to coupling constant of toluene. Molecules 2013, 18 6708 The literature provides a large number of papers concerning the simulation of ESR spectra of selected transition metal complexes, as e.g., considering the surrounding matrix of complexes of copper isotopes in their natural abundance [153]. 3.8.2.1. Spin Relaxation As mentioned in Section 2.2. the usage of isotopes is reflected in spin relaxation times T1 and T2. The findings of Du et al. [161] and Biller et al. [162] shall serve as examples for that. In the range of rapid tumbling (0.5–0.1 μs) the relaxation times T1 and T2 in liquids approach each other (T1~T2). Biller et al. [162] found larger values for T1 as well as T2 for selected 15N spin probes than for the respective 14N probes. This and the smaller line widths improve the signal-to-noise ratio, too. All this is based on the differences in the nuclear magnetic moments und the nuclear spin. Beside the thermal modulation of the anisotropic nitrogen hyperfine structure the modulation of the spin-rotation interaction contributes to T1. 3.8.1.3. Spin trapping The method is characterized by transferring short-living paramagnetic species, mainly radical intermediates, to longer living radicals by reacting with so-called spin traps (e.g., DMPO). For that, there are similar considerations as stated already [6,7]. Also in these cases the identification of the species by isotopic marking (1H → 2D, 14N → 15N) will be easier and the sensitivity will be enhanced [159]. The trapping of the superoxide anion and the identification of the trapped radical species could be improved after applying isotopically marked spin traps as it is shown in [160]. 3.8.2. Dynamic Effects 3.8.2. Dynamic Effects 3.8.2.2. Diffusion of Spin Probes and ESR Tomography The isotopic marking of the spin probes 15N-2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO) and 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPOL) allows the observation of their lateral diffusion in opposite directions in a one-phase system by means of ESR tomography [163]. Figure 13 displays the profiles of the signal amplitudes after 10 and 135 min, respectively, for the diffusion of the spin probes in the ionic liquid butylmethylimidazolium tetrafluoroborate (BuMeImBF4) [163]. By means of the marking the concentration profiles can be assigned unambiguously and specifically for the respective probe including their spatial distribution. The lateral diffusion of two spin probes in a two-phase system can be similarly tracked by 15N-marking and ESR tomographic examination as shown in Figure 14. For that purpose 15N-TEMPO was dissolved in Precirol®, while 14N-TEMPOL was dissolved in Miglyol® and brought into contact within a capillary tube. The spectral-spatial images and the respective spectra in different depths show the progress of the distribution after 5 and 240 min [37], respectively, are displayed in Figure 14. Particularly in biological systems, ESR imaging requires the usage of spin probes and conditions for measurements which give acceptable signal-to-noise ratios. Such systems are usually characterized by high dielectric losses due to microwave absorption and it might be generally advisable to perform the examinations at smaller microwave frequencies than to use the standard frequency of about 10 GHz. Molecules 2013, 18 6709 Another method for improving the signal-to-noise ratio will be the increase of the spin probe concentration. Unfortunately this is not always possible because of the toxicity of the spin probe and, moreover, an increase in line widths, whereas the isotopic substitution of 1H by 2H will improve the situation because the small nuclear moments of the 2H nuclei give smaller magnetic dipole-dipole contributions to the line widths [164]. The 14N-15N substitution in the nitroxyl spin probes is favourable, too, because to the reduction from three to two lines also improves the signal-to-noise ratio. Figure 13. Lateral diffusion of the spin probes 14N-TEMPOL and 15N-TEMPO dissolved in the ionic liquid BuMImBF4 at 300 K. The signal amplitudes are displayed as function of the spatial coordinates. 6710 Molecules 2013, 18 Figure 14. Reverse diffusion of the spin probes 15N-TEMPO and 14N-TEMPOL in Precirol and Miglyol, respectively, after 5 and 240 min. Spectra-spatial images including of selected ESR spectra in different depths of the two-phase object (black: 2.17 mm depth, red: 2.71 mm depth, blue: 2.85 mm depth, green: 3.25 mm depth). Figure 14. Reverse diffusion of the spin probes 15N-TEMPO and 14N-TEMPOL in Precirol and Miglyol, respectively, after 5 and 240 min. Spectra-spatial images including of selected ESR spectra in different depths of the two-phase object (black: 2.17 mm depth, red: 2.71 mm depth, blue: 2.85 mm depth, green: 3.25 mm depth). 3.8.2.3. Spin Exchange and Chemical Reactions 3.8.2.3. Spin Exchange and Chemical Reactions The direct determination of 3O2 in solution (e.g., in H2O) is very difficult due the small distribution coefficient of 3O2 and the relaxation times of the respective spin states are very short and, thus, the determination of 2 O p , too. Examples for this are the changes of 2 O p in the course of the Maillard reactions [165], the analysis of water samples, in which 2 O p provides environmental information about the sum of further dissolved substances and about activity and state of biosystems. Contrary to chemical or other spectroscopic procedures the investigation of the spin exchange between stable spin probe radicals and the paramagnetic 3O2 by means of ESR has the advantage that the concentration can be determined by observing the changes in line widths [166], i.e., the x axis is precisely calibrated in units of frequency is used for finding the actual 2 O p . Molecules 2013, 18 The marking of the mainly used nitroxyl spin probes with the isotope 15N instead of 14N is advantageous due to the increase in signal amplitude because the complete signal intensity is distributed over two lines only. Additional signal splittings can be avoided by substitution of 1H by 2H, which leads to smaller line widths. Altogether at comparable sensitivity the spin probe concentration can be reduced. Additionally the reduction of the dipole-dipole interactions between the probes leads to an increase of sensitivity of the 2 O p . Disadvantageous seems to be the case that only one parameter (K [165]) is used which usually even depends on the applied radical concentration. Moreover, the signal-to-noise ratio decreases because the spectra are recorded with small modulation amplitudes. E.g., the reduced information content results already from the fact that out of 18 hyperfine lines only two are used. In oximetry the influence of the spin-spin relaxation time T2 on the line widths of the spin probes with 15N and 2H marking the sensitivity could be increased. In their considerations Halpern et al. [167] went further. They reduced the contribution of the inhomogeneous component of the spectrum and used the advantage of a selective deuteration for an additional reduction of the line width. In this they could find a compromise between the usage of the 6711 Molecules 2013, 18 spectral “richness” and the reduction of the line widths. That compromise is of importance at low ESR frequencies (e.g., at 250 MHz) for in vivo investigations, including ESR imaging. E.g., after reduction of the contribution of the inhomogeneous component it could differentiated which part of line broadening results from interactions between the spin probe molecules themselves and the interactions with 3O2. The spin exchange of the dissolved spin probes TEMPO, CAT-1, and TEMPOL (see Figure 15) has been proven to be a valuable indicator for different interactions of the spin probes in H2O and D2O, respectively [96]. So, the different structure of the probes gave different rate constants ke for the spin exchange at 293 K (contrary to the values of the hyperfine coupling which differ on slightly between H2O and D2O solutions) and make a differentiation between H2O and D2O possible. Different zero-point and dissociation energies for both fluid systems are the reason for that. Moreover, these disparities are responsible for other characteristic mainly macroscopic properties of water [168]. 4. Conclusions It is not only necessary to consider effects of naturally occurring isotopes for the interpretation of ESR spectra, but further information about spin density distribution, reaction behaviour and other phenomena of paramagnetic molecules, organic systems, and solids will be accessible. Finally, the exchange of isotopes up to the synthesis of isotopically marked compounds is indispensable, despite the recent highly developed ESR technique. The conclusiveness of the interpretation is out of doubt. The variety of applications of isotopes extends via nearly all spin depending parameters, which provide a contribution to the ESR response. The relationship between electron spin and specific properties of isotopes such as nuclear spin, nuclear magnetic moment, mass and nuclear volume leads to characteristic phenomena such as electron spin, nuclear spin, vibronic couplings, tunnel effects and others which have a direct connection to the quantum mechanical basic effects. Well-planned experiments may approve the access to these phenomena. Further developments of the ESR technique, the chemical methods for stabilizing reactive paramagnetic species, good command of isotopic marking on a chemical way, the adaption and improvement of quantum mechanical techniques for the interpretation of experimental findings will make it possible to follow the course of dynamic processes and chemical and biological reactions. Molecules 2013, 18 With respect to the influence of isotopes on chemical reactions further examples are discussed in [1,33,155,169–171]. Furthermore it is of interest how the spin exchange in biradicals can be elucidated by using the symmetrical and non-symmetrical isotope exchange [34]. Figure 15. ESR spectra of different concentrations of the three spin probes TEMPO, CAT-1, and TEMPOL in H2O and D2O at 300 K. 6712 Molecules 2013, 18 Figure 15. Cont. 3.9. Indication of Some Sources Dealing with Non-cw Techniques and Modern Applications of ESR Non-stationary and multiple resonance techniques are described in [14] and [15]. Problems of the creation and handling of quantum bits are outlined in [172–176]. Figure 15. Cont. 3.9. Indication of Some Sources Dealing with Non-cw Techniques and Modern Applications of ESR 3.9. Indication of Some Sources Dealing with Non-cw Techniques and Modern Applicati Non-stationary and multiple resonance techniques are described in [14] and [15]. Problems of the creation and handling of quantum bits are outlined in [172–176]. Non-stationary and multiple resonance techniques are described in [14] and [15]. Problems of the creation and handling of quantum bits are outlined in [172–176]. Non-stationary and multiple resonance techniques are described in [14] and [15]. Problems of the creation and handling of quantum bits are outlined in [172–176]. Acknowledgements We would like to express our thanks to M. Päch, G. Scholz, H.-H. Borchert, Christian Kroll, Klaus-Peter Moll, Ulrike Hochkirch for their support to papers cited in this article. Molecules 2013, 18 Molecules 2013, 18 6713 Molecules 2013, 18 References 1. Isotope Effects in Chemistry and Biology; Kohen, A., Limbach, H.-H., Eds; CRC Press — Taylor & Francis: Boca Raton, FL, USA, 2006. 1. Isotope Effects in Chemistry and Biology; Kohen, A., Limbach, H.-H., Eds; CRC Press — Taylor & Francis: Boca Raton, FL, USA, 2006. 2. Bigeleisen, J. Gilbert N. Lewis and the beginnings of isotope chemistry. J. Chem. Educ. 1984, 61, 108–116. 2. Bigeleisen, J. Gilbert N. 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Molecules 2013, 18 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/). © 2013 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 license (http://creativecommons.org/licenses/by/3.0/).
https://openalex.org/W4285088845	https://www.researchsquare.com/article/rs-1844677/latest.pdf	English	null	SARS-CoV-2 S1 Protein Persistence in SARS-CoV-2 Negative Post-Vaccination Individuals with Long COVID/ PASC-Like Symptoms	Research Square (Research Square)	2,022	cc-by	4,643	SARS-CoV-2 S1 Protein Persistence in SARS-CoV-2 Negative Post-Vaccination Individuals with Long COVID/ PASC-Like Symptoms Bruce K. Patterson (  brucep@incelldx.com ) Rodrigo Mora Rodrigo Mora Lab of Tumor Chemosensitivity, CIET / DC Lab, Faculty of Microbiology, Universidad de Costa Rica Javier Mora Lab of Tumor Chemosensitivity, CIET / DC Lab, Faculty of Microbiology, Universidad de Costa Rica Rodrigo Mora Lab of Tumor Chemosensitivity, CIET / DC Lab, Faculty of Microbiology, Universidad de Costa Rica Javier Mora Lab of Tumor Chemosensitivity, CIET / DC Lab, Faculty of Microbiology, Universidad de Costa Rica g Lab of Tumor Chemosensitivity, CIET / DC Lab, Faculty of Microbiology, Universidad de Costa Rica Javier Mora b f Ch i i i C C b l f i bi l i id d d C i Short Report Short Report Short Report Page 1/14 Page 1/14 Page 1/14 Keywords: COVID-19, PASC, long COVID, long haulers, SARS CoV-2 S1 protein, non-classical monocytes, CCR5, fractalkine Posted Date: July 12th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-1844677/v1 Results We determined that post-vaccination individuals with PASC-like symptoms had similar symptoms to PASC patients. When analyzing their immune profile, post-vaccination individuals had statistically significant elevations of sCD40L, CCL5, IL-6, and IL-8. SARS-CoV-2 S1 and S2 protein were detected in CD16 + monocytes using flow cytometry and mass spectrometry on sorted cells. DOI: https://doi.org/10.21203/rs.3.rs-1844677/v1 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 2/14 Background We sought to determine the immunologic abnormalities in patients following SARS-CoV-2 vaccines who experience post-acute sequelae of COVID-19 (PASC)-like symptoms > 4 weeks post vaccination. In addition, we investigated whether the potential etiology was similar to PASC. Design: We enrolled 50 post-vaccination individuals who experience PASC-like symptoms, 10 healthy individuals, and 35 individuals post-vaccination without symptoms. We performed multiplex cytokine/chemokine profiling with machine learning as well as SARS-CoV-2 S1 protein detection on monocyte subsets using flow cytometry and mass spectrometry. Conclusions Post-vaccination individuals with PASC-like symptoms exhibit markers of platelet activation and pro- inflammatory cytokine production which may be driven by the persistence of SARS-CoV-2 S1 protein persistence in intermediate and non-classical monocytes. Flow Cytometric S1 Quantification in Monocyte Lineage Cells Flow Cytometric S1 Quantification in Monocyte Lineage Cells Peripheral blood mononuclear cells were collected in EDTA anticoagulated whole blood collection tubes. Whole blood (200 uL) was lysed using 4 mL of 1X Red Blood Cell (RBC) Lysis Buffer (Abcam Part #ab204733) for 10 min. and centrifuged at 400x g for 5 min. Cells were resuspended in 1 mL 2%BSA/D- PBS and incubated for 5 min at room Temperature. The cells were then pelleted at 300x g for 6 min and the supernatant was aspirated leaving 50–100 µL residual liquid. An antibody cocktail with the following reagents: 5 µL True-Stain Monocyte Blocker, 5 µL CD45 FITC, 3 µL CD14 PE, 5 µL CD3 PC5.5, 1.5 µL CD19 PC7,1.5 µL CD16 AF700, 5 µL COVIDS1-AF647, 74 µL 2%BSA/D-PBS. (100uL) was added to each tube and the mixture was incubated in the dark for 25 min. 37° C. Cells were washed in 1 mL 2% BSA, centrifuged at 300x g for 6 min and re-suspended in 200 µL D-PBS for analysis. Single cells were analyzed on a Beckman Coulter Cytoflex flow cytometer and gated as previously described8. Patients Fifty patients were enrolled following written informed consent. None of the patients reported any clinical signs and symptoms of previous SARS-CoV-2 infection, with some also having documented multiple negative polymerase chain reaction (PCR) tests. Lack of prior infection was determined using anti- nucelocapsid antibody testing or Food and Drug Administration (FDA) Emergency Use Authorization (EUA) T-Detect Test. Representative patients vaccinated with all four approved vaccines (Pfizer-BioNTech, Moderna, Johnson and Johnson, and Oxford/AstraZeneca), were included in the study as well as the duration of the symptoms post-vaccination. Thirty-five healthy controls all 90 days post-vaccination were enrolled as the control group following informed consent. Introduction Over 1.5 billion doses of SARS-CoV-2 vaccine have been delivered to individuals worldwide. Vaccines exhibit a > 80% protection against infection by the original alpha variant with some vaccines offering > 90% protection1. Most importantly, vaccines have protected against serious disease and death2. Since the launch of global vaccination programs, side effects of the vaccine have been reported including increased thrombosis, cardiomyopathy, and other vascular events3–5. The possibility that long hauler symptoms could occur following a breakthrough infection was published by the Center for Disease Control (CDC). This publication did not discuss the possibility that long hauler symptoms could occur in vaccinated individuals in the absence of infection6. Here, we report on 50 individuals with symptoms > 4 weeks following vaccination that resemble the spectrum of symptoms reported in long COVID or PASC7, 8. Page 3/14 Page 3/14 We used machine learning to analyze the immune profiles in these individuals. Further, we used high parameter, single cell analysis to determine if S1 protein generated by vaccination persisted in immune cells as previously described9. Multiplex Cytokine Quantification Fresh plasma was used for cytokine quantification using a customized 14-plex bead based flow cytometric assay (IncellKINE, IncellDx, Inc) on a CytoFlex flow cytometer as previously described8 using the following analytes: TNF-α, IL-4, IL-13,IL-2, GM-CSF, sCD40L, CCL5 (RANTES), CCL3 (MIP-1α),IL-6, IL-10, IFN-γ, VEGF, IL-8, and CCL4 (MIP-1β). For each patient sample, 25 µL of plasma was used in each well of a 96-well plate. Flow Cytometric Cell Sorting Cryopreserved PBMCs were quick-thawed, centrifuged, and washed in 2% BSA solution in D-PBS. Cells were blocked for 5 min. in 2% BSA and then incubated at room temperature for 30 min. with Alexa Fluor® Page 4/14 Page 4/14 488 Anti-CD45 antibody (IncellDx, 1/100 dilution), 2.5 ug of Alexa Fluor® 647 Anti-CD16 antibody (BD, Cat. # 55710), and 1 ug of PerCP/Cy5.5 Anti-human CD14 antibody (Biolegend, Cat. #325622). Cells were washed twice with 2% BSA/D-PBS, filtered, and kept on ice for the duration of the cell sort. Data was acquired on a Sony SH800, and only CD45 + cells staining positive for both CD14 + and CD16 + were sorted into test tubes with 100 uL 2% BSA solution. Sort purity of control PBMCs was confirmed to be > 99% by re-analyzing sorted PBMCs using the same template and gating strategy. 488 Anti-CD45 antibody (IncellDx, 1/100 dilution), 2.5 ug of Alexa Fluor® 647 Anti-CD16 antibody (BD, Cat. # 55710), and 1 ug of PerCP/Cy5.5 Anti-human CD14 antibody (Biolegend, Cat. #325622). Cells were washed twice with 2% BSA/D-PBS, filtered, and kept on ice for the duration of the cell sort. Data was acquired on a Sony SH800, and only CD45 + cells staining positive for both CD14 + and CD16 + were sorted into test tubes with 100 uL 2% BSA solution. Sort purity of control PBMCs was confirmed to be > 99% by re-analyzing sorted PBMCs using the same template and gating strategy. Single Cell Protein Identification Patient cells were sorted based on phenotypic markers (as above) and frozen at -80o C. Six patient samples with positive flow cytometry signal and sufficient cell counts were chosen for LCMS confirmation. Frozen cells were lysed with the IP Lysis/Wash Buffer from the kit according to the manufacturer’s protocol. 10 ug of anti-S1 mAb were used to immunoprecipitate the S1 Spike protein from cell lysate of each patient. After overnight incubation with end-over-end rotation at 4oC and then three washes with IP Lysis/Wash Buffer, bound S1 Spike protein was eluted with the elution buffer from the kit. IP elution fractions were dried in vacuo, resuspended in 20 uL of water, pooled, and purified by Agilent 1290 UPLC Infinity II on a Discovery C8 (3cm x 2.1 mm, 5 µm, Sigma-Aldrich, room temperature) using mobile phase solvents of 0.1% trifluoroacetic acid (TFA) in water or acetonitrile. The gradient is as follows: 5–75% acetonitrile (0.1% TFA) in 4.5 min (0.8 mL/min), with an initial hold at 5% acetonitrile (0.1% TFA) for 0.5 min (0.8 mL/min). The purified protein was dried in vacuo and resuspended in 50 µL of 100 mM HEPES, pH 8.0 (20% Acetonitrile). 1µL of TCEP (100 mM) was added and the samples were incubated at 37ºC for 30 min. 1 µL of chloroacetamide (500 mM) was added to the samples and incubated at room temperature for 30 min. 1 µL rAspN (Promega 0.5 µg/µL) and 1 µL of LysC (Pierce, 1 µg/µL) were added and the samples incubated at 37ºC for 16 h, prior to LCMS analysis. Immune Profile Determined using a 14-Plex Cytokine/Chemokine Panel. To determine whether the PASC-like symptoms may be associated with similar cytokine/chemokine abnormalities found in PASC8, we performed statistical analysis on the 50 post-vaccination patients with PASC-like symptoms and compared the cytokine expression levels to individuals post-vaccination without PASC-like symptoms. Post-vaccination patients with symptoms had statistically significant elevation in CCL5 (p=0.006), sCD40L (P<0.001), IL-8 (P<0.001), and IL-6 (P=0.04) (Fig. 3). Vaccinated individuals without PASC-like symptoms had elevated IL-2 (P=0.007) and reduced CCL4 (P=0.001) compared to the group of vaccinated individuals with PASC-like symptoms. Elevated IL-8 is a key difference compared to the elevated cytokines in PASC. Vaccine-Specific PASC-Like Symptoms We investigated 50 individuals that exhibited PASC-like symptoms similar to those previously published7,8. On average these symptoms were reported after 105 days post-vaccination (Table 1) and previously published in post-vaccination individuals10. There was variability in symptoms according to the vaccine administered, however, the predominant symptoms were fatigue, neuropathy, brain fog and headache. All of these symptoms were reported in all four of the vaccines administered in this cohort. Notably infrequent in the post-vaccination individuals with PASC-like symptoms compared to PASC itself were shortness of breath and loss of taste/smell. Liquid Chromatography/Mass Spectroscopy (LC-MS) analysis Digested recombinant SARS-CoV-2 Spike S1 protein was analyzed by a high accuracy mass spectrometer to generate a list of detectable peptides with retention time and accurate masses. An Agilent 1290 Infinity II high pressure liquid chromatography (HPLC) system and an AdvanceBio Peptide Mapping column (2.1 × 150 mm, 2.7 µm) were used for peptide separation prior to mass analysis. The mobile phase used for peptide separation consists of a solvent A (0.1% formic acid in H2O) and a solvent B (0.1% formic acid in 90% CH3CN). The gradient was as follows: 0–1 min, 3% B; 1– 30 min, to 40% B; 30–33 min, to 90% B; 33–35 min, 90% B; 37–39 min, 3% B. Eluted peptides were electrosprayed using a Dual JetStream ESI source coupled with the Agilent 6550 iFunnel time-of-flight MS analyzer. Data was acquired using the MS method in 2 GHz (extended dynamic range) mode over a mass/charge range of 50–1700 Daltons and an auto MS/MS method. Acquired data were saved in both centroid and profile mode using Agilent Masshunter Workstation B09 Data acquisition Software. The same analytical method was applied to immunoprecipitated samples from sorted patient cells except no ms/ms was acquired Page 5/14 Page 5/14 Discussion To date, clinical manifestations of post-vaccination side effects or injuries have been described but very few mechanisms have been offered to explain these findings. In the present study, we investigated whether an S1 protein mechanism of inflammation similar to what we published in PASC might underlie the persistent, PASC-like symptoms that remain for months following vaccination with currently available vaccines in the US. Given that ongoing viral replication may not be required for prolonged symptoms9 and given the overlap in symptomatology, we applied machine learning to a panel of previously published immune biomarkers to determine if an immune signature for post-vaccination PASC-like symptoms might exist. Using two algorithms (severity score and long hauler index) previously derived from these biomarkers8, we found that post-vaccination PASC-like symptoms were associated with an inflammatory profile with statistically significant elevations in CCL5, sCD40L, IL-6, and IL-8. Further these patients were classified as PASC using a single classifier and PASC with inflammation using a dual classifier. Elevated IL-8 was a unique marker relative to PASC in post-vaccination individuals with PASC-like symptoms. We recently found a statistically significant correlation between decreased IL-8 and improvement in the NYHA cardiac symptom score in PASC following treatment with a CCR5 antagonist and statin11. Because of the similarities between PASC and patients with post-vaccination PASC-like symptoms, we examined whether S1 protein persistence might also occur in patients with post-vaccination PASC-like symptoms. We demonstrated a statistically significant elevation of S1 protein containing non-classical monocytes (NCM) and in S1-containing intermediate monocytes (IM) in post-vaccination PASC-like patients compared to normal controls. We sorted these CD16 + monocytes as previously performed and used mass spectroscopy to interrogate whether S proteins (S1 and or S2) were present in these highly mobile cells. We confirmed the presence of S1 sequences as well as S2 sequences in these monocytic subsetss. Further, we found amino acid mutations in all six patients analyzed; the impact of which remains to be elucidated. NCM bind to fractalkine expressed on vascular endothelial cells through the expression of the fractalkine receptor CX3CR1 on the surface of NCM11. Fractalkine is also upregulated by IL-1, IFN-γ, and TNF-α12, cytokines that we have reported to be elevated in PASC9. CX3CR1 also provides a survival signal to non- classical monocytes through CX3CR1-dependent expression of the anti-apoptotic protein BCL213, 14. Stress and exercise mobilize non-classical monocytes including up to 4-fold with exercise15, 16. Analysis of S1 Protein Persistence Similar to our published findings of S1 persistence in non-classical and intermediate monocytes in PASC9, we investigated whether a similar mechanism could account for the PASC-like symptoms in post- vaccination individuals with PASC-like symptoms. We used flow cytometry to screen patients for S1 protein in their monocyte subsets. We screened 14 post-vaccination individuals with simultaneously collected PBMCs and with PACS-like symptoms and 10 normal controls. As demonstrated in figure 4, there was statistically significant elevation of S1 containing non-classical monocytes (13 of 14, P<0.001) and S1 containing intermediate monocytes (9/14, P=0.006). Of the S1 positive post-vaccination patients, we sorted the CD16+ cells from six patients as previously performed for PASC patients9. Upon isolation of the protein, we demonstrated by LC-MS that these CD16+ cells from post-vaccination patients also contained S1 protein months after vaccination (Fig 5A). Further analysis revealed that these S1 positive, CD16+ cells also contained peptide sequences of S2, and mutant S1 peptides (Fig 5B). Page 6/14 Discussion The interaction between fractalkine and CX3CR1 has been reported to be involved in the pathogenesis of atherosclerosis, vasculitis, vasculopathies, and inflammatory brain disorders17 and could also be contributing to a vascular endotheliitis in post-vaccination individuals with PASC-like symptoms. Vascular inflammation has been shown to expose the collagen surface and platelet activation/adherence by way of glycoprotein 1b-IX-V-receptor (GPIb-IX-V) with collagen-bound von Willebrand factor (vWF)18,19. Activated platelets also release soluble CD40 ligand (sCD40L) which leads to recruitment of both NCM bind to fractalkine expressed on vascular endothelial cells through the expression of the fractalkine receptor CX3CR1 on the surface of NCM11. Fractalkine is also upregulated by IL-1, IFN-γ, and TNF-α12, cytokines that we have reported to be elevated in PASC9. CX3CR1 also provides a survival signal to non- classical monocytes through CX3CR1-dependent expression of the anti-apoptotic protein BCL213, 14. Stress and exercise mobilize non classical monocytes including up to 4 fold with exercise15, 16 The Page 7/14 Page 7/14 neutrophils and monocytes to the sites of vascular inflammation, thus activating the coagulation cascade20. Activated platelets also release CCL5/RANTES which binds to endothelial cells, promoting monocyte adhesion to inflamed endothelial tissues21 and acting as a chemoattractant for inflammatory cells. Studies in atherosclerosis have shown that CCR5 antagonists reduce non-classical monocyte recruitment to sites of atherosclerosis21,22. In addition, accumulation of non-classical monocytes can be reduced by statin treatment through reduction in fractalkine expression23,24. Interfering with these pathways may hold potential therapeutic targets for PASC and post-vaccination individuals with PASC- like symptoms10. Further, activated platelets and endothelial cells also secrete (VEGF) which induces angiogenesis and microvascular hyperpermeability. VEGF contributes to vasculitic neuropathy and also promotes a pro- inflammatory-prothrombotic environment25. Given the elevated levels of sCD40L and the frequent co- expression of VEGF, this pathway may also contribute to the thrombosis seen in some individuals post- vaccination. Taken together, these findings suggest a possible mechanism for the debilitating symptoms found in some patients weeks and months following vaccination. The findings that the immune profile and persistent S1 protein in CD16 + monocytes suggest that S1 protein persistence is a major contributor not only of symptoms in post-vaccination individuals with PASC-like symptoms but also may be a major contributor of PASC itself given that S1 alone delivered by vaccination can cause similar pathologic features. References 1. 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N Engl J Med. 2022 Jan 27;386(4):340-350. doi: 10.1056/NEJMoa2115481. Epub 2022 Jan 12. PMID: 35021002; PMCID: PMC8781262. 3. Fox SE, Falgout L, Vander Heide RS: COVID-19 myocarditis: quantitative analysis of the inflammatory infiltrate and a proposed mechanism. Cardiovasc Pathol. 2021 Jun 24;54:107361. doi: 10.1016/j.carpath.2021.107361. Epub ahead of print. PMID: 34174415; PMCID: PMC8223028 3. Fox SE, Falgout L, Vander Heide RS: COVID-19 myocarditis: quantitative analysis of the inflammatory infiltrate and a proposed mechanism. Cardiovasc Pathol. 2021 Jun 24;54:107361. doi: 10.1016/j.carpath.2021.107361. Epub ahead of print. PMID: 34174415; PMCID: PMC8223028 3. Fox SE, Falgout L, Vander Heide RS: COVID-19 myocarditis: quantitative analysis of the inflammatory infiltrate and a proposed mechanism. Cardiovasc Pathol. 2021 Jun 24;54:107361. doi: 10.1016/j.carpath.2021.107361. Epub ahead of print. PMID: 34174415; PMCID: PMC8223028 Page 8/14 Page 8/14 4. Hunter PR. Thrombosis after covid-19 vaccination. BMJ. 2021 Apr 14;373:n958. doi: 10.1136/bmj.n958. PMID: 33853865. 5. Violi F, Cammisotto V, Pastori D, Pignatelli P. Thrombosis in pre- and post-vaccination phase of COVID-19, European Heart Journal Supplements, Volume 23, Issue Supplement_E, October 2021, Pages E184-188, 6. Centers for Disease Control and Prevention. Post-COVID Conditions. July 12, 2021 7. Davis HE, Assaf GS, McCorkell L, Wei H, Low RJ, Re'em Y, Redfield S, Austin JP, Akrami A. Characterizing long COVID in an international cohort: 7 months of symptoms and their impact. EClinicalMedicine. 2021 Aug;38:101019. doi: 10.1016/j.eclinm.2021.101019. Epub 2021 Jul 15. PMID: 34308300; PMCID: PMC8280690. 8. References Patterson BK, Guevara-Coto J, Yogendra R, Francisco EB, Long E, Pise A, Rodrigues H, Parikh P, Mora J, Mora-Rodríguez RA. Immune-Based Prediction of COVID-19 Severity and Chronicity Decoded Using Machine Learning. 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Targeting the Monocytic-Endothelial-Platelet Axis with Maraviroc and Pravastatin as a Therapeutic Option to Treat Post-Acute Sequelae of COVID (PASC). doi.org/10.21203/rs.3.rs-1344323/v1 11. Patterson BK, Yogendra, R, Guevarra-Coto J et al. Targeting the Monocytic-Endothelial-Platelet Axis with Maraviroc and Pravastatin as a Therapeutic Option to Treat Post-Acute Sequelae of COVID (PASC). doi.org/10.21203/rs.3.rs-1344323/v1 12. Kapellos TS, Bonaguro L, Gemünd I, Reusch N, Saglam A, Hinkley ER, Schultze JL. Human Monocyte Subsets and Phenotypes in Major Chronic Inflammatory Diseases. Front Immunol. 2019 Aug 30;10:2035. doi: 10.3389/fimmu.2019.02035. PMID: 31543877; PMCID: PMC6728754. 12. Kapellos TS, Bonaguro L, Gemünd I, Reusch N, Saglam A, Hinkley ER, Schultze JL. Human Monocyte Subsets and Phenotypes in Major Chronic Inflammatory Diseases. Front Immunol. 2019 Aug 30;10:2035. doi: 10.3389/fimmu.2019.02035. PMID: 31543877; PMCID: PMC6728754. 13. Matsumiya T, Ota K, Imaizumi T, Yoshida H, Kimura H, Satoh K. Characterization of synergistic induction of CX3CL1/fractalkine by TNF-alpha and IFN-gamma in vascular endothelial cells: an essential role for TNF-alpha in post-transcriptional regulation of CX3CL1. J Immunol. 2010 Apr 15;184(8):4205-14. doi: 10.4049/jimmunol.0903212. Epub 2010 Mar 15. PMID: 20231691. 13. Matsumiya T, Ota K, Imaizumi T, Yoshida H, Kimura H, Satoh K. Characterization of synergistic induction of CX3CL1/fractalkine by TNF-alpha and IFN-gamma in vascular endothelial cells: an essential role for TNF-alpha in post-transcriptional regulation of CX3CL1. J Immunol. 2010 Apr 15;184(8):4205-14. doi: 10.4049/jimmunol.0903212. Epub 2010 Mar 15. PMID: 20231691. 14. Wang H, Cai J, Du S, Guo Z, Xin B, Wang J, Wei W, Shen X. References Fractalkine/CX3CR1 induces apoptosis resistance and proliferation through the activation of the AKT/NF-κB cascade in pancreatic cancer cells. Cell Biochem Funct. 2017 Aug;35(6):315-326. doi: 10.1002/cbf.3278. PMID: 28845524. 14. Wang H, Cai J, Du S, Guo Z, Xin B, Wang J, Wei W, Shen X. Fractalkine/CX3CR1 induces apoptosis resistance and proliferation through the activation of the AKT/NF-κB cascade in pancreatic cancer cells. Cell Biochem Funct. 2017 Aug;35(6):315-326. doi: 10.1002/cbf.3278. PMID: 28845524. 15. Landsman L, Bar-On L, Zernecke A, Kim KW, Krauthgamer R, Shagdarsuren E, Lira SA, Weissman IL, Weber C, Jung S. CX3CR1 is required for monocyte homeostasis and atherogenesis by promoting cell survival. Blood. 2009 Jan 22;113(4):963-72. doi: 10.1182/blood-2008-07-170787. Epub 2008 Oct 29. PMID: 18971423. 15. Landsman L, Bar-On L, Zernecke A, Kim KW, Krauthgamer R, Shagdarsuren E, Lira SA, Weissman IL, Weber C, Jung S. CX3CR1 is required for monocyte homeostasis and atherogenesis by promoting cell survival. Blood. 2009 Jan 22;113(4):963-72. doi: 10.1182/blood-2008-07-170787. Epub 2008 Oct 29. PMID: 18971423. Page 9/14 Page 9/14 16. Heidt T, Sager HB, Courties G, et al. Chronic variable stress activates hematopoietic stem cells. Nat Med. 2014;20(7):754-758. doi:10.1038/nm.3589 17. Jajtner AR, Townsend JR, Beyer KS, Varanoske AN, Church DD, Oliveira LP, Herrlinger KA, Radom-Aizik S, Fukuda DH, Stout JR, Hoffman JR. Resistance Exercise Selectively Mobilizes Monocyte Subsets: Role of Polyphenols. Med Sci Sports Exerc. 2018 Nov;50(11):2231-2241. doi: 10.1249/MSS.0000000000001703. PMID: 29957728. 18. Umehara H, Bloom ET, Okazaki T, Nagano Y, Yoshie O, Imai T. Fractalkine in vascular biology: from basic research to clinical disease. Arterioscler Thromb Vasc Biol. 2004 Jan;24(1):34-40. doi: 10.1161/01.ATV.0000095360.62479.1F. Epub 2003 Sep 11. PMID: 12969992. 19. Gawaz M, Langer H, May AE. Platelets in inflammation and atherogenesis. J Clin Invest. 2005 Dec;115(12):3378-84. doi: 10.1172/JCI27196. PMID: 16322783; PMCID: PMC1297269. 20. Varga-Szabo D, Pleines I, Nieswandt B. Cell adhesion mechanisms in platelets. Arterioscler Thromb Vasc Biol. 2008 Mar;28(3):403-12. doi: 10.1161/ATVBAHA.107.150474. Epub 2008 Jan 3. PMID: 18174460. 21. Flierl U, Bauersachs J, Schäfer A. Modulation of platelet and monocyte function by the chemokine fractalkine (CX3 CL1) in cardiovascular disease. Eur J Clin Invest. 2015 Jun;45(6):624-33. doi: 10.1111/eci.12443. Epub 2015 Apr 24. PMID: 25832902. 21. Flierl U, Bauersachs J, Schäfer A. Modulation of platelet and monocyte function by the chemokine fractalkine (CX3 CL1) in cardiovascular disease. Eur J Clin Invest. 2015 Jun;45(6):624-33. doi: 10.1111/eci.12443. Epub 2015 Apr 24. PMID: 25832902. 22. von Hundelshausen P, Weber KS, Huo Y, Proudfoot AE, Nelson PJ, Ley K, Weber C. References RANTES deposition by platelets triggers monocyte arrest on inflamed and atherosclerotic endothelium. Circulation. 2001 Apr 3;103(13):1772-7. doi: 10.1161/01.cir.103.13.1772. PMID: 11282909. 23. Thomas G, Tacke R, Hedrick CC, Hanna RN. Nonclassical patrolling monocyte function in the vasculature. Arterioscler Thromb Vasc Biol. 2015 Jun;35(6):1306-16. doi: 10.1161/ATVBAHA.114.304650. Epub 2015 Apr 2. PMID: 25838429; PMCID: PMC4441550. 24. Sakoda K, Yamamoto M, Negishi Y, Liao JK, Node K, Izumi Y. Simvastatin decreases IL-6 and IL-8 production in epithelial cells. J Dent Res. 2006 Jun;85(6):520-3. doi: 10.1177/154405910608500608. PMID: 16723648; PMCID: PMC3001137. 24. Sakoda K, Yamamoto M, Negishi Y, Liao JK, Node K, Izumi Y. Simvastatin decreases IL-6 and IL-8 production in epithelial cells. J Dent Res. 2006 Jun;85(6):520-3. doi: 10.1177/154405910608500608. PMID: 16723648; PMCID: PMC3001137. 25. Cimato TR, Palka BA. Fractalkine (CX3CL1), GM-CSF and VEGF-a levels are reduced by statins in adult patients. Clin Transl Med. 2014 Jun 14;3:14. doi: 10.1186/2001-1326-3-14. PMID: 24995121; PMCID: PMC4067646. 25. Cimato TR, Palka BA. Fractalkine (CX3CL1), GM-CSF and VEGF-a levels are reduced by statins in adult patients. Clin Transl Med. 2014 Jun 14;3:14. doi: 10.1186/2001-1326-3-14. PMID: 24995121; PMCID: PMC4067646. 26. Cuadrado MJ, Buendía P, Velasco F, Aguirre MA, Barbarroja N, Torres LA, Khamashta M, López- Pedrera C. Vascular endothelial growth factor expression in monocytes from patients with primary antiphospholipid syndrome. J Thromb Haemost. 2006 Nov;4(11):2461-9. doi: 10.1111/j.1538- 7836.2006.02193.x. Epub 2006 Sep 12. PMID: 16968331. 26. Cuadrado MJ, Buendía P, Velasco F, Aguirre MA, Barbarroja N, Torres LA, Khamashta M, López- Pedrera C. Vascular endothelial growth factor expression in monocytes from patients with primary antiphospholipid syndrome. J Thromb Haemost. 2006 Nov;4(11):2461-9. doi: 10.1111/j.1538- 7836.2006.02193.x. Epub 2006 Sep 12. PMID: 16968331. Data and materials availability: All requests for materials and raw data should be addressed to the corresponding author All requests for materials and raw data should be addressed to the corresponding author Competing interests: B.K.P, A.P., E.L, and EBF are employees of IncellDx, Inc Author contributions: R.Y., J.B, M.K. E.O. organized the clinical study and actively recruited patients. B.K.P, A.P., E.L., E.B.F, performed experiments and analyzed the data. J.G-C., R.A.M., J.M., C.B. performed the statistics and bioinformatics B.K.P., J.M., E.B.F, J.G-C., R.A.M., R.V. wrote and edited the draft of the manuscript and all authors contributed to revising the manuscript prior to submission. B.K.P., J.M., E.B.F, J.G-C., R.A.M., R.V. wrote and edited the draft of the manuscript and all authors contributed to revising the manuscript prior to submission. Funding: None Tables Table 1. Demographics of post-vaccination individuals with PASC-like symptoms. Patient Demographics Gender-M 14 F 36 Average age 41.8 (range 13-65) Time since last vaccine (mean) 105 days (range 38-245 days Figures Ethics: Page 10/14 All the patients/participants provided their written informed consent to participate in this study. The study was approved by the IRB or the Chronic COVID Treatment Center. Data and materials availability: Tables raphics of post-vaccination individuals with PASC-like symptoms. Table 1. Demographics of post-vaccination individuals with PASC-like symptoms. Patient Demographics Gender-M 14 F 36 Average age 41.8 (range 13-65) Time since last vaccine (mean) 105 days (range 38-245 days Figures Table 1. Demographics of post-vaccination individuals with PASC-like symptoms. Patient Demographics Gender-M 14 F 36 Average age 41.8 (range 13-65) Time since last vaccine (mean) 105 days (range 38-245 days Figures Figures Figure 1 Statistically significant elevation of cytokines in post-vaccination individuals with PASC-like symptoms compared to vaccinated, healthy individuals. Figure 1 Figure 1 Statistically significant elevation of cytokines in post-vaccination individuals with PASC-like symptoms compared to vaccinated, healthy individuals. Page 12/14 Figure 2 Flow cytometric quantification of S1-containing monocyte subsets as previously performed9. Increased S1-conatining intermediate and non-classical monocytes was statistically significant compared to vaccinated, healthy controls. Flow cytometric quantification of S1-containing monocyte subsets as previously performed9. Increased S1-conatining intermediate and non-classical monocytes was statistically significant compared to vaccinated, healthy controls. Page 13/14 Figure 3 (A) Liquid chromatography/mass spectrometry confirmation of S1 and S2 protein in flow cytometrically isolated cells from post-vaccination individuals with PASC-like symptoms. (B) Protein sequences found in CD16+ sorted monocytes including S1-mutated sequences. Figure 3 (A) Liquid chromatography/mass spectrometry confirmation of S1 and S2 protein in flow cytometrically isolated cells from post-vaccination individuals with PASC-like symptoms. (B) Protein sequences found in CD16+ sorted monocytes including S1-mutated sequences. Page 13/14 Figure 4 Figure 4 Figure 5 Page 14/14
https://openalex.org/W4316469072	https://www.scielo.br/j/rmat/a/yzthsVW8Tf8sSSSPCTnycyS/?lang=en&format=pdf	English	null	Influence of strength behavior in brick masonry prism and wallette under compression	Matéria	2,023	cc-by	5,780	Influence of strength behavior in brick masonry prism and wallette under compression Soundar Rajan M1 , Jegatheeswaran D2 1Sengunthar Engineering College, Civil Engineering, Namakkal, Tamilnadu 637205, Indi 2Sona College of Technology, Civil Engineering, Salem, Tamilnadu 636005, India. e-mail: soundarrajan06@gmail.com, profdjpillai@gmail.com ABSTRACT For decades, prisms and wallette have been used to explore masonry’s strength and deformation capabilities under uniaxial compression; moreover, the link between these two main test methods needs to be better estab lished. Using standard masonry design expressions and data, the prism or wallette test can determine compres sion qualities. The researchers compared the standards and came to judgments on their conservatism or lack thereof, independent of how the rules for those design standards were formed. As a result, an attempt was made to link the behaviour of the enormous masonry prism to the wallette pressure behaviour. Prisms and wallette were made and tested by three different types of units and three mortar compositions. According to test results, the compressive strength of prism tests is consistently higher than that of identical wallette samples. It has remained demonstrated that a linear relationship concerning the compressive strengths of prisms and wallette is tolerable. To test data from the prisms and wallette, a simplified analytical model for the deformation properties of masonry is given. Keywords: Strength under Compression; Initial Rate of Absorption; Peak Strain; Ductility. Keywords: Strength under Compression; Initial Rate of Absorption; Peak Strain; Ductility. Corresponding Author: Soundar Rajan M Accepted on 07/12/2022 Received on 12/10/2022 1. INTRODUCTION The laboratory-constructed prisms were tested, and predictive expressions touching masonry compressive Received on 12/10/2022 Accepted on 07/12/2022 DOI: https://doi.org/10.1590/1517-7076-RMAT-2022-0260 SOUNDAR RAJAN, M.; JEGATHEESWARAN, D.; revista Matéria, v.28, n.1, 2023 strength to the constituent ingredient compressive strengths and the masonry modulus of elasticity were developed based on the experimental results. Masonry compression stress-strain numerical fashions have been additionally developed to predict masonry stress-strain behaviour [5, 6]. In-plane shear strength of unreinforced masonry partitions relies upon in most cases, shear/tensile bond strength, issue ratio and pre-compression levels; compressive strength of masonry solely influences when the last failure is dominated. Failure of traditional unreinforced masonry shear partitions is characterized by brit tle tensile/shear cracking via the susceptible bond between the unit and mortar [7]. The important mechanical properties of masonry partitions are the compression, tensile, flexural and shear strengths. Of these properties, the most essential is the compression strength because, in general, masonry partitions are submitted to greater excessive vertical load than horizontal load produced through wind or seismic activity. Concrete hollow block masonry buildings are currently being used conspicuously in many parts of the world. There is an urgent need to conquer experimental and numerical information of special bonding preparations [8]. As mentioned, figuring out the last strength has been a primary study of masonry studies. Some of the troubles that also want explanation consist of the implicit assumption that masonry failure takes place on the last strength and that there may be no enormous distinction between masonry built with a susceptible and a sturdy mortar. The observation herein indicates that the failure mechanism of whole concrete block masonry is a chain of consecutive outcomes and is no longer an unmarried last event. The observation indicates that masonry failure relies upon the relative strength of the mortar and block. This expertise might also assist engineers in deciding a preferred overall performance degree for their masonry shape in place of, without a doubt the last strength and very last mode of failure [9, 10]. Irrespective of the share of reinforcement, no proof of yielding of the steel bar steel bar with confining detail may be determined in the literature; this suggests that the confining factors used to date showcase very restricted cracking till the fall apart of the masonry, even though the masonry energy is superior through the confining factors. 2.1. Materials A set of three different types of units and three types of mortar compositions were used to construct the spec imens. These masonry units have been deliberately chosen to cover a range of geometric dimensions and strengths. This work used three types of mortar compositions to construct the samples in combination with five different types of units as per codes [19–22]. The cement-sand ratios of M1:3, M1:4 and M1:5 was used for the mortar mix as per IS 269. The properties of six samples with different cement mortar compositions are shown in Table 1. The Geometric property values of the units are shown in Table 2. Clay bricks are entitled in the subsequent descriptions C.B. charted by their sequential figure. Six samples were cast, tested and analyzed to determine the properties. 1. INTRODUCTION Since there’s clean proof within the literature that the steel bar with confining factors no longer take part significantly in this research and most uncomplicated and not using a shear tie has been used [11, 12]. Containment of cement-based materials is typically done using ribbons, spirals, or circular hoops. Such a configuration is usually very long in cross-section in one dimension and challenging to achieve with relatively thin masonry walls in the other dimension. For masonry, one approach to inclusion is to use thin galvanized steel sheets placed at the mortar joints during construction. Boundary plates serve the same purpose as the lateral rebar of standard concrete components. As the compressive strain of the masonry increases, the masonry grows, and the tensile strain of the panel upturns. Next, the masonry is in a 3-axis compressed state, improving the strength and elasticity of the masonry [13, 14]. Given the importance of seismic action and the potential seismic susceptibility of masonry structures, an essential part of these efforts will be devoted to numerical simulations of masonry rupture under tensile and shear forces [15, 16]. Due to changes in regulations and use, masonry buildings often require structural reassessment and possibly retrofit measures. In this context, the compressive response of masonry plays a decisive role in evaluating the strength of masonry buildings concerning both the effects of vertical loads caused by vertical and horizontal actions. The response properties of masonry to com pression include determining parameters such as the compressive strength, modulus of elasticity, and general curve of compressive deformation under static and cyclic loading of the composite material [17, 18]. 1. INTRODUCTION Masonry has traditionally been a frequent and profitable potential of cladding and load-bearing structures. Today, it constitutes a vast share of global constructions regularly of ancient and cultural signiﬁcance. Masonry is a heterogeneous cloth with a complex, nonlinear, anisotropic behavior which can be attributed to the unique fabric elements and the great interfaces. For centuries, masonry used to be specific with lime mortars. However, as most limes construct energy slowly via carbonation, they have been superseded; ﬁrst by using hydraulic limes and then via Portland cement which rapidly develops strength on hydration. However, over two decades, there has been a renewed focus on using hydrated and hydraulic lime mortars for repairs and new buildings. The experience of masonry energy and deformation traits is vital as these decide overall masonry performance over time and allowable stress and stiffness in diagram codes for new buildings [1]. The final years witnessed considerable advances in masonry mechanics, each with recognition of exper imental trying out and numerical modelling. Despite this, the composite behavior of whole concrete block masonry nevertheless represents a genuine challenge. Hollow concrete blocks are constructions constituted by thin walls that interact between themselves and usually feature one-of-a-kind geometries. Besides the difficul ties in symbolizing the mechanical property of mortar internal the composite, the mechanical strength of the concrete from the blocks are commonly no longer known because the checks are carried out on entire blocks [2]. The strength of masonry under compression is a fundamental property in the design of masonry structures. Although test specimens of wall panels for determining the compressive strength of masonry as per AS3700 of stack bonded prisms tests. The compression behaviour of masonry prisms depends on the thickness of mortar joints, the stiffness of masonry units and mortar, and the units’ shape [3]. Bond properties are essential for masonry walls’ integrity, serviceability, and flexural and shear response. Various investigations address improving the bond between the masonry assemblies and the mortar layers. The method of bond improvement commences as quickly as the clean mortar is utilized on the masonry units. The absorption of mortar fluids precedes the cement compounds’ subsequent placement into the masonry unit and the consequential transport of mortar fines in the joint to the mortar unit interface [4]. 2.2. Testing methodology The Bureau of Indian Standards codes [23] commends the examination of masonry prisms of least 40 cm in height with height-to-thickness proportion in the middle of 2 to 5 for defining the strength of the masonry under compression behaviour. The British standard [24] commends the analysis of masonry panels of range between SOUNDAR RAJAN, M.; JEGATHEESWARAN, D.; revista Matéria, v.28, n.1, 2023 Table 1: Properties of cement mortar compositions as per IS 269. DESCRIPTION M1:3 M1:5 Density (kg/m3) 1900 1800 Water absorption (%) 8.6 8.5 Compressive strength (MPa) 6.5 4.2 Flexural strength (MPa) 1.15 1.02 Elastic modulus (MPa) 4500 3500 Table 1: Properties of cement mortar compositions as per IS 269. Table 1: Properties of cement mortar compositions as per IS 269. Table 2: Properties of masonry unit assembly as per IS 1905. DESCRIPTION CB1 CB2 CB3 Density (kg/m3) 1900 2010 1700 Compressive strength (MPa) 5.3 15.8 3.8 Modulus of rupture (MPa) 1.83 3.66 1.26 Water absorption (%) 10.6 4.5 12.9 Table 2: Properties of masonry unit assembly as per IS 1905. Table 3: Details of casted specimens. Table 3: Details of casted specimens. DESCRIPTION DIMENSIONS (l × t × h) mm h/t RATIO Prism CB 1 225 × 150 × 490 3.27 CB 2 210 × 100 × 410 4.10 CB 3 200 × 90 × 440 4.89 Wallette CB 1 460 × 150 × 490 3.27 CB 2 430 × 100 × 410 4.10 CB 3 410 × 90 × 440 4.89 1.2 to 1.8 m in length and range between 2.4 to 2.7 m in height, which wants an extravagant test setup. Hence, masonry Wallets of size are part of the masonry prisms and masonry wall panels that have been tried out in this examination. The intention of taking such mortar compositions is to analyze the consequence of maximum strength-stiff mortar and minimum strength-soft mortar on the strength of masonry under compression behaviour. The wallets would characterize the behaviour of walls superior to prisms due to the occurrence of joints [25]. In this study, 30 prism and 20 wallette samples were designed and tested for the three different dimensions and with three cement mortar combinations to evaluate the strength and strain behavior of the masonry under compression. The complete test matrix and sample dimensions are given in Table 3. The dimensions and the number of courses between the combinations varied since their units’ dimensions differed. 2.2. Testing methodology However, the relationship between the height and thickness of the prisms and the corre sponding wallettes remained the same for each combination. The prisms were built and tested in accordance with ASTM C1314-16 and illustrate similar prism test methods for evaluating the compressive strength of masonry. The sample specimens were evaluated under the steady state in a Universal Testing Machine of 600kN capacity. A thin 3 mm plywood sheet was positioned on top and bottom of the sample specimen overloaded in the middle of 25 mm thick steel plates. The sample Wallets were evaluated by insertion them in a self-pulling loading frame and loaded using hydraulic jacks with capabilities of 500kN and 1000kN. The schematic diagram of the test setup for Prism/Wallets is shown in the Figures 1 and 2. Displacement transducers were attached to the samples to record the vertical deformation of the prisms and wallets under compression. SOUNDAR RAJAN, M.; JEGATHEESWARAN, D.; revista Matéria, v.28, n.1, 2023 The measurement of the mean deformation was carried out to eliminate possible eccentricities in the direction of the height and thickness of the samples. Loads and displacements were measured and recorded with a synchronized data acquisition system. The deformation measurements were carried out in the three test pieces [26]. 3. RESULT AND DISCUSSIONS 3.1. Compressive strength Figure 1: Test setup for masonry prism. Figure 2: Test setup for masonry wallette. Figure 1: Test setup for masonry prism. Figure 1: Test setup for masonry prism. Figure 2: Test setup for masonry wallette. The measurement of the mean deformation was carried out to eliminate possible eccentricities in the direction of the height and thickness of the samples. Loads and displacements were measured and recorded with a synchronized data acquisition system. The deformation measurements were carried out in the three test pieces [26]. 3.1. Compressive strength The strength of the prisms and Wallets in compression was determined under a uniaxial pressure load arrangement. The average compressive strengths of the tested prisms and wallets are given in Table 4 and SOUNDAR RAJAN, M.; JEGATHEESWARAN, D.; revista Matéria, v.28, n.1, 2023 SOUNDAR RAJAN, M.; JEGATHEESWARAN, D.; revista Matéria, v.28, n.1, 2023 However, evaluation of the compressive strength of masonry has been reported in many studies in the past and has behaviored limited research on prism and wallette testing methods. The specimens exhibited a quasi-linear behaviour for a low load level and reduced their stiffness when they exceeded approximately 50% of the maximum load [29]. The equation arrived from the correlation between the compressive strength of Masonry prisms and wallettes Mortar Mix. The correlation between compressive strength of masonry prism and wallette graphical figure is shown in Figures 5 and 6. as there may be inherent differences. However, evaluation of the compressive strength of masonry has been reported in many studies in the past and has behaviored limited research on prism and wallette testing methods. The specimens exhibited a quasi-linear behaviour for a low load level and reduced their stiffness when they exceeded approximately 50% of the maximum load [29]. The equation arrived from the correlation between the compressive strength of Masonry prisms and wallettes Mortar Mix. The correlation between compressive strength of masonry prism and wallette graphical figure is shown in Figures 5 and 6. For Mix M1:3 Y = 0.9238x − 0.2704 R2 = 0.9947 For Mix M1:5 Y = 0.9304x − 0.2217 R2 = 0.9949 For Mix M1:3 Y = 0.9238x − 0.2704 R2 = 0.9947 For Mix M1:5 Y = 0.9304x − 0.2217 R2 = 0.9949 SOUNDAR RAJAN, M.; JEGATHEESWARAN, D.; revista Matéria, v.28, n.1, 2023 SOUNDAR RAJAN, M.; JEGATHEESWARAN, D.; revista Matéria, v.28, n.1, 2023 graphical representation format as shown in Figures 3 and 4. Uniform strength has dominated the compressive strength of wall prisms and wallets. However, a slight increase in the compressive strength of the masonry prisms and wallets while changing the strength of the mortar was caused. Therefore, the results authenticate that the compressive strength of mortar has a negligible influence on the compressive strength of masonry, which is consistent with the results of preceding studies [27]. In some cases, high-strength bonding can cause block failure. The thin mortar layer and high adhesion contributed to increasing the compressive strength of the bricks due to the low lateral expansion of the mortar layer [28]. In the combinations tested, it can be seen that the compressive strength of the prism is higher than that of the wallette. Prism-to-wallette compressive strength ratios range from 1.05 to 1.26, depending on the ratio of filler to mortar. Although the lowest and highest ratios were recorded for the CB1 (M1:3) and CB3 (M1:3) mor tar combinations, the reason for the discrepancy in strength values of the prism and wallette cannot be explained Figure 3: Compressive strength of masonry prism. Figure 4: Compressive strength of masonry wallette Table 4: Compressive strength of specimens. DESCRIPTION CATEGORY MEAN COMPRESSIVE STRENGTH M1:3 M1:5 CB 1 Prism 2.35 2.18 Wallette 1.75 1.59 CB 2 Prism 7.47 6.92 Wallette 6.65 6.25 CB 3 Prism 1.58 1.31 Wallette 1.32 1.18 Table 4: Compressive strength of specimens. Figure 3: Compressive strength of masonry prism. Figure 3: Compressive strength of masonry prism. Figure 4: Compressive strength of masonry wallette Figure 4: Compressive strength of masonry wallette. Figure 4: Compressive strength of masonry wallette. SOUNDAR RAJAN, M.; JEGATHEESWARAN, D.; revista Matéria, v.28, n.1, 2023 Figure 5: Correlation between compressive strength of masonry prism and wallettes (M1:3). Figure 6: Correlation between compressive strength of masonry prism and wallettes (M1:5). Figure 5: Correlation between compressive strength of masonry prism and wallettes (M1:3). Figure 5: Correlation between compressive strength of masonry prism and wallettes (M1:3). Figure 6: Correlation between compressive strength of masonry prism and wallettes (M1:5). Figure 6: Correlation between compressive strength of masonry prism and wallettes (M1:5). as there may be inherent differences. 3.2. Stress strain curve The axial stress values were calculated by dividing the recorded axial displacements by the gauge length (usually more than one-third of the sample height). The matching stress values from the load measurement were fitted to record the deformation of the stress curves of the sample. Under uniaxial compression, both masonry prisms and Wallets displayed quasi-brittle behaviour. All samples had a nearly linear stress-strain response until they reached 25 to 50 per cent of their break ing strength, then a nonlinear pattern until the break was detected and it was shown in the Figures 7–9. The emergence of vertical fissures in the specimens was primarily responsible for their nonlinear behaviour. As a result, the elasticity modulus of the bricks CB1, CB2, and CB3 is 750 MPa, 5300 MPa, and 1500 MPa, respec tively. As a result, the brick’s strength and deformation qualities have significantly impacted the masonry’s overall stress-strain behaviour. The stress-strain curves of the samples were impacted by the type of mortar used, particularly beyond the linear elastic portions of the curves. The M1:5 mortar ratio has a lesser compressibility than other mortar ratios. Furthermore, there was no apparent separation between the samples in the lower half of the stress-strain curves. In most cases, the testing was stopped because the samples were significantly damaged, posing a risk to the apparatus. SOUNDAR RAJAN, M.; JEGATHEESWARAN, D.; revista Mat Figure 7: Stress – strain curve for CB1 type prism and wallette. SOUNDAR RAJAN, M.; JEGATHEESWARAN, D.; revista Matéria, v.28, n.1, 2023 Figure 7: Stress – strain curve for CB1 type prism and wallette. Figure 8: Stress – strain curve for CB2 type prism and wallette. Figure 9: Stress – strain curve for CB3 type prism and wallette. Figure 7: Stress – strain curve for CB1 type prism and wallette. Figure 8: Stress – strain curve for CB2 type prism and wallette. Figure 8: Stress – strain curve for CB2 type prism and wallette. Figure 9: Stress – strain curve for CB3 type prism and wallette. Figure 9: Stress – strain curve for CB3 type prism and wallette. As a result, the stress-strain curves’ whole downward regions need to be adequately recorded. The com pressibility of the Wallets is higher than that of the matching prisms, as observed. Where the prisms are solely in a stacked bond, including bed joints and pre-mortar in Wallets, has enhanced axial deformability. 3.2. Stress strain curve At one- third of the maximum stress of the samples, the modulus of elasticity of the prisms and wallets was calculated and tabulated in Table 5. The corresponding strain measured at one-third of maximum tension is called elastic elongation. The maximum strain of the investigated samples was calculated based on their maximum stress. Simultaneously, the relatively elastic and peak strain shown in Tables 6 and 7 are observed and listed. The strain at break was calculated based on 80% of the post-peak stress. The stress-strain curve, modelled as an elastic-per fect-plastic curve, was used to determine the elasticity of the prisms and wallet [30, 31]. The intersection point of the bilinear approximation corresponds to the yield strain. The mortar proportion may not affect the elasticity modulus of the brickwork and its characteristics. This is especially true for the low-strength brick-and-mortar combinations studied in this study. Because ductility is defined as the link between strain at break and elastic limit, there was no systematic difference in ductility tested between prisms and Wallets; the ductility values listed in Table 8 support this con clusion. Consequently, averaging the ten possibilities yielded a value of 1.7 for the elasticity of the brickwork evaluated under pressure in this investigation. It should be noted, however, that every other standard data, with the exception, is based on prism testing. As a result of the findings of this study, the relationships between the modulus of elasticity and the compressive strength of prisms and Wallets have been established and are depicted SOUNDAR RAJAN, M.; JEGATHEESWARAN, D.; revista Matéria, v.28, n.1, 2023 Table 5: Elastic modulus of tested specimens. Table 5: Elastic modulus of tested specimens. DESCRIPTION CATEGORY ELASTIC MODULUS (MPA) M1:3 M1:5 CB 1 Prism 1880 1567 Wallette 996 839 CB 2 Prism 4989 4912 Wallette 3962 3705 CB 3 Prism 1035 933 Wallette 696 506 Table 6: Elastic strain of tested specimens. Table 6: Elastic strain of tested specimens. Table 6: Elastic strain of tested specimens. DESCRIPTION CATEGORY ELASTIC STRAIN M1:3 M1:5 CB 1 Prism 0.00039 0.00043 Wallette 0.00058 0.00065 CB 2 Prism 0.00041 0.00043 Wallette 0.00049 0.00054 CB 3 Prism 0.00045 0.00051 Wallette 0.00063 0.00087 Table 7: Peak strain of tested specimens. 3.2. Stress strain curve DESCRIPTION CATEGORY PEAK STRAIN M1:3 M1:5 CB 1 Prism 0.0028 0.0028 Wallette 0.0030 0.0031 CB 2 Prism 0.0020 0.0021 Wallette 0.0023 0.0025 CB 3 Prism 0.0030 0.0032 Wallette 0.0033 0.0035 Table 7: Peak strain of tested specimens. Table 8: Ductility of tested specimens. DESCRIPTION CATEGORY DUCTILITY AVERAGE DUCTILITY M1:3 M1:5 CB 1 Prism 1.8 1.5 1.65 Wallette 1.7 1.8 1.75 CB 2 Prism 1.6 1.4 1.50 Wallette 1.3 1.2 1.25 CB 3 Prism 1.9 2.1 2.00 Wallette 2.0 1.5 1.75 Table 8: Ductility of tested specimens. Table 8: Ductility of tested specimens. and expressed in pictorial representation in Figures 10–13. For the prism and wallette combinations, excellent correlations were found. The elastic modulus of the prism is almost 34% larger than that of the comparable wallettes elastic modulus. and expressed in pictorial representation in Figures 10–13. For the prism and wallette combinations, excellent correlations were found. The elastic modulus of the prism is almost 34% larger than that of the comparable wallettes elastic modulus. Furthermore, the strain peak is one of the primary characteristics that determine the stress-strain curve of brickwork under pressure (p). The maximum elongation varies between 0.002 and 0.0032 for prisms and SOUNDAR RAJAN, M.; JEGATHEESWARAN, D.; revista Matéria, v.28, n.1, 2023 Figure 10: Comparison of elastic modulus of masonry prisms and wallette. Figure 10: Comparison of elastic modulus of masonry prisms and wallette. Figure 11: Comparison of elastic strain of masonry prisms and wallette. Figure 12: Comparison of peak strain of masonry prisms and wallette. 0.0023-0.0035 for wallets. As a result, various scholars have proposed formulas for calculating maximal defor mations based on masonry strength, mortar strength, and modulus of elasticity [31–33]. Figure 10: Comparison of elastic modulus of masonry prisms and wallette. Figure 11: Comparison of elastic strain of masonry prisms and wallette. Figure 11: Comparison of elastic strain of masonry prisms and wallette. Figure 12: Comparison of peak strain of masonry prisms and wallette. Figure 12: Comparison of peak strain of masonry prisms and wallette. 0.0023-0.0035 for wallets. As a result, various scholars have proposed formulas for calculating maximal defor mations based on masonry strength, mortar strength, and modulus of elasticity [31–33]. 0.0023-0.0035 for wallets. As a result, various scholars have proposed formulas for calculating maximal defor mations based on masonry strength, mortar strength, and modulus of elasticity [31–33]. 4. CONCLUSIONS The experimental examination of masonry prisms and Wallets under uniaxial compression is presented in this article. To evaluate the strength and strain behavior, 30 prisms and 24 Wallets with three different units and three types of mortar were tested. The experimental data acquired in this inquiry can be used to derive the following findings. • According to the empirical connection determined using linear regression analysis, the prism’s compressive strength is roughly 25% more than the wallette strength. Taking the property factor into consideration, the compressive strength of the wallette may be safely estimated to be 0.67 times that of the prism why Masonry standards overstate masonry’s compressive strength, mainly when low-strength units (less than 5 MPa) are applied. • Prisms and Wallets’ modulus of elasticity may be predicted using relationships based on their compressive strength. Other formulas are proposed to predict the maximum elongation of the prism and the bag under compression based on the piece and mortar’s compressive strengths. • Prisms and Wallets’ modulus of elasticity may be predicted using relationships based on their compressive strength. Other formulas are proposed to predict the maximum elongation of the prism and the bag under compression based on the piece and mortar’s compressive strengths. • A simple stress-strain analytical model for the ascending section of the brick prism and Wallets under pressure is provided, with a second-order polynomial function for the ascending part and a linear model for the descending part. The suggested model requires the unit and mortar compressive strengths to create the stress-strain curves. • A simple stress-strain analytical model for the ascending section of the brick prism and Wallets under pressure is provided, with a second-order polynomial function for the ascending part and a linear model for the descending part. The suggested model requires the unit and mortar compressive strengths to create the stress-strain curves. • Using the wallette strength to prism connection presented in this publication, you may convert prism strength to wallette strength and apply design standards to incorporate it into genuine load-bearing wall construction. Solid blocks and bricks are the only materials that can be converted, while hollow block masonry supported by a shell was not considered. SOUNDAR RAJAN, M.; JEGATHEESWARAN, D.; revista Matéria, v.28, n.1, 2023 SOUNDAR RAJAN, M.; JEGATHEESWARAN, D.; revista Matéria, v.28, n.1, 2023 Figure 13: Comparison of ductility of masonry prisms and wallette. Figure 13: Comparison of ductility of masonry prisms and wallette. Figure 13: Comparison of ductility of masonry prisms and wallette. case, the bricks will be beneath triaxial compression, and the mortar will be beneath uniaxial compression and bilateral tension. In such a condition, the miscarriage of masonry is started by the tensile excruciating of the mortar in the joint if the brick–mortar bond is unbroken. The mortar miscarriage will then extend to the brick, causing masonry. case, the bricks will be beneath triaxial compression, and the mortar will be beneath uniaxial compression and bilateral tension. In such a condition, the miscarriage of masonry is started by the tensile excruciating of the mortar in the joint if the brick–mortar bond is unbroken. The mortar miscarriage will then extend to the brick, causing masonry. The regular letdown of solid masonry under pressure is well understood from previous studies. The incompatible elastic properties of the element and the mortar cause cracks in the masonry or mortar elements parallel to the direction of loading under pressure. All prisms and Wallets failed with vertical cracks parallel to the load direction on the front and side surfaces of the specimens. Most of the time, the cracks in the mortar interfaces started around 70–85% of the final failure of the samples. For the safety of the instrumentation, the test was cancelled after the maximum load was reduced by almost 20–50% due to the samples showing severe cracks. The axial stress values were calculated by dividing the recorded axial displacements by the gauge length. The matching stress values from the load measurement were fitted to record the stress curves deformation of the sample. 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https://openalex.org/W4281491665	https://edukatif.org/index.php/edukatif/article/download/2716/pdf	Indonesian	null	Landasan Filsafat Pendirian dan Pendidikan Karakter Kemandirian Anak Yatim di Lembaga Kesejahteraan Sosial Anak	Edukatif	2,022	cc-by-sa	6,736	Abstrak Anak yatim - piatu dan anak terlantar mereka merupakan seorang anak. Mereka memperlukan pendampingan dan pengawasan. Menyantuni anak yatim, yatim piatu dan terlantar merupakan perintah agama. Namun santunan yang berlebihan akan menjadikan anak memiliki karakter kemandirian rendah. Negara mengemban amanat memelihara dan melindungi segenap dan seluruh anak yang berada dalam kondisi kurang pendampingan. Masyarakat juga memiliki peran penting dengan membentuk lembaga sosial. Tujuan penelitian ini berupaya mengetahui landasan filsafat pendirian lembaga sosial dan mengetahui praktek lembaga sosial yang tidak hanya mengurusi masalah kesejahteraan, namun juga dapat berperan aktif dalam pendidikan karakter kemandirian. Dengan pendekatan kualitatif dan metode studi kasus, penelitian ini menyimpulkan bahwa pendirian lembaga sosial merupakan aktualisasi dari filsafat nilai-nilai moral dan ajaran agama serta sebagai jalan melaksanakan amanat pendiri bangsa. Sebagai pendampingan alternatif lembaga kesejahteraan sosial anak berdasarkan landasan filsafat dapat melakukan inovasi pembinaan anak asuhnya dengan prinsip akuntabilitas dan transparansi dalam pengelolaan. g p p p p g Kata Kunci: karekater kemandirian, Anak yatim, lembaga residensial, landasan filsafat. Edukatif : Jurnal Ilmu Pendidikan Volume 4 Nomor 3 Tahun 2022 Halm 3979 - 39 EDUKATIF: JURNAL ILMU PENDIDIKAN Research & Learning in Education https://edukatif.org/index.php/edukatif/index Edukatif : Jurnal Ilmu Pendidikan Volume 4 Nomor 3 Tahun 2022 Halm 3979 - 3989 EDUKATIF: JURNAL ILMU PENDIDIKAN Research & Learning in Education https://edukatif.org/index.php/edukatif/index Landasan Filsafat Pendirian dan Pendidikan Karakter Kemandirian Anak Yatim di Lembaga Kesejahteraan Sosial Anak Arif Hidayat1, Abdul Wachid Bambang Suharto2 UIN SAIZU Purwokerto, Indonesia1 UINSAIZU Purwokerto, Indonesia2 E-mail : rif99rif@gmail.com1, abdulwachidbs33@gmail.com2 PENDAHULUAN Disekitar kita masih banyak calon generasi penerus bangsa yang hidup dalam keadaan memprihatinkan. Ada yang secara ekonomi serba kekurangan. Ada yang harus hidup sebatang kara karena orang tuanya meninggal. Ada juga anak-anak terlantar akibat permasalahan orang tuanya sehingga perhatian terhadap anak- anak sangat kurang. Permasalahan-permasalahan tersebut dapat menghambat pertumbuhan kepribadian mereka baik mental maupun spiritual. Kementerian sosial merilis data per tanggal 7 September 2021 mengungkap jumlah anak yatim maupun anak yatim-piatu korban pandemi Covid-19 berjumlah 252.021 anak. Jumlah tersebut berdasarkan data pemerintah kabupaten kota di provinsi seluruh Indonesia. Anak yatim, piatu dan anak dari kaum dhuafa serta anak-anak terlantar karena faktor lain tentu jumlahnya lebih banyak lagi. Pasal 334 ayat Undang-Undang Dasar 1945 menyebutkan bahwa tanggung jawab anak-anak terlantar dipelihara oleh negara. Negara tidak dapat lepas dari persoalan sosial tersebut. Salah satu kebijakan negara dalam hal ini adalah Kementerian Sosial adalah dengan mengeluarkan kebijakan tentang Lembaga Kesejahteraan Sosial Anak yang disingkat dengan LKSA. Dalam surah al- Baqarah ayat 220 dijelaskan tentang anak yatim (Ikhsan, 2015). Mendampingi dan mengurus anak yatim merupakan perbuatan yang baik. Mengurus anak yatim tentu sama seperti mengurus anak-anak pada umumnya. Berdasarkan ayat ini, pendampingan anak yatim tidak cukup negara yang mengurus, namun masyarakat juga memiliki kewajiban mengurus anak yatim. Kebiasaan masyarakat, setiap bulan Muharram, dibentuk panitia-panitia dalam rangka santunan anak yatim. Panitia tersebut mengumpulkan dana dari masyarakat yang berniat untuk memberikan sumbangan kepada anak yatim. Kegiatan seperti ini tentu sangat baik. Tidak ada yang salah dalam tradisi santunan anak yatim pada bulan Muharram. Namun tidak sedikit keluarga anak yatim dan anak yatim menjadikan bantuan tersebut tidak mandiri. Masa anak adalah masa berkembang, dan memiliki sifat keterganungan kepada orang lain. Situasi dan kondisi keadan mutlak masih membutuhkan bantuan, bimbingan dari orang tua, keluarga atau pengganti dari orang tua. Mendidik, membimbing dan mengarahkan adalah beberapa tugas dari orang tua(Desmita, 2009). Lembaga panti asuhan merupakan lembaga pendidikan komunal bagi anak-anak terlantar, keterlantaran anak terjadi karena anak tidak mendapat penghidupan atas keluarga yang tidak layak, sehingga kebutuhan anak tidak terpenuhi secara memadai dalam ekonomi dan sosial. Dalam melaksanakan fungsinya, lembaga kesejahteraan sosial anak harus menyesuaikan lingkungan kelembagaan untuk menciptakan sebuah suasana, pola kehidupan di dalam sebuah keluarga yang harmonis. Penyelenggaraan kesejahteraan sosial memiliki tujuan untuk menaikan taraf kualitas, kualitas hidup, dan kesejahteraan serta kelangsungan hidup. 3980 Landasan Filsafat Pendirian dan Pendidikan Karakter Kemandirian Anak Yatim di Lembaga Kesejahteraan Sosial Anak – Arif Hidayat, Abdul Wachid Bambang Suharto DOI : https://doi.org/10.31004/edukatif.v4i3.2716 3980 Landasan Filsafat Pendirian dan Pendidikan Karakter Kemandirian Anak Yatim di Lembaga Kesejahteraan Sosial Anak – Arif Hidayat, Abdul Wachid Bambang Suharto DOI : https://doi.org/10.31004/edukatif.v4i3.2716 Abstract Orphans and their abandoned children are children. They need assistance and supervision. Caring for orphans, orphans and displaced people is a religious order. But excessive compensation will make children have a low independence character. The State carries out the mandate to maintain and protect all and all children who are in a state of under-assistance. Society also has an important role by forming social institutions. The purpose of this research seeks to find out the foundation of the philosophy of the establishment of social institutions and know the practice of social institutions that not only deal with welfare problems, but can also play an active role in the education of the character of self-reliance. With a qualitative approach and case study methods, the study concluded that the establishment of social institutions is an actualization of the philosophy of moral values and religious teachings and as a way of carrying out the mandate of the nation's founder. As an alternative assistance, children's social welfare institutions based on the foundation of philosophy can innovate foster children with the principles of accountability and transparency in management. Keywords: the character of independence, orphans, residential institutions, philosophical foundations. Edukatif : Jurnal Ilmu Pendidikan Vol 4 No 3 Tahun 2022 p-ISSN 2656-8063 e-ISSN 2656-8071 Copyright (c) 2022 Arif Hidayat, Abdul Wachid Bambang Suharto  Corresponding author Email : rif99rif@gmail.com ISSN 2656-8063 (Media Cetak) DOI : https://doi.org/10.31004/edukatif.v4i3.2716 ISSN 2656-8071 (Media Online) Copyright (c) 2022 Arif Hidayat, Abdul Wachid Bambang Suharto Copyright (c) 2022 Arif Hidayat, Abdul Wachid Bambang Suharto  Corresponding author Email : rif99rif@gmail.com DOI : https://doi.org/10.31004/edukatif.v4i3.2716  Corresponding author Email : rif99rif@gmail.com DOI : https://doi.org/10.31004/edukatif.v4i3.2716 ISSN 2656-8063 (Media Cetak) ISSN 2656-8071 (Media Online) ISSN 2656-8063 (Media Cetak) ISSN 2656-8071 (Media Online) Edukatif : Jurnal Ilmu Pendidikan Vol 4 No 3 Tahun 2022 p-ISSN 2656-8063 e-ISSN 2656-8071 Edukatif : Jurnal Ilmu Pendidikan Vol 4 No 3 Tahun 2022 p-ISSN 2656-8063 e-ISSN 2656-8071 3980 Landasan Filsafat Pendirian dan Pendidikan Karakter Kemandirian Anak Yatim di Lembaga Kesejahteraan Sosial Anak – Arif Hidayat, Abdul Wachid Bambang Suharto DOI : https://doi.org/10.31004/edukatif.v4i3.2716 PENDAHULUAN Selain itu memiliki arah untuk memulihkan fungsi sosial dalam rangka mencapai kemandirian serta meningkatkan sosial masyarakat dalam mencegah , mengontrol dan menangani masalah terkait kesejahteraan sosial(Kuntjorowati, 2018). Terlebih saat pandemi covid, perhatian dalam bidang pendidikan terhadap anak yatim cenderung terabaikan. Namun dengan adanya lembaga kesejahteraan sosial anak, pemberian pendidikan kepada anak yatim dapat diatasi dengan baik. Beberapa LKSA menggunakan model semi homeschooling anak asuhnya. Dengan model ini anak asuh masih dapat melaksanakan pembelajaran daring dengan baik (Hidayat & Fauzi, 2022). Penempatan anak-anak yatim-piatu di Lembaga Kesejahteraan Sosial Anak adalah sebuah usaha terakhir. Artinya keberadaan lembaga ini merupakan alternatif terakhir, jika tidak ada keluarga dekat maupun keluarga lain yang tidak menghendaki mengasuh anak tersebut. Pertumbuhan dan perkembangan anak-anak yatim miskin dalam segi biaya hidup, mental, maupun rohani perlu diberi perlakukan dan perhatian yang sesuai dan sebaik mungkin sebagaimana anak-anak umumnya atau sebaya dengan mereka. Dengan adanya Lembaga Kesejahteraan anak dapat mengurangkan beban pemerintah dalam menjaga dan melindungi anak- anak yatim miskin ini dari belenggu kemiskinan atau masalah-masalah sosial lainnya. Edukatif : Jurnal Ilmu Pendidikan Vol 4 No 3 Tahun 2022 p-ISSN 2656-8063 e-ISSN 2656-8071 Edukatif : Jurnal Ilmu Pendidikan Vol 4 No 3 Tahun 2022 p-ISSN 2656-8063 e-ISSN 2656-8071 3981 Landasan Filsafat Pendirian dan Pendidikan Karakter Kemandirian Anak Yatim di Lembaga Kesejahteraan Sosial Anak – Arif Hidayat, Abdul Wachid Bambang Suharto DOI : https://doi.org/10.31004/edukatif.v4i3.2716 3981 Landasan Filsafat Pendirian dan Pendidikan Karakter Kemandirian Anak Yatim di Lembaga Kesejahteraan Sosial Anak – Arif Hidayat, Abdul Wachid Bambang Suharto DOI : https://doi.org/10.31004/edukatif.v4i3.2716 Keberadaan LKSA sangat penting untuk pendidikan karakter kemandirian. Ikhwani dalam kajiannya menyebutkan bahwa lembaga panti asuhan atau LKSA (lembaga kesejahteraan sosial anak) memiliki hubungan yang signifikan dalam penanganan anak yang mengalami masalah seperti pelecehan, bullying dan penelantaran (Ikhwani, 2020). Penguatan pembinaan karakter kemandirian pada lksa atau yatim piatu juga oleh Najmi, bahwa lksa ataupun panti asuhan lebih mengembangkan aspek-aspek kemandirian dan membuat standar pembinaan kemandirian. Dengan demikian lembaga seperti LKSA sangat diperlukan bagi anak yatim dan anak dhuafa, sebagai tempat pengasuhan alternatif(Kirana, 2017). LKSA memiliki peran membentuk life skill pada anak asuh, dengan pelatihan kewirausahaan baik keterampilan soft skill maupun keterampilan hard skill. Hal ini sesuai dengan penelitian yang dilakukan oleh Patimah. Dalam penelitian tersebut pelatihan life skill bertujuan agar anak asuh memiliki kemandirian dan mempunyai bekal untuk mereka kelak saat dewasa. PENDAHULUAN Dengan kemandirian yang ada pada peserta didik atau anak yatim dapat mendorong terlecutnya motivasi untuk belajar secara mandiri, atas kesadaran sendiri, sehingga timbul tanggung jawab dan percaya diri dalam menghadapi masalah yang ada pada diri sendiri(Patimah & Sumartini, 2022). Hasil skripsi yang disusun oleh Syamsul Bahri menjelaskan tentang keberadaan Panti Asuhan Al- Khairiyah yang memberikan pendidikan karakter di daerah Cilandak Barat. Hasil pendidikan di panti asuhan tersebut terbukti melahirkan remaja-remaja yang mandiri. Model panti asuhan tersebut dalam pembinaan karakter kemandirian adalah melalui pendidikan agama, seperti membaca AL-Quran, mengkaji kitab, pendidikan akhlak dan ceramah. Keberhasilan tersebut juga atas dukungan dari masyarakat sekitar serta dari pemerintah(Bahri, 2017). Wahidin dalam tesisnya yang berlatar belakang problem minimnya perhatian terhadap pendidikan karakter di lembaga sosial. Penelitian berlokasi di Panti Asuhan Muhammadiyah Ajibarang Banyumas, menyimpulkan tiga tahapan dalam pengembangan pendidikan karakter di lembaga sosial. Pertama, menentukan tujuan pendidikan karakter, kedua menentukan sasaran pendidikan karakter, dan yang ketiga adalah tahap pengembangan. Jadi hasil temuan dalam tesis ini pendidikan karakter juga harus dirancang dan diprogram(Wahidin, 2020). Penelitian tentang kemandirian anak asuh juga ditulis oleh Darmawati, dilatarbelakangi pentingnya pendidikan karakter mandiri anak berkebutuhan khusus yang tinggal di Panti Asuhan Bina Siwi Bantul, menyimpulkan ada dua bentuk penguatan karakter, yaitu penguatan emosi dan sosial (Darmawati & Indriawati, 2020). Peran penting lembaga sosial dalam menanamkan pendidikan karakter anak asuh, ditulis oleh Winda Anggunita Lestari, Ia menyebutkan bahwa salah satu faktor penghambat pendidikan karakter di panti asuhan adalah kecenderungan pengurus melakukan pengasuhan sebatas pada tataran kognitif(Lestari, 2016). Selain itu pengurus atau pengasuh masih menomorduakan pendidikan karakter. Hal yang sama terkait faktor pengurus lembaga sosial yang menjadi hambatan juga ditemukan dalam penelitian oleh Judika Yona Silvia dalam penelitian tentang pendidikan karakter di panti asuhan Aisyiyah Kabupaten Agam. Silvia menemukan faktor penghambat pembinaan karakter di panti tersebut adalah ketidak kompakan pengurus(Silvia & Angraini, 2018). Penelitian ini memiliki topik yang sama dengan penelitian yang sudah diuraikan sebelumnya bahwa pendidikan karakter dan kemandirian di lembaga sosial anak, merupakan hal penting. Namun kajian penelitian ini berbeda dengan penelitian-penelitian sebelumnya. Dalam tulisan hasil penelitian ini mengkaji latar belakang secara filsafati pendirian dan menguatkan pentingnya pendidikan karakter kemandirian bagi anak asuh terutama anak yatim, yatim piatu berdasarkan standar pengasuhan sesuai kebijakan pemerintah. Mengacu pada salah satu faktor penghambat kemandirian yaitu anak terbiasa menerima bantuan yang berlebihan dari orang tua ataupun dari orang dewasa lainnya (Ferawati & Multahada, 2021). PENDAHULUAN Bantuan berlebihan dapat memberi kesempatan anak memanipulasi bantuan, anak cenderung tidak berusaha dikala mengalami kesulitan. Maka penelitian ini bermaksud untuk menjawab rumusan masalah, apakah pendirian dan pembinaan karakter kemandirian anak asuh LKSA Iklil Mahya Insani sesuai standar pengasuhan berdasarkan landasan filsafat?. Dari rumusan masalah tersebut penelitian ini bertujuan mengetahui landasan pendirian lembaga sosial sebagai Edukatif : Jurnal Ilmu Pendidikan Vol 4 No 3 Tahun 2022 p-ISSN 2656-8063 e-ISSN 2656-8071 3982 Landasan Filsafat Pendirian dan Pendidikan Karakter Kemandirian Anak Yatim di Lembaga Kesejahteraan Sosial Anak – Arif Hidayat, Abdul Wachid Bambang Suharto DOI : https://doi.org/10.31004/edukatif.v4i3.2716 3982 Landasan Filsafat Pendirian dan Pendidikan Karakter Kemandirian Anak Yatim di Lembaga Kesejahteraan Sosial Anak – Arif Hidayat, Abdul Wachid Bambang Suharto DOI : https://doi.org/10.31004/edukatif.v4i3.2716 pondasi utama pembinaan karakter anak asuh di LKSA Iklil Mahya Insani. Sehingga kebijakan Permensoso RI No. 30/HUK/2011, menyebutkan bahwa anak anak yatim terjamin pengasuhannya dan keterikatan dengan keluarga inti tetap terjamin pada lembaga kesejahteraan sosial(Wijayanti et al., 2015). pondasi utama pembinaan karakter anak asuh di LKSA Iklil Mahya Insani. Sehingga kebijakan Permensoso RI No. 30/HUK/2011, menyebutkan bahwa anak anak yatim terjamin pengasuhannya dan keterikatan dengan keluarga inti tetap terjamin pada lembaga kesejahteraan sosial(Wijayanti et al., 2015). METODE PENELITIAN Pendekatan yang digunakan dalam penelitian ini menggunakan pendekatan kualitatif. Pendekatan kualitatif adalah pendekatan yang diwarnai dengan pemikiran filsafat platonik, yang berkembang dari ilmu- ilmu sosial di Jerman. Filsafat platonik tergambar seperti pemikiran Kant dan Hegel. Paradigma penelitian kualitatif menurut Muhadjir dalam (Ginting; 2008,81) diilhami falsafah rasionalisme yang bertujuan adanya pembahasan holistik, sistemik dan mengungkap makna di balik fakta empiris sensual. Selanjutnya metode yang digunakan dalam penelitian ini adalah studi kasus. Studi kasus menurut Nugrahani(2018) yaitu penelitian yang memandu secara rinci dan mendalam dalam bentuk pendeskripsian terhadap konteks yang terjadi di lapangan. Lokasi penelitian ini adalah Lembaga Kesejahteraan Sosial Anak (LKSA) Iklil Mahya Insani Desa Dagan Kecamatan Bobotsari, Purbalingga, Jawa Tengah. Untuk menguji hasil penelitian dengan teknik triangulasi data sebagai uji kredibilitas. Penelitian ini mengkaji tentang latar belakang pembentukan LKSA Iklil Mahya Insani yang memiliki tujuan dalam hal pembinaan karakter kemandirian anak yatim dan dhuafa. Untuk memperinci pembahasan, dalam pembahasan ini diuraikan dalam beberapa sub judul yaitu: perspektif filsafat (ontologis, epistemologis, aksiologis) LKSA Iklil Mahya Insani, pendidikan karakter kemandirian anak asuh di LKSA Iklil Mahya Insani. Edukatif : Jurnal Ilmu Pendidikan Vol 4 No 3 Tahun 2022 p-ISSN 2656-8063 e-ISSN 2656-8071 HASIL DAN PEMBAHASAN PENELITIAN Kedua kata tersebut asalnya dari bahasa Yunani. Hal tersebut merupakan pengertian secara istilah dari epistemologi. Berdasarkan maksud tersebut, maka epistemologi secara etimologi adalah teori pengetahuan. Epistemologi jika diterapkan dalam kajian lembaga sosial anak maka pembahasannya antara lain seluk beluk lembaga sosial. Arah membangun lembaga sosial, unsur unsur lembaga sosial, sasaran dan tujuan, serta macam macam bentuk lembaga sosial. Epistemologi merupakan sebutan yang berbeda dari logika material disebut juga logika mayor. Istilah tersebut membahas dari sumber akal manusia yaitu pikiran yang melahirkan pengetahuan. Dengan adanya pengetahuan berupaya menjawab persoalan-persoalan yang terjadi. Manusia merupakan bagian dari masyarakat. Manusia adalah makhluk yang berakal, makhluk yang senantiasa ingin memecahkan persoalan yang ada dan nyata ada disekitar. Manusia selalu ikut merancang dan mencipta kehidupan sesuai dengan apa yang dikehendaki. Menurutnya manusia tidak sekedar ‘makhluk yang berada’ “being creature”, akan tetapi ia adalah makhluk menjadi “becoming creature”. Artinya eksistensi manusia adalah eksistensi yang selalu berubah dinamik dari waktu ke waktu. Manusia mengubah lingkungan sera kreatif sesuai dengan keinginannya. Manusia memiliki kompetensi mengubah nature menjadi culture (Nuddin, 2017). Lembaga kesejahteraan sosial anak, atau istilah/pengertian lain yang memiliki fungsi dan tujuan sama, merupakan fenomena sosial objektif. Dalam fenomena sosial objektif memiliki eksistensi riil dan material seperti aktor, tindakan, interaksi, struktur birokrasi, hukum dan aparatur negara. Disamping fenomena sosial objektif, ada juga fenomena sosial yang hanya dalam ranah gagasan atau subjektif. Fenomena sosial subjektif tidak memiliki eksistensi materi seperti proses mental, konstruksi sosial atas realitas, norma dan nilai serta elemen kebudayaan lainnya. Baik fenomena objektif maupun subjektif adalah dua elemen yang membentuk pola kehidupan masyarakat dan berhubungan secara dialektis. Lahir dari pemikiran yang skeptis, bahwa sumbangan yang diberikan donatur kepada anak yatim menjadikan anak memiliki jiwa pengemis. Maka perlu orang atau lembaga yang harus memenej kuangan mereka, agar mereka tidak salah jalan. Skeptis merupakan salah satu kajian bidang epistemologis. Dari pemikiran ini maka yayasan iklil mahya insani melakukan kegiatan pendidikan karakter kepada anak yatim atau anak asuh. Pendidikan karakter bagi anak yatim dan anak asuh merupakan hal yang mendasar. Karakter anak yatim harus kuat karena ketiadaan orang tua menjadikan mereka harus siap menerima apapun yang ada di kehidupan mereka kelak saat mereka dewasa. Secara kebahasaan istilah yatim memiliki dua pengertian. Pertama istilah yatim berasal dari perkataan al-yummu yang memiliki pengertian terputusnya anak yang belum mencapai usia baligh dengan orang tua. Pengertian yang kedua mengacu pada pengertian seseorang yang sendiri, menyendiri, tunggal, dalam istilah kebahasaan sering disebut dengan sebatang kara. HASIL DAN PEMBAHASAN PENELITIAN Keberadaan anak yatim dan piatu merupakan hal nyata ada di dalam masyarakat. Keberadaan anak yatim adalah sebuah realitas yang konkret yang ada dalam masyarakat. Sebagai bentuk rasa kemanusiaan lembaga kesejahteraan sosial anak merupakan wujud dari aspek ontologis anak yatim piatu yang tidak memiliki ayah, ibu atau kedua duanya. Mereka tentu tidak menginginkan hidup sendiri ditinggal orang tuanya. Pengasuhan bagi anak yatim piatu, adalah hal yang sangat penting dilakukan. Lembaga sosial anak adalah lembaga yang bergerak dalam bidang sosial. Ia ada karena adanya anak yatim piatu. Lembaga ini terbentuk atas respon sosial atas keberadaan anak yatim. Tujuan LKSA adalah mengasuh, melindungi dan memberikan pelayanan akses kebutuhan anak. LKSA Iklil Mahya Insani berdiri atas dasar respon anak anak yatim yang menerima berbagai santunan , namun karena santunan tersebut, beberapa dari mereka tidak dapat mengelola uang, sehingga jika dibiarkan akan berdampak pada permasalahan sosial lainnya yaitu berjiwa konsumerisme. Jika mereka tidak menerima santunan akan timbul rasa kesal. Pengasuhan, pembinaan, pendidikan anak yatim dan dhuafa tidak bisa diserahkan sepenuhnya kepada negara. Artinya negara tidak mungkin mampu menjalankan amanat pasal 34 empat ayat dua Undang-Undang Dasar(UUD) tahun 1945, tanpa peran serta masyarakat. Berdasarkan pasal Peraturan Menteri Sosial, masyarakat dapat membentuk LKSA sebagai tempat pembinaan anak yatim dan dhuafa. Berdasarkan tulisan yang dikemukakan oleh (Hasibuan, 2018), bahwa anak yatim yang hanya dipenuhi kebutuhan hidupnya, tanpa memberikan pembinaan, dapat menimbulkan karakter yang jauh dari karakter kemandirian. Pengasuhan anak terlantar di LKSA adalah alternatif terakhir, jika tidak ada keluarga yang berkenan menjadi orang tua asuh. Tentunya keberadaan LKSA sebagai alternatif terakhir harus benar benar menjadi sebuah tempat yang layak untuk mendampingi anak yatim dan anak keluarga dhuafa. Landasan moral keberadaan LKSA harus dicermati. Dari landasan inilah tentu LKSA dalam pembinaan anak yatim dan Edukatif : Jurnal Ilmu Pendidikan Vol 4 No 3 Tahun 2022 p-ISSN 2656-8063 e-ISSN 2656-8071 3983 Landasan Filsafat Pendirian dan Pendidikan Karakter Kemandirian Anak Yatim di Lembaga Kesejahteraan Sosial Anak – Arif Hidayat, Abdul Wachid Bambang Suharto DOI : https://doi.org/10.31004/edukatif.v4i3.2716 3983 Landasan Filsafat Pendirian dan Pendidikan Karakter Kemandirian Anak Yatim di Lembaga Kesejahteraan Sosial Anak – Arif Hidayat, Abdul Wachid Bambang Suharto DOI : https://doi.org/10.31004/edukatif.v4i3.2716 dhuafa. LKSA yang asal-asalan dalam mengelola kelembagaan lebih baik ditutup dan dilarang. Keberanian inilah yang harus dimiliki kementerian sosial dalam mengambil keputusan akan adanya lembaga sosial anak yang berlaku sewenang-wenang. Epistemologi bersumber dari kata yaitu episteme. Epistime memiliki arti pengetahuan. Sedangkan logos yang memiliki makna teori. HASIL DAN PEMBAHASAN PENELITIAN Aksiologi adalah abang filsafat yang membahas berkaitan tentang prinsip dasar dan tujuan manfaat pengetahuan atau menyelidiki hakekat value. Dalam kajian lembaga maka landasan aksiologi adalah mengkaji tentang nilai nilai atau kegunaan dari lembaga sosial anak baik bagi anak, masyarakat, maupun negara. Cabang filsafat yang membahas dan mendiskusikan tentang hakikat nilai disebut dengan aksiologi. Penjelasan Frondizi dalam Wijaya (1993: 38), bahasan dalam aksiologi yang merupakan permasalahan pokok psikologi merangkum empat hal, yaitu: persoalan nilai dalam lingkungan sehari-hari, persoalan nilai yaitu objektif dan juga nilai subjektif, persoalan cara pemahaman nilai, dan persoalan bagaimana kita memahami dan mengetahui nilai. Pembahasan nilai dalam kehidupan sehari-hari terkait dengan pembentukan LKSA Iklil Mahya Insani tidak lepas dari persoalan ini. Ironi dari keadaan anak yatim dan anak dhuafa yang mendapat bantuan, sumbangan, donasi dari masyarakat saat bulan Muharram. Pada bulan ini anak yatim dan dhuafa mendapat Edukatif : Jurnal Ilmu Pendidikan Vol 4 No 3 Tahun 2022 p-ISSN 2656-8063 e-ISSN 2656-8071 Edukatif : Jurnal Ilmu Pendidikan Vol 4 No 3 Tahun 2022 p-ISSN 2656-8063 e-ISSN 2656-8071 3984 Landasan Filsafat Pendirian dan Pendidikan Karakter Kemandirian Anak Yatim di Lembaga Kesejahteraan Sosial Anak – Arif Hidayat, Abdul Wachid Bambang Suharto DOI : https://doi.org/10.31004/edukatif.v4i3.2716 3984 Landasan Filsafat Pendirian dan Pendidikan Karakter Kemandirian Anak Yatim di Lembaga Kesejahteraan Sosial Anak – Arif Hidayat, Abdul Wachid Bambang Suharto DOI : https://doi.org/10.31004/edukatif.v4i3.2716 sumbangan atau santunan. Tentu ini tidak lepas dari ajaran agama, bahwa menyantuni anak fakir miskin adalah perbuatan yang baik. Namun bantuan tanpa pengawasan, dapat menjadikan anak memiliki sifat mengharap. Mengharap akan adanya santunan. Dari pokok persoalan inilah LKSA Iklil Mahya Insani dibentuk. sumbangan atau santunan. Tentu ini tidak lepas dari ajaran agama, bahwa menyantuni anak fakir miskin adalah perbuatan yang baik. Namun bantuan tanpa pengawasan, dapat menjadikan anak memiliki sifat mengharap. Mengharap akan adanya santunan. Dari pokok persoalan inilah LKSA Iklil Mahya Insani dibentuk. Aksiologi merupakan salah satu bagian dari trilogi, selain ontologi dan epistemologi. Dalam perspektif filsafat, aksiologi menunjuk pada ilmu pengetahuan yang menyelidiki hakikat nilai dilihat dari sudut kefilsafatan. Aksiologi dapat diartikan dengan teori nilai yang berhubungan dengan manfaat dari pengetahuan yang diselidiki atau diperoleh. Dengan penjelasan tersebut aksiologi dapat diartikan sebagai nilai kegunaan pengetahuan (Giri, 2020) . Berdasarkan hal tersebut landasan aksiologi kegunaan atau fungsi atau keberadaan LKSA Iklil Mahya Insani melihat dari kegunaan lembaga tersebut. LKSA Iklil Mahya Insani memiliki kegunaan untuk membantu penanganan anak yatim piatu. Tanpa peran lembaga tersebut, anak yatim dan piatu akan hidup. HASIL DAN PEMBAHASAN PENELITIAN Kedudukan anak yatim dalam Islam merupakan hal yang mendapat perhatian khusus. Dalam ayat-ayat Al-Quran kaum muslimin diharapkan untuk menyantuni, membela, dan melindungi anak yatim. Mengapa Al- Quran sangat konsen terhadap anak yatim, karena pada diri anak yatim terdapat beberapa kelemahan dan kekurangan yang memerlukan pihak lain untuk membantu. Dalam al-Quran terdapat ayat yang jelas-jelas memerintahkan segenap umat Islam untuk melindungi, menjaga, dan menyantuni serta memelihara anak yatim, seperti pada surat al-Maun ayat satu sampai tiga yang menerangkan sebutan bagi kaum muslimin yang menzalimi anak yatim. Pada surah al-Baqoroh ayat 220 dan surah an-Nisa ayat dua berisi tentang pemeliharaan anak yatim. Berdasarkan hal tersebut tentu keberadaan lembaga kesejahteraan sosial jika dilihat dalam sudut pandang agama, dalam hal ini agama Islam adalah bentuk perintah Tuhan. Perintah Tuhan jika dilaksanakan dengan tulus ikhlas termasuk ibadah. Selain sebagai perintah ajaran agama, Lembaga kesejahteraan sosial anak juga bentuk pengabdian masyarakat terhadap negara. Dan negara dengan segala kewenangannya memberikan kebijakan berupa peraturan baik peraturan menteri maupun peraturan kepala daerah sebagai langkah melaksanakan amanat Undang-Undang Dasar 1945 khususnya pasal 34 (Grasindo, 2017). Dengan pembentukan lembaga ini diharapkan anak yatim dan dhuafa dapat memiliki karakter kemandirian sebagai bekal ia dewasa kelak. Pembentukan LKSA tentu berdasarkan nilai-nilai moral, dan ini yang semestinya. Sebagaimana LKSA Iklil Mahya Insani dibentuk berdasarkan pertimbangan-pertimbangan nilai-nilai moral. Nilai moral yang menjadi pertimbangan pendirian LKSA antara lain: 1) agar anak yatim dan dhuafa selalu terpenuhi kebutuhan baik fisik maupun non fisik, 2) agar donatur dapat memberikan santunan yang lebih bermanfaat, 3) agar anak yatim dan dhuafa memiliki pikiran positif terhadap santunan yang diberikan oleh donatur. Secara aksiologis, pengembangan LKSA harus selalu mengacu pada sumber yang murni dan berwawasan kemanusiaan. Dengan kata lain penekanan pengembangan lembaga sosial anak yatim harus memiliki ‘value’ secara sosial masyarakat dan juga tidak lepas dari nilai tanggung jawab kepada Tuhan sang pencipta. Dua hal tersebut sebagai landasan iman dan landasan kemaslahatan. Untuk itu, dalam konteks ini, agama, etika, estetika, dan aturan adat harus tetap terjaga. Aksiologis meliputi nilai-nilai (value) yang bersifat normatif. Nilai-nilai yang bersifat normatif adalah nilai yang menentukan tingkah laku sesuai harapan dalam sistem masyarakat disekitar, dengan mengesampingkan fungsi bagian- bagiannya. Secara umum, rujukan normatif disandarkan pada sesuatu yang ideal atau model yang seharusnya berada dalam masyarakat. Pernyataan normatif dapat diartikan adalah suatu pertimbangan nilai baik dan buruk, salah atau betul, diterima atau ditolak. HASIL DAN PEMBAHASAN PENELITIAN Lebih dari itu nilai-nilai yang ditonjolkan pada aspek aksiologi sebagai suatu conditio since quanon yang wajib ditaati oleh lingkungan masyarakat(Musliichah, n.d.). Edukatif : Jurnal Ilmu Pendidikan Vol 4 No 3 Tahun 2022 p-ISSN 2656-8063 e-ISSN 2656-8071 Edukatif : Jurnal Ilmu Pendidikan Vol 4 No 3 Tahun 2022 p-ISSN 2656-8063 e-ISSN 2656-8071 3985 Landasan Filsafat Pendirian dan Pendidikan Karakter Kemandirian Anak Yatim di Lembaga Kesejahteraan Sosial Anak – Arif Hidayat, Abdul Wachid Bambang Suharto DOI : https://doi.org/10.31004/edukatif.v4i3.2716 Dalam membentuk kepribadian karakter, warga masyarakat harus patuh dan mengikuti norma-norma yang berlaku didalam masyarakat. Norma merupakan aturan yang diwariskan secara turun temurun. Masyarakat dengan interaksi yang kuat dapat memberikan penguatan karakter kepada anak. Masyarakat melahirkan komunitas-komunitas yang berbasis kepedulian moral tumbuh dan berkembang. Dalam menumbuhkan karakter pada anak perlu pembiasaan dan bukan hanya sekedar teoritis, sebagaimana menurut Al-Ghazali dalam kitab Ayyuha al-walad menyebutkan bahwa salah satu cara agar anak memiliki karakter mandiri adalah dengan metode pembiasaan(Aminuddin & Wahidin, 2022). Pembentukan LKSA Ilkil Mahya Insani tidak lain sebagai perwujudan pembiasaan karakter baik bagi anak asuhnya. Berdasarkan wawancara dengan ketua LKSA Iklil Mahya Insani desa Dagan Kecamatan Bobotsari, di dapat informasi bahwa faktor pertama didirikannya LKSA ini adalah keadaan dimana setiap bulan Muharram, para donatur memberikan sumbangan yang tidak sedikit kepada anak-anak yatim yang ada di desa Dagan. Dari beberapa anak yatim piatu tersebut didapat menggunakan uang pemberian atau uang santunan tersebut dipergunakan hal-hal bersifat konsumerisme. Bahkan dengan barang-barang yang dibeli dari hasil uang santunan tersebut, menjadikan mereka jauh dari nilai-nilai moral yang ada di dalam masyarakat. Faktor kedua landasan didirikannya LKSA ini adalah bagaimana uang hasil sumbangan para donatur dapat bermanfaat lebih baik. Dengan adanya lembaga ini, anak yatim yang ada tidak menerima secara langsung dari para donatur. Donatur memberikan sumbangan lewat LKSA ini. Kemudian dengan manajemen yang sudah disepakati oleh pengurus, diputuskan kebutuhan-kebutuhan yang diperlukan oleh anak-anak. Alasan ketiga pendirian LKSA ini adalah LKSA Iklil Mahya Insani menjadi wadah para sukarelawan untuk mengabdikan diri sebagai salah satu bentuk tanggung jawab moral warga masyarakat. Berdasarkan dokumen profil LKSA Iklil Mahya Insani saat penelitian ini, diketahui sebanyak 11 orang. Sebelas orang tersebut masuk dalam kepengurusan atas inisiatif pribadi, untuk mengabdikan diri dalam lembaga sosial. Pendidikan kemandirian merupakan salah satu peran lembaga pendidikan untuk membina generasi muda bangsa agar berperilaku baik dan benar sesuai dengan nilai-nilai yang berlaku sebagaimana mestinya dalam masyarakat. Kita sadari begitu penting pendidikan karakter. HASIL DAN PEMBAHASAN PENELITIAN Selain itu perkembangan kemandirian juga dipengaruhi oleh dua faktor yang mempengaruhi tingkat kemandirian anak, yaitu; pertama faktor internal, yaitu faktor yang ada di dalam diri Edukatif : Jurnal Ilmu Pendidikan Vol 4 No 3 Tahun 2022 p-ISSN 2656-8063 e-ISSN 2656-8071 Edukatif : Jurnal Ilmu Pendidikan Vol 4 No 3 Tahun 2022 p-ISSN 2656-8063 e-ISSN 2656-8071 3986 Landasan Filsafat Pendirian dan Pendidikan Karakter Kemandirian Anak Yatim di Lembaga Kesejahteraan Sosial Anak – Arif Hidayat, Abdul Wachid Bambang Suharto DOI : https://doi.org/10.31004/edukatif.v4i3.2716 3986 Landasan Filsafat Pendirian dan Pendidikan Karakter Kemandirian Anak Yatim di Lembaga Kesejahteraan Sosial Anak – Arif Hidayat, Abdul Wachid Bambang Suharto DOI : https://doi.org/10.31004/edukatif.v4i3.2716 anak dalam bentuk perasaan emosi pribadi anak dan intelektual yang dimiliki anak, yang kedua adalah faktor yang berasal dari luar (eksternal) berupa: lingkungan tempat tinggal, status ekonomi dan kesejahteraan keluarga, rangsangan, pola asuh, kasih sayang, kualitas informasi dan interaksi anak dengan orang tua, dan status mata pencaharian atau pekerjaan orang tua. Perilaku yang tidak mencerminkan karakter kemandirian pada diri anak yatim piatu perlu diatasi, agar perilaku tidak mandiri tidak dibawa sampai ia dewasa. anak dalam bentuk perasaan emosi pribadi anak dan intelektual yang dimiliki anak, yang kedua adalah faktor yang berasal dari luar (eksternal) berupa: lingkungan tempat tinggal, status ekonomi dan kesejahteraan keluarga, rangsangan, pola asuh, kasih sayang, kualitas informasi dan interaksi anak dengan orang tua, dan status mata pencaharian atau pekerjaan orang tua. Perilaku yang tidak mencerminkan karakter kemandirian pada diri anak yatim piatu perlu diatasi, agar perilaku tidak mandiri tidak dibawa sampai ia dewasa. Anak yatim -piatu adalah anak yang memerlukan pendamping atau pengasuh. Pendamping atau pengasuh alternatif terakhir adalah dengan mendapat pengasuhan lembaga kesejahteraan sosial anak. LKSA sebagai kepanjangan tangan dari orang tua, LKSA tentu memiliki kewenangan untuk mengatur kebutuhan anak yatim -piatu sesuai kebutuhan anak pada umumnya. Berdasarkan Peraturan Menteri Sosial Republik Indonesia Nomor: 30/HUK/2011 Tentang Standar Nasional Pengasuhan Anak untuk Lembaga Kesejahteraan Sosial Anak menekankan pada aspek pendekatan(Astuti, 2014). Salah satunya adalah prinsip pengakuan Lembaga Kesejahteraan Sosial Anak(LKSA). Pengakuan terhadap LKSA dapat melahirkan kekuatan untuk mendukung terbangunnya sistem pengasuhan anak yang mendukung pengasuhan berbasis keluarga. LKSA Iklil Mahya Insani menggunakan metode pengasuhan anak luar panti. Pengasuhan anak luar panti artinya anak yatim-piatu yang tergabung dalam LKSA tersebut, tidak diasramakan. Anak yatim yang ada di LKSA tersebut adalah warga desa Dagan. Artinya mereka masih berada dilingkungan keluarga sendiri. HASIL DAN PEMBAHASAN PENELITIAN Dalam Islam karakter disebut akhlak mulia atau akhlak karimah. Kecerdasan akademik tanpa dibarengi dengan karakter yang kuat adalah sesuatu yang tidak ada gunanya. Akhlak dan kemampuan akademik adalah dua hal yang saling berhubungan dan mensubtitusi. Manusia yang jauh dari jiwa berkarakter baik atau tidak berakhlak mulia dikatakan sebagai manusia atau orang tidak bermoral atau beradab dan tidak memiliki harga atau nilai. Akhlak harus dibangun, untuk membangun akhlak salah satunya dengan pendidikan. Pendidikan dapat meliputi pendidikan di dalam keluarga, di sekolah maupun di masyarakat. Pendidikan karakter yang diselenggarakan pada lembaga sosial anak seperti halnya LKSA termasuk bentuk pendidikan karakter dalam lingkup masyarakat (Sajadi, 2019). Dengan bahasa lain perilaku baik atau buruk manusia pada intinya lahir dari respon atau rangsangan berupa keturunan dan lingkungannya. Hasil kolaborasi karena adanya hubungan saling timbal balik antara unsur pembawa hidup dan pengaruh lingkungan membentuk perilaku manusia(Rukiyati & Purwastuti, 2015). Hal senada disampaikan oleh Berns(1997), ia menyebutkan bahwa pengasuhan adalah proses interaksi yang berlangsung terus menerus, yang saling mempengaruhi antara orang tua asuh dan anak yatim-piatu. Keduanya saling mempengaruhi. Hal tersebut juga berlaku pada anak yatim atau anak piatu dan para donatur. Anak yatim dan atau anak piatu yang selalu diberi santunan oleh para donatur menimbulkan interaksi. Setiap tahun khususnya bulan Muharram, anak akan memiliki harapan mendapat bantuan atau santunan. Santunan yang diberikan ‘Cuma- Cuma’ tersebut dapat menimbulkan efek yang tidak baik bagi perkembangan anak. Menurut Izzaty dalam Suryani salah satu faktor penyebab anak memiliki karakter mandiri rendah adalah anak terbiasa dan atau membiasakan anak menerima bantuan dari orang tua, orang tua asuh atau orang dewasa lainnya melebihi dari kebutuhan anak (Suryani, 2016). Edukatif : Jurnal Ilmu Pendidikan Vol 4 No 3 Tahun 2022 p-ISSN 2656-8063 e-ISSN 2656-8071 HASIL DAN PEMBAHASAN PENELITIAN Sehingga akses anak yatim terhadap lembaga maupun sebaliknya sangat terjangkau dalam segi waktu maupun biaya. Dengan anak yatim tidak diasramakan seperti kebanyakan LKSA, setidaknya memberikan dukungan kepada keluarga besar untuk turut serta dalam pengasuhan berbasis keluarga. Hal ini sejalan dengan pendekatan-pendekatan ekologi dan psikososial, bahwa anak tetap dan terus menerus terjalin dengan keluarga, baik keluarga inti yaitu keluarga penuh maupun keluarga besar (saudara) bagi anak yatim piatu, walaupun anak-anak tinggal dalam Lembaga Kesejahteraan Sosial Anak. Dalam prakteknya pengurus LKSA Iklil Mahya Insani desa Dagan berstatus sebagai orang tua asuh. Pengurus dengan musyawarah diberi amanat menjadi orang tua asuh bagi anak yatim-piatu atau anak asuh. Orang tua asuh yang sekaligus sebagai pengurus dengan jadwal yang telah disusun dan disepakati antara pengurus, anak dan orang tua asuh mengadakan kunjungan ke rumah anak. Tujuan kunjungan ke tempat tinggal anak antara lain, memantau perkembangan mental anak asuh, mengetahui kebutuhan-kebutuhan anak dan juga sebagai bentuk asesmen terhadap anak dan keluarga. Asesmen tersebut berfungsi untuk mendapat pemahaman tentang situasi pengasuhan oleh keluarga besar maupun keluarga inti yang masih ada. Dengan melakukan kunjungan kerumah anak, orang tua asuh kemudian menyampaikan atau meneruskan di dalam rapat dewan pengurus LKSA yang diadakan setiap hari Jumat. Dalam rapat setiap satu minggu sekali tersebut, selain menyampaikan temuan-temuan yang didapat dari hasil kunjungan orang tua asuh, orang tua asuh juga menyampaikan uang sumbangan atau santunan yang diterimanya dari para donatur. Dalam aturan yang tidak tertulis, setiap pengurus diperbolehkan menerima uang sumbangan dari para donatur. Hal ini menurut wakil ketua LKSA Iklil Mahya Insani sebagai bentuk efektifitas kerja secara organisasi. Kebijakan lain dari Lembaga Kesejahteraan Sosial Anak ini adalah, tidak menerima sumbangan atas nama ‘Hamba Allah’ atau istilah lain yang bertujuan untuk menyembunyikan nama. Alasan tidak menerima sumbahan dari atas nama ‘Hamba Allah’ atau atas nama yang tidak sebenarnya bertujuan agar, terciptanya transparansi keuangan, baik antar pengurus maupun dengan anak dan juga keluarga besar anak asuh berasal. Lembaga Kesejahteraan Sosial Anak merupakan lembaga non profit, namun demikian sebagai sebuah lembaga LKSA ini memiliki kegiatan manajemen . Baik manajemen berkaitan dengan kegiatan operasional, sumber daya maupun keuangan(Fahmi, 2016). Salusu menjelaskan bahwa organisasi non profit dalam pelayanan terhadap masyarakat tidak menjadikan keuntungan sebagai tujuannya. HASIL DAN PEMBAHASAN PENELITIAN Sanusi menyamakan organisasi non profit seperti LKSA sebagai korporasi, namun tidak memberikan keuntungan sekecil apapun Edukatif : Jurnal Ilmu Pendidikan Vol 4 No 3 Tahun 2022 p-ISSN 2656-8063 e-ISSN 2656-8071 Edukatif : Jurnal Ilmu Pendidikan Vol 4 No 3 Tahun 2022 p-ISSN 2656-8063 e-ISSN 2656-8071 3987 Landasan Filsafat Pendirian dan Pendidikan Karakter Kemandirian Anak Yatim di Lembaga Kesejahteraan Sosial Anak – Arif Hidayat, Abdul Wachid Bambang Suharto DOI : https://doi.org/10.31004/edukatif.v4i3.2716 3987 Landasan Filsafat Pendirian dan Pendidikan Karakter Kemandirian Anak Yatim di Lembaga Kesejahteraan Sosial Anak – Arif Hidayat, Abdul Wachid Bambang Suharto DOI : https://doi.org/10.31004/edukatif.v4i3.2716 yang diberikan kepada pengurus atau karyawan dan juga para petingginya (Hidayat & Machali, 2012). Terlepas dari hal tersebut, semua hal yang menyangkut keuangan baik uang yang masuk maupun keluar harus dilaporkan dalam laporan keuangan(Fahmi; 2016). Hal demikian juga diterapkan dalam manajemen LKSA Iklil Mahya Insani, sebagai bentuk akuntabilitas dan transparansi lembaga sosial. Anak adalah harapan masa depan, tidak hanya bagi keluarga, namun juga masyarakat, dan juga bangsa. Anak adalah pewaris peradaban, dengan demikian membekali anak dengan berbagai karakter dan pengetahuan adalah sebuah keniscayaan. Dengan demikian dengan anak asuh yang berstatus yatim piatu. Mereka harus berada pada lingkungan yang mendukung tumbuh kembang ke arah positif. Salah satu bentuk perkembangan yang harus ditanamkan sejak dini adalah sikap karakter kemandirian. Masalah karakter anak adalah tantangan yang krusial di tengah era teknologi informasi saat ini (Jalil, 2016). Betapa krusialnya pendidikan karakter, sampai pemerintah mengeluarkan kebijakan berupa Peraturan Presiden Nomor 87 Tahun 2017 tentang Penguatan Pendidikan Karakter(PPK). Isi dari Peraturan Presiden tersebut tidak membebankan pendidikan karakter pada lembaga pendidikan formal saja. Tanggung jawab pendidikan karakter juga diperankan oleh keluarga, dan masyarakat. Kerjasama lembaga pendidikan formal/nonformal, keluarga dan masyarakat diharapkan terwujud karakter anak yang mencerminkan harmonisasi olah hati, olah rasa, olah pikir, dan olah raga. Sebagaimana menurut Ikhwani(2020) bahwa pelaksanaan dan penyelenggaraan pendidikan tidak hanya menjadi tugas lembaga pendidikan formal. Selanjutnya ia menekankan bahwa di luar lembaga pendidikan formal terbuka peluang sebagai andil mewujudkan pendidikan karakter anak bangsa. Agar terwujud karakter kuat pada diri anak, Koesoema dalam Subianto menawarkan tiga metode sebagai basis desain program pendidikan karakter. Ketiga basis tersebut, pertama : desain pendidikan karakter berbasis kelas, kedua pendidikan karakter berbasis kultur sekolah, dan yang ketiga adalah pendidikan karakter berbasis komunitas. Dari tiga desain yang ditawarkan tersebut, sekolah atau lembaga formal tidak berjuang sendiri, dalam membentuk karakter bagi anak didik. Masyarakat, Keluarga dan Negara memiliki andil yang sama (Subianto, 2013). HASIL DAN PEMBAHASAN PENELITIAN Lembaga Kesejahteraan Sosial Anak Iklil Mahya Insani adalah bagian dari masyarakat, khususnya desa Dagan. Dengan memberikan pendampingan dan pembinaan karakter pada anak asuh adalah wujud kepedulian masyarakat atas masa depan anak asuh dalam hal ini anak yatim dan piatu. LKSA ini fokus pada pembinaan karakter kemandirian. Desmita dalam Wirawati merumuskan tentang pengertian kemandirian, yaitu kemampuan untuk mengendalikan atau mengontrol dan mengatur keseimbangan pikiran, perasaan dan perilaku atau tindakan yang dilakukan sendiri secara merdeka serta berusaha sendiri untuk mengatasi apa yang ada dalam perasaan malu dan keragu raguan yang dimiliki (Desmita, 2009). Dalam prakteknya anak asuh yang ada di LKSA Iklil Mahya Insani mendapat pembinaan keterampilan. Pembinaan keterampilan dilaksanakan setiap satu minggu sekali. Pembinaan ketrampilan ini sebagai wujud kemandirian LKSA maupun anak asuh agar mereka mendapat penghasilan dari keterampilan yang dimiliki. KESIMPULAN Berdasarkan uraian sebelumnya keberadaan Lembaga Kesejahteraan Sosial Anak atau LKSA merupakan tindak lanjut pemerintah dalam melaksanakan amanat luhur pendiri bangsa yang tertuang dalam pasal 34. Bahwa negara menjamin pemeliharaan dan perlindungan bagi anak-anak terlantar termasuk anak yatim-piatu. Bagi masyarakat yang berbudaya dan beragama, keberadaan lembaga sosial merupakan bentuk pengabdian dan respon sosial masyarakat terhadap anak anak yang perlu perhatian khusus. Selain itu lembaga kesejahteraan sosial anak (LKSA) merupakan bentuk ibadah kepada Tuhan Yang Maha Esa, karena dalam ajaran khususnya agama Islam, umatnya diperintahkan untuk melindungi dan mengasuh anak yatim-piatu. Lembaga sosial dalam prakteknya tidak hanya mengurusi tentang kesejahteraan anak saja. Peran aktif lembaga sosial yang turut serta membina mendidik karakter kemandirian anak yatim, adalah bentuk tanggung jawab Edukatif : Jurnal Ilmu Pendidikan Vol 4 No 3 Tahun 2022 p-ISSN 2656-8063 e-ISSN 2656-8071 3988 Landasan Filsafat Pendirian dan Pendidikan Karakter Kemandirian Anak Yatim di Lembaga Kesejahteraan Sosial Anak – Arif Hidayat, Abdul Wachid Bambang Suharto DOI : https://doi.org/10.31004/edukatif.v4i3.2716 3988 Landasan Filsafat Pendirian dan Pendidikan Karakter Kemandirian Anak Yatim di Lembaga Kesejahteraan Sosial Anak – Arif Hidayat, Abdul Wachid Bambang Suharto DOI : https://doi.org/10.31004/edukatif.v4i3.2716 3988 Landasan Filsafat Pendirian dan Pendidikan Karakter Kemandirian Anak Yatim di Lembaga Kesejahteraan Sosial Anak – Arif Hidayat, Abdul Wachid Bambang Suharto DOI : https://doi.org/10.31004/edukatif.v4i3.2716 3988 Landasan Filsafat Pendirian dan Pendidikan Karakter Kemandirian Anak Yatim di Lembaga Kesejahteraan Sosial Anak – Arif Hidayat, Abdul Wachid Bambang Suharto DOI : https://doi.org/10.31004/edukatif.v4i3.2716 moral, bahwasanya lembaga sosial adalah lembaga yang diberikan kewenangan sebagai orang tua. Orang tua tentu tidak hanya memberikan makan, minum dan kesenangan semata kepada anaknya. Karena jika demikian anak akan memiliki karakter pemalas yang selalu berharap mendapat bantuan atau santunan. Kehadiran Lembaga Kesejahteraan Sosial Anak jika didirikan dan dikelola berdasar pandangan filsafat maka kebijakan permensos agar anak yatim tetap dalam pengasuhan keluarga inti tidak akan menjadi kenadala. Sebagai pendampingan alternatif lembaga kesejahteraan sosial anak dapat melakukan inovasi pembinaan anak asuhnya dengan prinsip akuntabilitas dan transparansi dalam pengelolaan. UCAPAN TERIMA KASIH Terima kasih kami ucapkan kepada Abdul Wachid Bambang Soeharto, sebagai dosen pembimbing dalam penulisan artikel ini, sehingga artikel ini dapat tersusun dan dapat disajikan di jurnal Edukatif ini. Terima kasih pula kami sampaikan kepada tim editor jurnal Edukatif yang bersedia mempublikasikan artikel ini. Kepada Istri, dan teman-teman pascasarjana PGMI UIN SAIZU Purwokerto, atas masukan dan koreksinya serta dukungan, kami sampaikan Jazakumullah Khairan Katsiran. Edukatif : Jurnal Ilmu Pendidikan Vol 4 No 3 Tahun 2022 p-ISSN 2656-8063 e-ISSN 2656-8071 DAFTAR PUSTAKA Aminuddin, A., & Wahidin, K. (2022). Metode Pendidikan Karakter Al Gozali Dalam Kitab Ayyuhal Walad. Edukatif: Jurnal Ilmu Pendidikan, 4(1), 195–200. Astuti, M. (2014). Implementasi Kebijakan Kesejahteraan Dan Perlindungan Anak. Sosio Konsepsia: Jurnal Penelitian Dan Pengembangan Kesejahteraan Sosial, 4(1), 215–235. Bahri, S. (2017). Upaya Pembinaan Kepribadian Dan Kemandirian Anak Asuh Dalam Pelayanan Kesejahteraan Sosial Pada Psaa Al-Khairiyah Cilandak Barat. Fakultas Dakwah Dan Ilmu Komunikasi. Darmawati, I., & Indriawati, R. (2020). Peningkatan Kemandirian Anak Berkebutuhan Khusus Di Panti Asuhan Binasiwi, Bantul. Prosiding Seminar Nasional Program Pengabdian Masyarakat. Desmita, D. (2009). Psikologi Perkembangan Peserta Didik. Remaja Rosdakarya. Https://Difarepositories.Uin-Suka.Ac.Id/24/ Fahmi, R. A. (2016). Penerapan Laporan Keuangan Pada Yayasan Panti Asuhan Yatim Piatu Dan Fakir Miskin Daarul Aytam Situbondo. Ferawati, D., & Multahada, A. (2021). Upaya Guru Dalam Melatih Kemandirian Anak Usia Dini. Primearly: Jurnal Kajian Pendidikan Dasar Dan Anak Usia Dini, 4(1), 27–34. Grasindo, T. (2017). Uud 1945 & Amandemennya Untuk Pelajar Dan Umum. Gramedia Widiasarana Indonesia. Hasibuan, K. B. (2018). Implementasi Program Kerja Panti Asuhan Puteri ‘Aisyiyah Dalam Pembentukan Karakter Kemandirian Anak Asuh. Universitas Islam Negeri Sumatea Utara Medan. Hidayat, A., & Fauzi, F. (2022). Pendampingan Residensial Semi Homeschooling Pembelajaran Daring Anak Yatim-Piatu Di Era New Normal. Jurnal Basicedu, 6(3), 4101–4110. Hidayat, A., & Machali, I. (2012). Pengelolaan Pendidikan: Konsep, Prinsip, Dan Aplikasi Dalam Mengelola Sekolah Dan Madrasah. Kaukaba. Ikhsan, M. (2015). Pengasuhan Anak Yatim Qs. Al-Baqarah Ayat 220 Dalam Tafsir Jami’al-Bayan Fi Takwil Al-Qur’an Karya Al-Thabari. Stain Kudus. Ikhwani, I. (2020). Pembinaan Karakter Mandiri Anak Asuh (Studi Realitas Pembinaan Karakter Mandiri Anak Asuh Di Panti Asuhan Putri Muhammadiyah Kecamatan Pekuncen Kabupaten Banyumas). Jurnal Tawadhu, 4(2), 1114–1127. Edukatif : Jurnal Ilmu Pendidikan Vol 4 No 3 Tahun 2022 p-ISSN 2656-8063 e-ISSN 2656-8071 3989 Landasan Filsafat Pendirian dan Pendidikan Karakter Kemandirian Anak Yatim di Lembaga Kesejahteraan Sosial Anak – Arif Hidayat, Abdul Wachid Bambang Suharto DOI : https://doi.org/10.31004/edukatif.v4i3.2716 Jalil, A. (2016). Karakter Pendidikan Untuk Membentuk Pendidikan Karakter. Nadwa: Jurnal Pendidikan Islam, 6(2), 175–194. Kirana, V. T. (2017). Kemandirian Alumni Pasca Pelayanan Pengasuhan Di Panti Asuhan (Studi Kasus Di Panti Asuhan Putri’aisyiyah Kel. Dinoyo Kec. Lowokwaru Kota Malang). University Of Muhammadiyah Malang. Kuntjorowati, E. (2018). 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Pelaksanaan Pendidikan Karakter Di Panti Asuhan Aisyiah Kabupaten Agam. Journal Of Civic Education, 1(2), 88–97. Subianto, J. (2013). Peran Keluarga, Sekolah, Dan Masyarakat Dalam Pembentukan Karakter Berkualitas. Edukasia: Jurnal Penelitian Pendidikan Islam, 8(2). Suryani, N. (2016). Pengembangan Media Pembelajaran Sejarah Berbasis It. Jurnal Sejarah Dan Budaya, 10(2), 186–196. Wahidin, W. (2020). Pengembangan Pendidikan Karakter Di Panti Asuhan Muhammadiyah Ajibarang Banyumas. Iain Purwokerto. Wijayanti, R., Rengga, A., & Santoso, R. S. (2015). Implementasi Standar Pelayanan Berbasis Lembaga Kesejahteraan Sosial Anak (Lksa) Oleh Dinas Sosial, Pemuda Dan Olahraga Kota Semarang. Journal Of Public Policy And Management Review, 4(2), 265–275. Edukatif : Jurnal Ilmu Pendidikan Vol 4 No 3 Tahun 2022 p-ISSN 2656-8063 e-ISSN 2656-8071 Edukatif : Jurnal Ilmu Pendidikan Vol 4 No 3 Tahun 2022 p-ISSN 2656-8063 e-ISSN 2656-8071
https://openalex.org/W4384200149	https://figshare.com/articles/journal_contribution/Supplementary_Table_S2_from_Associations_between_AI-Assisted_Tumor_Amphiregulin_and_Epiregulin_IHC_and_Outcomes_from_Anti-EGFR_Therapy_in_the_Routine_Management_of_Metastatic_Colorectal_Cancer/23676008/1/files/41550128.pdf	English	null	Supplementary Table S2 from Associations between AI-Assisted Tumor Amphiregulin and Epiregulin IHC and Outcomes from Anti-EGFR Therapy in the Routine Management of Metastatic Colorectal Cancer	null	2,023	cc-by	527	Biomarker 96 hour fixation: Block 1 96 hour fixation: Block 2 48 hour fixation Patient 1 AREG 64.9 64.2 59.5 EREG 60.1 64.6 56.3 Patient 2 AREG 81.3 90.6 41.3 EREG 96.1 97.4 77.8 Patient 3 AREG 46.6 44.4 31.2 EREG 84.2 87.8 73.4 Patient 4 AREG 15.9 20.7 16.8 EREG 0.2 0.1 0.5 Supplementary Table S2 Pre-study assessment of the effect of variation in tissue fixation time on AREG/EREG tumor cell percentage positivity. AREG and EREG scores (expressed as the percentage of tumor cells in the tumor area staining biomarker positive) for three formalin-fixed, paraffin- embedded tumor blocks from different regions of the same tumor in four individual patients. Two blocks were taken from tumor tissue fixed for 96 hours, and one block from tumor tissue fixed for the standard fixation time of 48 hours. Biomarker 96 hour fixation: Block 1 96 hour fixation: Block 2 48 hour fixation Patient 1 AREG 64.9 64.2 59.5 EREG 60.1 64.6 56.3 Patient 2 AREG 81.3 90.6 41.3 EREG 96.1 97.4 77.8 Patient 3 AREG 46.6 44.4 31.2 EREG 84.2 87.8 73.4 Patient 4 AREG 15.9 20.7 16.8 EREG 0.2 0.1 0.5 Supplementary Table S2 Pre-study assessment of the effect of variation in tissue fixation time on AREG/EREG tumor cell percentage positivity. AREG and EREG scores (expressed as the percentage of tumor cells in the tumor area staining biomarker positive) for three formalin-fixed, paraffin- embedded tumor blocks from different regions of the same tumor in four individual patients. Two blocks were taken from tumor tissue fixed for 96 hours, and one block from tumor tissue fixed for the standard fixation time of 48 hours. Biomarker 96 hour fixation: Block 1 96 hour fixation: Block 2 48 hour fixation Patient 1 AREG 64.9 64.2 59.5 EREG 60.1 64.6 56.3 Patient 2 AREG 81.3 90.6 41.3 EREG 96.1 97.4 77.8 Patient 3 AREG 46.6 44.4 31.2 EREG 84.2 87.8 73.4 Patient 4 AREG 15.9 20.7 16.8 EREG 0.2 0.1 0.5 Supplementary Table S2 Pre-study assessment of the effect of variation in tissue fixation time on AREG/EREG tumor cell percentage positivity. AREG and EREG scores (expressed as the percentage of tumor cells in the tumor area staining biomarker positive) for three formalin-fixed, paraffin- Supplementary Table S2 Pre-study assessment of the effect of variation in tissue fixation time on AREG/EREG tumor cell percentage positivity. AREG and EREG scores (e pressed as the percentage of t mor cells in the AREG and EREG scores (expressed as the percentage of tumor cells in the tumor area staining biomarker positive) for three formalin-fixed, paraffin- embedded tumor blocks from different regions of the same tumor in four individual patients. Two blocks were taken from tumor tissue fixed for 96 hours, and one block from tumor tissue fixed for the standard fixation time of 48 hours. AREG and EREG scores (expressed as the percentage of tumor cells in the tumor area staining biomarker positive) for three formalin-fixed, paraffin- embedded tumor blocks from different regions of the same tumor in four individual patients. Two blocks were taken from tumor tissue fixed for 96 hours, and one block from tumor tissue fixed for the standard fixation time of 48 hours.
https://openalex.org/W3162743095	https://surgicalcasereports.springeropen.com/counter/pdf/10.1186/s40792-021-01206-7	English	null	Transperineal total mesorectal excision for rectal cancer on the residual rectum after multiple abdominal surgeries in a patient with Crohn’s disease: a case report	Surgical case reports	2,021	cc-by	5,431	Background As the treatment of Crohn’s disease (CD) has progressed, its prognosis has improved. Accordingly, the opportunity for patients with CD to undergo surgical treatment has increased. However, the longer duration of Crohn’s coli- tis may increase the risk of cancer [1]. The relative risk of developing colorectal cancer is reportedly 13 times higher in patients with the colonic type of CD than in Correspondence: shin_mto0509@yahoo.co.jp 1 Department of Gastroenterological Surgery I, Hokkaido University Graduate School of Medicine, Kita 15 Nishi 7, Kita‑ku, Sapporo, Hokkaido 060‑8638, Japan Full list of author information is available at the end of the article Full list of author information is available at the end of the article © 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/. Abstract Background: The improved prognosis of Crohn’s disease may increase the opportunities of surgical treatment for patients with Crohn’s disease and the risk of development of colorectal cancer. We herein describe a patient with Crohn’s disease and a history of multiple surgeries who developed rectal stump carcinoma that was treated laparo- scopically and transperineally. Case presentation: A 51-year-old man had been diagnosed with Crohn’s disease 35 years earlier and had under- gone several operations for treatment of Crohn’s colitis. Colonoscopic examination was performed and revealed rectal cancer at the residual rectum. The patient was then referred to our department. The tumor was diagnosed as clini- cal T2N0M0, Stage I. We treated the tumor by combination of laparoscopic surgery and concomitant transperineal resection of the rectum. While the intra-abdominal adhesion was dissected laparoscopically, rectal dissection in the correct plane progressed by the transperineal approach. The rectal cancer was resected without involvement of the resection margin. The duration of the operation was 3 h 48 min, the blood loss volume was 50 mL, and no intraopera- tive complications occurred. The pathological diagnosis of the tumor was type 5 well- and moderately differentiated adenocarcinoma, pT2N0, Stage I. No recurrence was evident 3 months after the operation, and no adjuvant chemo- therapy was performed. Conclusion: The transperineal approach might be useful in patients with Crohn’s disease who develop rectal cancer after multiple abdominal surgeries. Keywords: Rectal cancer, Crohn’s disease, History of surgery, Laparoscopic surgery, Transperineal total mesorectal excision Transperineal total mesorectal excision for rectal cancer on the residual rectum after multiple abdominal surgeries in a patient with Crohn’s disease: a case report Shin Emoto1 , Shigenori Homma1, Tadashi Yoshida1, Nobuki Ichikawa1, Yoichi Miyaoka1, Hiroki Matsui1, Ryo Takahashi1, Keita Ishido1, Takuya Otsuka2, Tomoko Mitsuhashi2, Takehiko Katsurada3 and Akinobu Taketomi1 Emoto et al. surg case rep (2021) 7:122 https://doi.org/10.1186/s40792-021-01206-7 Emoto et al. surg case rep (2021) 7:122 https://doi.org/10.1186/s40792-021-01206-7 Open Access *Correspondence: shin_mto0509@yahoo.co.jp 1 Department of Gastroenterological Surgery I, Hokkaido University Graduate School of Medicine, Kita 15 Nishi 7, Kita‑ku, Sapporo, Hokkaido 060‑8638, Japan Full list of author information is available at the end of the article Emoto et al. surg case rep (2021) 7:122 Page 2 of 7 Page 2 of 7 abdomen and a scar from a midline incision extending from the epigastric to suprapubic region were observed. Digital examination could not be performed because of stenosis of the anus secondary to the previous anal fis- tula, which had cured and showed no evidence of can- ceration. On hematological examination, his blood urea nitrogen concentration was 43 mg/dL, creatinine con- centration was 12.16 mg/dL (before hemodialysis), carci- noembryonic antigen concentration was 1.8 ng/mL, and CA19-9 concentration was 24.4 ng/mL. Colonoscopy showed a circumferential tumor at the remaining rec- tum (Fig. 1a), and biopsy revealed well-differentiated ade- nocarcinoma. A contrast enema revealed a 6-cm defect at the remaining rectum around the pritoneal reflection (Fig. 1b). Computed tomography examination revealed wall thickening of the remaining rectum but no swollen regional lymph nodes or distant metastasis. Magnetic resonance imaging showed that the estimated depth of wall invasion by the tumor was T2 because the muscle layer was continuous (Fig. 1c). The preoperative diagno- sis of the rectal cancer was clinical T2N0M0, Stage I. the general population [2]. Moreover, cancerization at the residual rectum or rectal stump after total colectomy may become a concern in patients with colonic CD [3, 4]. Surgery for rectal stump carcinoma may be techni- cally demanding because of patients’ history of multiple surgeries. Local recurrence is a potential complication after sur- gery for low rectal cancer. Complete total mesorectal excision (TME) with a negative circumferential resec- tion margin (CRM) is important to prevent local recur- rence. However, achieving TME with a negative CRM is difficult in patients with obesity, a contracted pelvis, or a bulky tumor [5, 6]. Transanal TME (Ta-TME) is con- sidered to resolve these difficulties. Ta-TME is a tech- nique known as the “bottom up” procedure of TME. This approach provides clear visualization of the dissection plane from the anus or low rectum, which may facilitate improvement of TME quality and reduction of a positive CRM and postoperative complications [7, 8]. Ta-TME is also reportedly useful for patients with a history of mul- tiple abdominal surgeries [9], which causes abdominal adhesion and makes the abdominal approach difficult. Especially, the approach from the bottom was called as “transperineal approach” when the anus was removed [10]. Severe intra-abdominal adhesions were expected because of the patient’s history of multiple surgeries; therefore, we performed an operation with both laparo- scopic transabdominal and transperineal approaches. The transabdominal procedure and transperineal approach were carried out concomitantly. For the transabdominal approach, three ports were placed in the abdomen. One of the ports was placed in the umbilicus for scope insertion, and the other two were placed on the left side of abdomen for the laparoscopic operator. The transperineal approach was concurrently implemented. First, two purse-string sutures were placed to close the rectal lumen and prevent cancer cell dissemination. The scar due to the previous anal fistula existed, but could be easily dissected. After dissecting the perianal skin and perirectal soft tissue, a GelPOINT Path transanal access platform (Applied Medical, Rancho Santa Margarita, CA, USA) was inserted and an AirSeal System (CONMED, Utica, NY, USA) was used for carbon dioxide insufflation during the transperineal approach. In the transabdomi- nal approach, adhesion of the small intestine and greater omentum to the abdominal wall was observed. Even after dissection of the intestinal and omental adhesion, it was difficult to recognize the wall of the remaining rectum because of the pelvic adhesion due to previous operations. Careful dissection of the rectum progressed laparoscopically. In the transperineal approach, the cor- rect plane could be recognized and TME smoothly pro- gressed. The influences of the inflammation of CD or chronic renal failure were none or very little to dissect the correct plane. The rectal dissection progressed along with the levator ani muscle, and the anococcygeal ligament In this report, we describe a patient with CD who developed rectal cancer at the residual rectum after mul- tiple abdominal surgeries. The rectal cancer was success- fully treated with transperineal(Tp)-TME. Discussion We have herein presented a case of CD with rectal can- cer at the residual rectum after multiple abdominal sur- geries; the cancer was successfully treated with Ta-TME. Approximately 70 to 80% of patients with CD report- edly undergo surgical treatment [11, 12] and the rate of clinical anastomotic recurrence without drug therapy is around 20 to 25% per year [13]. Although the long-term impact of improvements in pharmaceutical and biologi- cal therapy on surgical outcomes is still unknown, it is considered that many patients with CD might undergo several surgical treatments. It is clear that the more fre- quently patients undergo surgical treatment, the more difficult transabdominal operations become because of the development of intra-abdominal adhesions. The patient in this report underwent three abdominal opera- tions before rectal cancer surgery. The difficulty of the operation for the residual rectal cancer was easily pre- dicted, and severe adhesion was in fact observed during the laparoscopic surgery. On gross examination of the resected specimen, the tumor was a flat and villiform mass classified as type 5 (Fig. 3a). The size of the tumor was 80 × 67 mm. Histo- logically, the tumor was a well- to moderately differenti- ated adenocarcinoma with foci of a poorly differentiated component (Fig. 3b). The background contained char- acteristic findings of CD: chronic inflammation through the rectal wall and noncaseous epithelioid cell granulo- mas with multinucleated giant cells in the submucosa Fig. 1 Preoperative examination findings. a Colonoscopy showed a circumferential tumor in the residual rectum. b Contrast enema showed the tumor located at the low rectum (black arrow). c Magnetic resonance imaging revealed the circumferential tumor at the low rectum (dashed circle). The depth of wall invasion by the tumor was estimated as T2 because the muscle layer of the rectum was continuous Cancer at the defunctioning residual rectum or rectal stump is also reportedly problematic in patients with Crohn’s coloproctitis after total colectomy [3, 4, 14], although colorectal cancer rarely occurs in the entire cohort of patients with CD. In patients with CD, end ile- ostomy with a closed rectal stump is sometimes selected for treatment of Crohn’s colitis because perianal com- plications or impaired healing of a perianal wound may occur [15, 16]. Although fecal diversion may reduce the rectal inflammation, rectal cancer at the residual rectum can develop. Lutgens et al. Case presentationh The patient was a 51-year-old man with a history of renal failure due to acute rapidly progressive glomerulonephri- tis that had been diagnosed 2 years earlier and required treatment with artificial hemodialysis. He had been diag- nosed with CD 35 years earlier and underwent ileocecal resection. He developed a CD-induced vesicoileal fistula and vesicosigmoid fistula and underwent right colectomy, sigmoid colectomy, closure of the bladder fistula, small bowel resection, and ileostomy 23 years earlier. After this operation, the estimated length of the remaining small bowel was 1 m. Since then, he had received home par- enteral nutrition because of short bowel syndrome. He also underwent total colectomy 13 years earlier because he had developed extensive colitis. He currently desired renal transplantation for chronic renal failure and was examined in our hospital. Pre-transplantation colono- scopic examination led to a diagnosis of rectal cancer of the remaining rectum. He was then referred to our department. On physical examination, his height was 150.7 cm, weight was 38.1 kg (dry weight), and body mass index was 16.8 kg/m2. A single ileostomy at the right lower Emoto et al. surg case rep (2021) 7:122 Page 3 of 7 Page 3 of 7 (Fig. 3c). Therefore, we diagnosed the tumor as colitic cancer associated with CD. The pathological stage was pT2N0M0, Stage I. The resection margin was negative. During the postoperative course, the patient devel- oped a fever due to an intra-abdominal infection 3 days after the operation, which might be due to a retrograde drainage tube infection estimated by CT, and it was treated with antibiotics. He was discharged 21 days after the operation. No recurrence was evident 3 months after the operation, and no adjuvant chemotherapy was performed. was resected in the transperineal approach (Fig. 2a). Rendezvous of the abdominal space with the transper- ineal space was achieved on the dorsal side of the rectum. TME progressed from the posterior to lateral side of the rectum laparoscopically and transperineally. Preserva- tion of the S4 nerve was confirmed in the transperineal approach. Finally, the anterior wall of the rectum was completely dissected after confirming the posterior wall of the prostate in the laparoscopic approach (Fig. 2b). The specimen was extracted from the perineal incision. Fig- ure 2c shows the pelvic space after extraction of the spec- imen. Strong adhesion was observed in the pelvic floor. Case presentationh The operation was finished without conversion from lap- aroscopy to open surgery (Fig. 2d). The duration of the operation was 3 h 48 min, and the blood loss volume was 50 mL. No intraoperative complications occurred. (Fig. 3c). Therefore, we diagnosed the tumor as colitic cancer associated with CD. The pathological stage was pT2N0M0, Stage I. The resection margin was negative. h During the postoperative course, the patient devel- oped a fever due to an intra-abdominal infection 3 days after the operation, which might be due to a retrograde drainage tube infection estimated by CT, and it was treated with antibiotics. He was discharged 21 days after the operation. No recurrence was evident 3 months after the operation, and no adjuvant chemotherapy was performed. Discussion The prostate was explored laparoscopically, and the layer between the prostate and the residual rectum was dissected with support from the transanal approach to avoid injuring the rectourethral muscle and the urethra. c Transabdominal (laparoscopic) view after resection of the residual rectum. Strong adhesion of the small intestine to the pelvic wall was observed, and the natural pelvic anatomy was unclear because of the history of multiple surgeries. d Schema of the intraoperative overview of the abdomen. During the operation, the end ileostomy was covered by gauze and plastic film. A drainage tube was inserted from the left lower incision, through which a port was inserted, to the pelvic cavity after the operation accessibility from the abdominal cavity to the distal rec- tum is reduced by the forward angle of the low rectum [8]. In addition, obesity, a narrow pelvis, a bulky tumor, and pelvic irradiation make access to the low rectum and completion of TME more difficult [5, 6]. The transa- nal approach may provide clear visualization and detec- tion of the correct dissection layer, facilitating complete TME [7]. The potential benefits of Ta-TME are expected to include higher-quality TME with a better CRM, less morbidity, a lower frequency of conversion, and more sphincter-saving resection [8]. Indeed, some of these benefits have been reported [6, 7, 21, 22]. Ta-TME is also considered to be useful in patients with a history of mul- tiple abdominal surgeries. Narihiro et al. [9] reported a case of rectal cancer treated with Ta-TME in a patient who had undergone cholecystectomy, appendectomy, and Hartmann’s operation for sigmoid colon cancer. They concluded that they could overcome the difficul- ties of the transabdominal approach by Ta-TME; that is, the risks of bleeding and organ injury, longer duration of surgery, poor visual field due to intra-abdominal adhe- sion, poor surgical maneuverability, and loss of curability. In our case, although severe intra-abdominal adhesion was observed as predicted before the operation, Tp-TME incident ratio of rectal cancer in patients with CD who developed a perianal inflammatory lesion was 0.77 cases per 1000 patient-years and that the risk of rectal cancer in patients with CD who developed a perianal lesion was 5.11 times higher than that in patients without a peri- anal lesion [18]. These data indicate that a longer dura- tion of extensive Crohn’s coloproctitis increases the risk of rectal cancer. Discussion [3] reviewed the literature of rectal stump cancer, including 29 patients with CD. They showed that one of the risk factors for rectal stump can- cer was the duration of the disease. von Roon et al. [2] reported that the risk of developing colorectal cancer in patients younger than 30 years of age at the time of diag- nosis of CD was 9.50 to 21.46 times higher than that in the general population. Whereas the risk of CD-associ- ated rectal cancer was reportedly comparable with the risk of rectal cancer in the general population [2, 17], a multicenter investigation in France revealed that the Fig. 1 Preoperative examination findings. a Colonoscopy showed a circumferential tumor in the residual rectum. b Contrast enema showed the tumor located at the low rectum (black arrow). c Magnetic resonance imaging revealed the circumferential tumor at the low rectum (dashed circle). The depth of wall invasion by the tumor was estimated as T2 because the muscle layer of the rectum was continuous Page 4 of 7 Emoto et al. surg case rep (2021) 7:122 Emoto et al. surg case rep Fig. 2 Operative views. a Transanal view. After dissecting the levator ani muscle, the anococcygeal ligament was explored. b Transabdominal (laparoscopic) view. After rendezvous of the transanal and transabdominal space, the residual rectum was dissected from the dorsal side to lateral side. The prostate was explored laparoscopically, and the layer between the prostate and the residual rectum was dissected with support from the transanal approach to avoid injuring the rectourethral muscle and the urethra. c Transabdominal (laparoscopic) view after resection of the residual rectum. Strong adhesion of the small intestine to the pelvic wall was observed, and the natural pelvic anatomy was unclear because of the history of multiple surgeries. d Schema of the intraoperative overview of the abdomen. During the operation, the end ileostomy was covered by gauze and plastic film. A drainage tube was inserted from the left lower incision, through which a port was inserted, to the pelvic cavity after the operation Fig. 2 Operative views. a Transanal view. After dissecting the levator ani muscle, the anococcygeal ligament was explored. b Transabdominal (laparoscopic) view. After rendezvous of the transanal and transabdominal space, the residual rectum was dissected from the dorsal side to lateral side. Discussion c Pathological examination of the resected residual rectum with hematoxylin and eosin staining (× 40 and × 200). At low magnification, mononuclear cells (white arrow) were found to have migrated into the submucosal layer. At high magnification, noncaseous epithelioid cell granulomas (black arrowhead) and multinucleated giant cells (black arrow) were seen. These findings are typical pathological observations in patients with Crohn’s disease facilitated detection of the correct plane of TME from the anal approach, reduced the duration of surgery by a two-team approach, avoided injury of organs adjacent to the rectum, and achieved negative pathological resec- tion margin. The two-team approach also helped with the rectal dissection after rendezvous of the transabdominal and transperineal approaches. The appropriate dissec- tion layer can sometimes be identified by applying ten- sion to the tissue from both above and below. In addition, the advantages of Tp-TME also maximized in our case although the dissection between the rectum and the prostate was eventually performed laparoscopically. To dissect between the rectum and the prostate safely, the dorsal side of the prostate should be exposed from the lateral side. Although we finally dissected anterior side of the rectum laparoscopically, the transperineal approach played an important role to identify the correct layer between the rectum and the prostate. facilitated detection of the correct plane of TME from the anal approach, reduced the duration of surgery by a two-team approach, avoided injury of organs adjacent to the rectum, and achieved negative pathological resec- tion margin. The two-team approach also helped with the rectal dissection after rendezvous of the transabdominal and transperineal approaches. The appropriate dissec- tion layer can sometimes be identified by applying ten- sion to the tissue from both above and below. In addition, the advantages of Tp-TME also maximized in our case although the dissection between the rectum and the prostate was eventually performed laparoscopically. To dissect between the rectum and the prostate safely, the dorsal side of the prostate should be exposed from the lateral side. Although we finally dissected anterior side of the rectum laparoscopically, the transperineal approach played an important role to identify the correct layer between the rectum and the prostate. revealed that misdirection of the correct layer occurred in 7.8% of cases and that injury to adjacent organs, includ- ing the urethra, occurred in 1.5% of cases [23]. Discussion We previously described a patient with CD who developed colitic cancer in the residual rectum following subtotal colectomy [19]. He had developed CD at 20 years of age and underwent subtotal colectomy at 37 years of age; the residual rectal cancer developed 8 years after subtotal colectomy. The risk of residual rec- tal cancer was deemed to be high in our patient because he had been diagnosed with CD at 16 years of age and had a history of perianal inflammation. Although his cancer was not derived from the scar of the anal fistula, pathological findings showed inflammation of the rem- nant rectum; this inflammation may have caused his rec- tal cancer.i Since Ta-TME was at first reported by Lacy and Adelsdorfer in 2011 [20], this procedure has become widespread throughout the world. It is difficult to achieve TME for mid- or low-rectal cancer because the Emoto et al. surg case rep (2021) 7:122 Page 5 of 7 Emoto et al. surg case rep Fig. 3 Pathological findings. a Resected specimen. White arrowheads show the tumor at the residual rectum. b Pathological examination of the rectal tumor with hematoxylin and eosin staining (× 40 and × 200). The depth of tumor invasion was pathologically diagnosed as T2. Columnar cells with hyperchromatic nuclei proliferating in tubular patterns were seen. Focal non-glandular irregular nests were also observed. The pathological diagnosis was well- to moderately differentiated adenocarcinoma with foci of a poorly differentiated component. MP: muscularis propria. c Pathological examination of the resected residual rectum with hematoxylin and eosin staining (× 40 and × 200). At low magnification, mononuclear cells (white arrow) were found to have migrated into the submucosal layer. At high magnification, noncaseous epithelioid cell granulomas (black arrowhead) and multinucleated giant cells (black arrow) were seen. These findings are typical pathological observations in patients with Crohn’s disease Fig. 3 Pathological findings. a Resected specimen. White arrowheads show the tumor at the residual rectum. b Pathological examination of the rectal tumor with hematoxylin and eosin staining (× 40 and × 200). The depth of tumor invasion was pathologically diagnosed as T2. Columnar cells with hyperchromatic nuclei proliferating in tubular patterns were seen. Focal non-glandular irregular nests were also observed. The pathological diagnosis was well- to moderately differentiated adenocarcinoma with foci of a poorly differentiated component. MP: muscularis propria. Declarations 15. Sher ME, Bauer JJ, Gorphine S, Gelernt I. Low Hartmann’s procedure for severe anorectal Crohn’s disease. Dis Colon Rectum. 1992;35(10):975–80. Acknowledgements g We thank Angela Morben, DVM, ELS, from Edanz Group (https://en-author- services.edanz.com/ac), for editing a draft of this manuscript. We thank Angela Morben, DVM, ELS, from Edanz Group (https://en-author- services.edanz.com/ac), for editing a draft of this manuscript. 9. Narihiro S, Ohdaira H, Takeuchi H, Kamada T, Marukuchi R, Suzuki N, et al. Transanal total mesorectal excision (Ta-TME) in a rectal cancer patient with a history of abdominal surgery: a case report. J Anus Rectum Colon. 2020;4(1):41–6. 9. Narihiro S, Ohdaira H, Takeuchi H, Kamada T, Marukuchi R, Suzuki N, et al. Transanal total mesorectal excision (Ta-TME) in a rectal cancer patient with a history of abdominal surgery: a case report. J Anus Rectum Colon. 2020;4(1):41–6. Discussion Recently, data from the national registry of Norway showed a high rate of local recurrence in patients who had undergone Ta-TME for rectal cancer [24]. In this report, the 2-year local recurrence rate was > 10%, which was signifi- cantly higher than that associated with the laparoscopic approach. The authors speculated that gas pressure and surgical manipulation stressed the purse-string suture used to close the rectal lumen, resulting in spread of the cancer cells from the rectal lumen to the intra-abdominal cavity. In our department, a double purse-string suture is applied to tightly close the rectal rumen and prevent the spread of tumor cells. The international Ta-TME educa- tional collaborative group provided recommendations for surgeons and centers as well as a training curriculum of Ta-TME [25]. Surgeons should carefully introduce this procedure to their own institute, determine the indica- tions for Ta-TME, and train their own clinicians accord- ing to the expert guidance. Ta-TME is technically demanding and requires a proper anatomical perspective from the anal side. The data of the international registry of first Ta-TME cases Emoto et al. surg case rep (2021) 7:122 Emoto et al. surg case rep (2021) 7:122 Page 6 of 7 Availability of data and materials 13. Luglio G, Rispo A, Imperatore N, Giglio MC, Amendola A, Tropeano FP, et al. Surgical prevention of anastomotic recurrence by excluding mes- entery in Crohn’s disease: the SuPREMe-CD study—a randomized clinical trial. Ann Surg. 2020;272(2):210–7. The data are not available for public access because of patient privacy con- cerns, but are available from the corresponding author on reasonable request. g 14. Cirincione E, Gorfine SR, Bauer JJ. Is Hartmann’s procedure safe in Crohn’s disease? Report of three cases. Dis Colon Rectum. 2000;43(4):544–7. Abbreviations CD C h ’ di 7. Roodbeen SX, Penna M, Mackenzie H, Kusters M, Slater A, Jones OM, et al. Transanal total mesorectal excision (TaTME) versus laparoscopic TME for MRI-defined low rectal cancer: a propensity score-matched analysis of oncological outcomes. Surg Endosc. 2019;33(8):2459–67. 7. Roodbeen SX, Penna M, Mackenzie H, Kusters M, Slater A, Jones OM, et al. Transanal total mesorectal excision (TaTME) versus laparoscopic TME for MRI-defined low rectal cancer: a propensity score-matched analysis of oncological outcomes. Surg Endosc. 2019;33(8):2459–67. CD: Crohn’s disease; CRM: Circumferential resection margin; Ta-TME: Transanal total mesorectal excision; TME: Total mesorectal excision; Tp-TME: Transper- ineal total mesorectal excision. 8. Deijen CL, Velthuis S, Tsai A, Mavroveli S, de Lange-de Klerk ES, Sietses C, et al. COLOR III: a multicentre randomised clinical trial comparing transanal TME versus laparoscopic TME for mid and low rectal cancer. Surg Endosc. 2016;30(8):3210–5. 8. Deijen CL, Velthuis S, Tsai A, Mavroveli S, de Lange-de Klerk ES, Sietses C, et al. COLOR III: a multicentre randomised clinical trial comparing transanal TME versus laparoscopic TME for mid and low rectal cancer. Surg Endosc. 2016;30(8):3210–5. Funding 12. Bernstein CN, Nabalamba A. Hospitalization, surgery, and readmission rates of IBD in Canada: a population-based study. Am J Gastroenterol. 2006;101(1):110–8. This study was supported by no financial source. Ethics approval and consent to participate Not applicable. 16. Yamamoto T, Keighley MR. Long-term outcome of total colectomy and ileostomy for Crohn disease. Scand J Gastroenterol. 1999;34(3):280–6. Consent for publication 17. Sjödahl RI, Myrelid P, Söderholm JD. Anal and rectal cancer in Crohn’s disease. Colorectal Dis. 2003;5(5):490–5. Written informed consent was obtained from the patient for publication of this case report and publication of his data. 18. Beaugerie L, Carrat F, Nahon S, Zeitoun JD, Sabaté JM, Peyrin-Biroulet L, et al. High risk of anal and rectal cancer in patients with anal and/or peri- anal Crohn’s disease. Clin Gastroenterol Hepatol. 2018;16(6):892-9.e2. Authors’ contributions SE wrote the manuscript. SE, SH, TY, NI, YM, HM, RT, KI, and AT contributed to performance of the operation and postoperative management. TK contributed to the preoperative diagnosis and managed the preoperative and postoperative treatment. TO and TM contributed to the postoperative pathological diagnosis. All authors critically reviewed, read and approved the final manuscript. 10. Yasukawa D, Hori T, Kadokawa Y, Kato S, Aisu Y, Hasegawa S. Trans-per- ineal minimally invasive surgery during laparoscopic abdominoperineal resection for low rectal cancer. Surg Endosc. 2019;33(2):437–47. 11. Hancock L, Windsor AC, Mortensen NJ. Inflammatory bowel disease: the view of the surgeon. Colorectal Dis. 2006;8(Suppl 1):10–4. Competing interests The authors declare no financial nor non-financial competing interests. 19. Shibuya K, Homma S, Yoshida T, Ohno Y, Ichikawa N, Kawamura H, et al. Carcinoma in the residual rectum of a long-standing Crohn’s disease patient following subtotal colectomy: a case report. Mol Clin Oncol. 2018;9(1):50–3. References Three-year nationwide experience with transanal total mesorectal excision for rectal cancer in the Netherlands: a propen- sity score-matched comparison with conventional laparoscopic total mesorectal excision. J Am Coll Surg. 2019;228(3):235-44.e1. References 1. Freeman HJ. Colorectal cancer risk in Crohn’s disease. World J Gastroen- terol. 2008;14(12):1810–1. 1. Freeman HJ. Colorectal cancer risk in Crohn’s disease. World J Gastroen- terol. 2008;14(12):1810–1. We experienced a case of residual rectal cancer treated by combination of a laparoscopic and transperineal approach in a patient who had undergone several surgeries for Crohn’s enterocolitis. The development of rectal can- cer might be a concern in patients with extensive Crohn’s coloproctitis, especially in those who were diagnosed with CD at < 30 years of age or who have had a long duration of CD. Increasingly more patients with CD are undergo- ing multiple abdominal operations as the prognosis of CD has improved, and Ta-TME or Tp-TME might be useful in patients with CD who develop rectal cancer after undergo- ing multiple abdominal surgeries. 2. van Roon AC, Reese G, Teare J, Constantinides V, Darzi AW, Tekkis PP. The risk of cancer in patients with Crohn’s disease. Dis Colon Rectum. 2007;50(6):839–55. 2. van Roon AC, Reese G, Teare J, Constantinides V, Darzi AW, Tekkis PP. The risk of cancer in patients with Crohn’s disease. Dis Colon Rectum. 2007;50(6):839–55. 3. Lutgens MW, van Oijen MG, Vleggaar FP, Siersema PD, Broekman MM, Oldenburg B. Risk factors for rectal stump cancer in inflammatory bowe disease. Dis Colon Rectum. 2012;55(2):191–6. 3. Lutgens MW, van Oijen MG, Vleggaar FP, Siersema PD, Broekman MM, Oldenburg B. Risk factors for rectal stump cancer in inflammatory bowel disease. Dis Colon Rectum. 2012;55(2):191–6. 4. Ten Hove JR, Bogaerts JMK, Bak MTJ, Laclé MM, Meij V, Derikx L, et al. Malignant and nonmalignant complications of the rectal stump in patients with inflammatory bowel disease. Inflamm Bowel Dis. 2019;25(2):377–84. 5. Cecil TD, Taffinder N, Gudgeon AM. A personal view on laparoscopic rectal cancer surgery. Colorectal Dis. 2006;8(Suppl 3):30–2. 5. Cecil TD, Taffinder N, Gudgeon AM. A personal view on laparoscopic rectal cancer surgery. Colorectal Dis. 2006;8(Suppl 3):30–2. 6. Detering R, Roodbeen SX, van Oostendorp SE, Dekker JT, Sietses C, Bemelman WA, et al. Three-year nationwide experience with transanal total mesorectal excision for rectal cancer in the Netherlands: a propen- sity score-matched comparison with conventional laparoscopic total mesorectal excision. J Am Coll Surg. 2019;228(3):235-44.e1. 6. Detering R, Roodbeen SX, van Oostendorp SE, Dekker JT, Sietses C, Bemelman WA, et al. Emoto et al. surg case rep (2021) 7:122 Page 7 of 7 Page 7 of 7 i g 24. Wasmuth HH, Faerden AE, Myklebust T, Pfeffer F, Norderval S, Riis R, et al. Transanal total mesorectal excision for rectal cancer has been suspended in Norway. Br J Surg. 2020;107(1):121–30. 23. Penna M, Hompes R, Arnold S, Wynn G, Austin R, Warusavitarne J, et al. Transanal total mesorectal excision: international registry results of the first 720 cases. Ann Surg. 2017;266(1):111–7. 24. Wasmuth HH, Faerden AE, Myklebust T, Pfeffer F, Norderval S, Riis R, et al. Transanal total mesorectal excision for rectal cancer has been suspended in Norway. Br J Surg. 2020;107(1):121–30. 25. Francis N, Penna M, Mackenzie H, Carter F, Hompes R. Consensus on structured training curriculum for transanal total mesorectal excision (TaTME). Surg Endosc. 2017;31(7):2711–9. 23. Penna M, Hompes R, Arnold S, Wynn G, Austin R, Warusavitarne J, et al. Transanal total mesorectal excision: international registry results of the first 720 cases. Ann Surg. 2017;266(1):111–7. 25. Francis N, Penna M, Mackenzie H, Carter F, Hompes R. Consensus on structured training curriculum for transanal total mesorectal excision (TaTME). Surg Endosc. 2017;31(7):2711–9. Author details 1 1 Department of Gastroenterological Surgery I, Hokkaido University Graduate School of Medicine, Kita 15 Nishi 7, Kita‑ku, Sapporo, Hokkaido 060‑8638, Japan. 2 Department of Surgical Pathology, Hokkaido University Hospital, Kita 14 Nishi 5, Kita‑ku, Sapporo, Hokkaido 060‑8648, Japan. 3 Department of Gastroenterology and Hepatology, Hokkaido University Faculty of Medicine and Graduate School of Medicine, Kita 15 Nishi 7, Kita‑ku, Sapporo, Hokkaido 060‑8638, Japan. 20. Lacy AM, Adelsdorfer C. Totally transrectal endoscopic total mesorectal excision (TME). Colorectal Dis. 2011;13(Suppl 7):43–6. 21. Veltcamp Helbach M, Koedam TWA, Knol JJ, Diederik A, Spaargaren GJ, Bonjer HJ, et al. Residual mesorectum on postoperative magnetic reso- nance imaging following transanal total mesorectal excision (TaTME) and laparoscopic total mesorectal excision (LapTME) in rectal cancer. Surg Endosc. 2019;33(1):94–102. Received: 19 March 2021 Accepted: 10 May 2021 Received: 19 March 2021 Accepted: 10 May 2021 22. Ma B, Gao P, Song Y, Zhang C, Zhang C, Wang L, et al. Transanal total mesorectal excision (taTME) for rectal cancer: a systematic review and meta-analysis of oncological and perioperative outcomes compared with laparoscopic total mesorectal excision. BMC Cancer. 2016;16:380. Emoto et al. surg case rep (2021) 7:122 Emoto et al. surg case rep (2021) 7:122 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations.
https://openalex.org/W3021032566	https://www.frontiersin.org/articles/10.3389/fphar.2020.00559/pdf	English	null	Comparative Efficacy and Acceptability of Pharmaceutical Management for Adults With Post-Traumatic Stress Disorder: A Systematic Review and Meta-Analysis	Frontiers in pharmacology	2,020	cc-by	14,047	Keywords: post-traumatic stress disorder, pharmacotherapy, selective serotonin reuptake inhibitors, efﬁcacy, core symptoms, all-cause discontinuation SYSTEMATIC REVIEW published: 08 May 2020 doi: 10.3389/fphar.2020.00559 Edited by: Tahir Mehmood Khan, University of Veterinary and Animal Sciences, Pakistan Edited by: Tahir Mehmood Khan, University of Veterinary and Animal Sciences, Pakistan Zhen-Dong Huang †, Yi-Fan Zhao †, Shuang Li, Hui-Yun Gu, Lu-Lu Lin, Zhi-Yan Yang, Yu-Ming Niu, Chao Zhang and Jie Luo* Reviewed by: Kurt Neumann, Independent Researcher, Kere´ kteleki, Hungary Mmamosheledi Elsie Mothibe, Sefako Makgatho Health Sciences University, South Africa Center for Evidence-Based Medicine and Clinical Research, Taihe Hospital, Hubei University of Medicine, Shiyan, China The current clinical guidelines on post-traumatic stress disorder (PTSD) recommend selective serotonin reuptake inhibitors (SSRIs) and serotonin and norepinephrine reuptake inhibitors (SNRIs) of drugs. However, there is uncertainty about the efﬁcacy of other drugs and selecting which treatments work best for which patients. This meta-analysis evaluated efﬁcacy and acceptability of pharmaceutical management for adults with PTSD. Randomized-controlled trials, which reported active comparators and placebo- controlled trials of pharmaceutical management for adults with PTSD, from the Ovid Medline, EMBase, CENTRAL, PsycINFO, Ovid Health and Psychosocial Instruments, and ISIWeb of Science, were searched until June 21, 2019. In terms of efﬁcacy, all active drugs demonstrated superior effect than placebo (SMD = −0.33; 95% CI, −0.43 to −0.23). The medications were superior to placebo in reducing the symptom of re-experiencing, avoidance, hyperarousal, depression, and anxiety. For acceptability, medicine interventions for PTSD showed no increase in all-cause discontinuation compared with placebo. Nevertheless, in terms of safety, medicine interventions indicated a higher risk of adverse effect compared with placebo (RR = 1.47, 95% CI: 1.24 to 1.75). Compared with placebo, the SSRIs and atypical antipsychotics drugs had signiﬁcant efﬁcacy whether in patients with severe or extremely severe PTSD status. However, only atypical antipsychotics (SMD = −0.29, 95% CI: −0.48 to −0.10) showed superior efﬁcacy than placebo in veterans. Medication management could be effective in intervention of PTSD, which demonstrated a sufﬁcient improvement in the core symptoms. This meta-analysis supports the status of SSRIs and SNRIs as recommended pharmacotherapy. However, patients with different clinical characteristics of PTSD should consider individualized drug management. Correspondence: Jie Luo taihehospital@yeah.net Yu-Ming Niu niuyuming@yeah.net Chao Zhang zhangchao0803@126.com †These authors have contributed equally to this work Correspondence: Jie Luo taihehospital@yeah.net Yu-Ming Niu niuyuming@yeah.net Chao Zhang zhangchao0803@126.com †These authors have contributed equally to this work Specialty section: This article was submitted to Pharmaceutical Medicine and Outcomes Research, a section of the journal Frontiers in Pharmacology Received: 04 March 2020 Accepted: 14 April 2020 Published: 08 May 2020 Specialty section: This article was submitted to Pharmaceutical Medicine and Outcomes Research, a section of the journal Frontiers in Pharmacology Received: 04 March 2020 Accepted: 14 April 2020 Published: 08 May 2020 Citation: Huang Z-D, Zhao Y-F, Li S, Gu H-Y, Lin L-L, Yang Z-Y, Niu Y-M, Zhang C and Luo J (2020) Comparative Efﬁcacy and Acceptability of Pharmaceutical Management for Adults With Post- Traumatic Stress Disorder: A Systematic Review and Meta-Analysis. Front. Pharmacol. 11:559. doi: 10.3389/fphar.2020.00559 Specialty section: This article was submitted to Pharmaceutical Medicine and Outcomes Research, a section of the journal Frontiers in Pharmacology Received: 04 March 2020 Accepted: 14 April 2020 Published: 08 May 2020 Specialty section: This article was submitted to Pharmaceutical Medicine and Outcomes Research, a section of the journal Frontiers in Pharmacology Received: 04 March 2020 Accepted: 14 April 2020 Published: 08 May 2020 Citation: Huang Z-D, Zhao Y-F, Li S, Gu H-Y, Lin L-L, Yang Z-Y, Niu Y-M, Zhang C and Luo J (2020) Comparative Efﬁcacy and Acceptability of Pharmaceutical Management for Adults With Post- Traumatic Stress Disorder: A Systematic Review and Meta-Analysis. Front. Pharmacol. 11:559. doi: 10.3389/fphar.2020.00559 May 2020 \| Volume 11 \| Article 559 1 Frontiers in Pharmacology \| www.frontiersin.org Pharmaceutical Management for PTSD Pharmaceutical Management for PTSD Huang et al. INTRODUCTION Although there are a few studies that have provided the most effective interventions for particular patients under speciﬁc conditions, they do not address the awkward situation of choosing an appropriate drug for different types of PTSD patients (Association, 2017) in clinical practice. Considering the uncertainty of existing evidence and the lack of information on a particular type of trauma (e.g., the severity of trauma), clinical guidelines have not yet provided a clear intervention scheme for PTSD management. This meta- analysis evaluated efﬁcacy, acceptability, and safety of pharmacological treatments while considering patients’ clinical characteristics; it provides the latest evidence that can help make decisions for pharmaceutical management of PTSD in adults. Post-traumatic stress disorder (PTSD) is a mental disorder that can occur after a person has experienced a traumatic event, such as physical abuse, sexual relationship violence, combat exposure, witnessing death or serious injury (Association, 2013). In Diagnostic and Statistical Manual of Mental Disorders (DSM)- 5, PTSD is characterized by intrusion, avoidance, hyperarousal, and negative thinking in cognition and mental; all these characteristics have a certain degree of impact on a patient’s life, occupation, and interpersonal dysfunction (Association, 2013). Patients with lifetime PTSD have developed one comorbid psychiatric disorder (Maher et al., 2006; Association, 2013; Rytwinski et al., 2013), such as depression, dissociation, anxiety, and sleep disturbance. As of 2017, a survey (Koenen et al., 2017) estimated the cross-national lifetime prevalence of PTSD at 3.9% and about 5.6% of the population were exposed to trauma events. In comparison, the National Comorbidity Survey Replication (NCS-R) (Kessler et al., 2005) estimated the lifetime prevalence of PTSD among American adults in 2005 (Ofﬁce, 2012) at 6.8%. The costs of managing PTSD are substantial. In 2012 (Committee on the Assessment of Ongoing Efforts in the Treatment of Posttraumatic Stress et al., 2014), the Department of Defense (DOD) and Veterans Affairs (VA) spent about USD 3 billion and USD 294 million for PTSD treatments of veterans and related service members, respectively. PTSD brings serious health-related and economic burden for patients and society. METHODS Search Strategy and Selection Criteria We used the guidelines from the Preferred Reporting Items for Systematic Review and Meta-Analyses (PRISMA) statement (Moher et al., 2009). All studies were obtained by searching the Ovid Medline, EMBase, CENTRAL, PsycINFO, Ovid Health and Psychosocial Instruments, and ISIWeb of Science for articles that were published until June 21, 2019. Four reviewers (Z-DH, H-YG, Y-FZ, and SL) independently assessed the abstracts and potentially eligible articles identiﬁed during literature selection. Discrepancies were resolved in discussions. If necessary, a ﬁnal reviewer (CZ) was involved when faced with a disagreement. Detailed search strategies are shown in Supplemental Method 1. p y A meta-analysis (Bromis et al., 2018) of structural magnetic resonance imaging (MRI) studies found that PTSD is associated with a decreased volume of hippocampus and structural brain abnormalities. Other reports (Geracioti et al., 2001; Milani et al., 2017) have demonstrated that PTSD patients have a greater central nervous system (CNS) noradrenergic activity under baseline conditions. PTSD is characterized by a series of neuroendocrine symptoms that may be responsive and sensitive to medication. The efﬁcacy of selective serotonin reuptake inhibitor (SSRIs) was afﬁrmed in previous meta- analyses (Stein et al., 2006; Hoskins et al., 2015; Puetz et al., 2015; Gu et al., 2016; Lee et al., 2016). At present, the American Psychological Association (APA) guideline (Association, 2017) suggests the use of ﬂuoxetine, paroxetine, sertraline, and venlafaxine. Although pharmacological treatments (Association, 2017) are currently considered as an important part of clinical guidelines of PTSD management, only sertraline and paroxetine drugs are approved for PTSD by the Food and Drug Administration (FDA) to date. There is insufﬁcient evidence to recommend for or against offering risperidone and topiramate. Nevertheless, there is no recommended ﬁrst-line treatment drug for PTSD because of sufﬁcient evidence from comparative effectiveness studies in the APA guideline (Association, 2017). More importantly, the APA guideline also indicated future research must assess the effectiveness of treatment for speciﬁc groups, such as gender differences, racial or cultural groups, and persons exposed to a particular type and severity of trauma (e.g., combat trauma, sexual assault, and community violence). Inclusion and Exclusion Criteria Inclusion and Exclusion Criteria Studies were included when they met the following criteria: (1) Adults (≥18 years old) with a primary diagnosis of PTSD according to diagnostic criteria (DSM-III, DSM-III-R, DSM-IV, DSM-IV-TR, DSM-V, or ICD-10 (santé Omdl et al., 1992) (2) Interventions: pharmacological treatments whether oral or intravenous infusion for adults with PTSD, such as monotherapy, adjunctive, or augmentation interventions; (3) Comparisons: placebo or other active drugs; (4) Outcomes: efﬁcacy (change in PTSD total symptoms using clinician rating scales or interview instruments (Association, 2018); reduction rate of core symptoms, including re-experiencing, avoidance, and hyperarousal; reduction rate of other symptoms, including depression and anxiety) and acceptability (all-cause discontinuation and discontinuation due to adverse effects). All-cause discontinuation was used as a measure for the acceptability of treatments because it encompasses efﬁcacy and tolerability; (5) type of studies: randomized controlled trials (RCTs). Studies were excluded for the following reasons: (1) The presence of a schizophrenia, schizoaffective disorder or bipolar disorder in particular cases participants with current depression or anxiety were included provided that their sub-symptom was secondary to PTSD; (2) Cognitive disorder or at immediate risk May 2020 \| Volume 11 \| Article 559 Frontiers in Pharmacology \| www.frontiersin.org 2 Pharmaceutical Management for PTSD Huang et al. of suicide; (3) Prevention or prevention of relapse trials; (4) Combination therapy with two or more drugs as main intervention, or psychological treatment combined with medication; (5) Data were not available or not convertible from the original research; (6) Duplicate publication. therapeutic regimens (monotherapy and adjunctive drugs). We also conducted stratiﬁed analyses to explore the special efﬁcacy of different drugs for patient ages (older adults, older than 60 years and non-older adults), gender (male and female), races, populations (veterans or civilians), and severity of trauma based only on CAPS scores (severe and extreme PTSD symptomatology); the outcome of changes in PTSD total symptoms, which were based on a comparison of active drugs with placebo, was used. Meta-regression analysis was performed to explore the effects of modiﬁers on the efﬁcacy of overall PTSD symptoms. The analysis included mean age at onset, baseline severity, publication year, and sponsorship. The trim-and-ﬁll method base on funnel plots (Duval and Tweedie, 2000) was used to evaluate the possible publication bias of the efﬁcacy of the drug. Funnel plots were used only for at least 10 studies to ensure adequate test performance (Higgins and Green, 2011). Frontiers in Pharmacology \| www.frontiersin.org Inclusion and Exclusion Criteria All statistical analyses were performed using R 3.5.2 software. Data Extraction and Quality Assessment Information and data were extracted by four independent authors (ZDH, HYG, FYZ, and ZYY); a ﬁnal investigator (CZ) proofread and handled any arguments. To reduce the heterogeneity of research in a variety of clinical measurement tools (Fervaha et al., 2015), we used clinician rating scales or interview instruments (Association, 2018) to assess PTSD total symptoms. We only used the clinically administered PTSD scale (Association, 2013) to assess the severity of trauma (Weathers et al., 2001), which is deﬁned as few symptoms (0–19), mild PTSD (20–39), moderate PTSD (40–59), severe PTSD symptomatology (60–79), and extreme PTSD symptomatology (≥80). Objective drugs that we study were added to an ongoing pharmacotherapy regimen that is, adjunctive treatment. To avoid over-optimistic estimates of the efﬁcacy, data were extracted from the intention to treat (ITT) sample whenever possible (Gupta, 2011). We used the change values from the baseline as much as possible in all the continuous outcomes. If change values from the baseline were not mentioned, we used a comparison of ﬁnal measurements according to Cochrane Handbook (Higgins and Green, 2011), which is a randomized trial estimating the same baseline value in theory. We prioritized the results of 8–12 weeks after drug treatment if a study reported different stages of treatment outcomes to reduce the associated heterogeneity impact. Two authors (ZDH and HYG) independently assessed the risk of bias in accordance with the Cochrane risk of bias tool (Higgins and Green, 2011). Study Characteristics Table 1 summarizes patient characteristics (i.e., author, sample, age, gender, race, population, severity of trauma, type of trauma, baseline score, diagnostic criteria, and drug dosage during treatment). In total, there were 14 trials of all-male populations, Three RCTs of drug treatment for all-female patients, and the rest were of mixed gender population. Although the DSM-5 indicates four core symptoms for PTSD, all of included studies only evaluated three core symptoms of PTSD (i.e., re-experiencing, avoidance, and hyperarousal) and other symptoms (depression and anxiety), therefore, these chosen symptoms were investigated to select the best pharmaceutical management regimens in this study. The average age range of population in all trials was 20 to 60 years. A total of 31 trials involving 1,987 veterans (1,831 patients combat-related PTSD) were included in PTSD pharmacological treatments trials. The remaining trials were contained in the population for civilians or mixed (civilians and veterans) (Table 1). Among the 66 included studies (Davidson et al., 1990; Kosten et al., 1991; Katz et al., 1994; van der Kolk et al., 1994; Baker et al., 1995; Connor et al., 1999; Hertzberg et al., 1999; Brady et al., 2000; Hertzberg et al., 2000; Butterﬁeld et al., 2001; Davidson et al., 2001; Marshall et al., 2001; Tucker et al., 2001; Martenyi et al., 2002; Stein et al., 2002; Zohar et al., 2002; Davidson et al., 2003; Hamner et al., 2003; Monnelly et al., 2003; Literature Identiﬁcation Literature Identiﬁcation The Ovid Medline, Ovid EMBASE, CENTRAL, and Web of Science were systematically searched until June 21, 2019. The search resulted in 8,237 articles. After initial evaluation, 1,056 studies were removed for being duplicates, 7,017 for being irrelevant (as determined by reading the title and abstracts), and 66 studies for reasons determined by reading the full text. The ﬁnal 66 studies, with 78 trials, were used in this study. Figure 1 shows the work ﬂow for the selection of studies. Change in Total PTSD Symptoms Based on a Clinician-Assessed Scale Compared with placebo, all active drugs (SMD, −0.33; 95% CI, −0.43 to −0.23), different drug classiﬁcations like atypical antipsychotics (SMD = −0.30, 95% CI: −0.46 to −0.13), SNRIs (SMD = −0.29, 95% CI: −0.44 to −0.14), SSRIs (SMD = −0.33, 95% CI: −0.40 to −0.25), and TeCAs (SMD = −1.87, 95% CI: −2.58 to −0.89) had signiﬁcant efﬁcacy in the change of clinician- assessed scale (Supplemental Table 4). However, the following speciﬁc drug branches presented more beneﬁcial effects than placebo (Supplemental Table 4): quetiapine (SMD = −0.49, 95% CI: −0.93 to −0.04), risperidone (SMD = −0.23, 95% CI: −0.42 to −0.03), ﬂuoxetine (SMD = −0.27, 95% CI: −0.42 to −0.12), hydroxyzine (SMD = −1.56, 95% CI: −2.11 to −1.02), mirtazapine (SMD = −1.87, 95% CI: −2.85 to −0.89), olanzapine (SMD = −0.66, 95% CI: −1.19 to −0.13), paroxetine (SMD = −0.48, 95% CI: −0.60 to −0.36), sertraline (SMD = −0.22, 95% CI: −0.35 to −0.10), and venlafaxine (SMD = −0.29, 95% CI: −0.44 to −0.14). Statistical Analysis We performed pooled risk ratios (RR) with 95% conﬁdence intervals (CI) using the Mantel-Haenszel statistical method for dichotomous data. Continuous data (Hedges, 1981) were analysed as the mean difference (MD) or standardized mean difference (SMD) with 95% CI. The SMD was used when the studies assessed the same outcome but different unit measurements; otherwise, MD was employed. SD was obtained from standard errors (SE) and CI for group means by appropriate statistical methods based on Cochrane Handbook (Higgins and Green, 2011). If studies only reported the median and range of the samples or the ﬁrst and third quartiles, we estimated the sample mean and SD (Wan et al., 2014; Luo et al., 2018). The I2 statistics (Higgins and Green, 2011) were used to assess the heterogeneity of each analysis (Higgins and Green, 2011). When I2 < 40%, a ﬁxed-effect model was used. If I2 ≥40, we assumed that there was statistical heterogeneity, therefore the pooled effect size was calculated by the random effects model. We performed pooling analysis of different outcomes based on placebo-controlled and active-comparators trials. Subgroup analyses were based on different classiﬁcations of pharmacological mechanisms and speciﬁc drug branches and May 2020 \| Volume 11 \| Article 559 3 Pharmaceutical Management for PTSD Huang et al. FIGURE 1 \| Study selection. FIGURE 1 \| Study selection. of the allocation concealment; and 29 studies with sufﬁcient information on the blinding of outcome assessment. of the allocation concealment; and 29 studies with sufﬁcient information on the blinding of outcome assessment. Symptoms of Re-Experiencing Compared with placebo, all active drugs (SMD = −0.32, 95% CI: −0.41 to −0.23) and different drug classiﬁcations, such as atypical antipsychotics (SMD = −0.37, 95% CI: −0.54 to −0.19), MAOI (SMD = −1.07, 95% CI: −1.77 to −0.38), SNRIs (SMD = −0.20, 95% CI: −0.35 to −0.05), and SSRIs (SMD = −0.28, 95% CI: −0.36 to −0.20), signiﬁcantly reduced the symptoms of re-experiencing Outcomes for Active Drug vs. Placebo Supplemental Table 4 shows the results of analysis of active drugs and placebo for different classiﬁcation of pharmacological mechanisms and speciﬁc drug branches. Supplemental Table 5 shows the results of the stratiﬁcation analysis. Overall, active drugs could signiﬁcantly reduce the PTSD total severity, symptoms of re-experiencing, avoidance, hyperarousal, depression, and anxiety relative to other active-comparators. Statistical Analysis Tucker et al., 2003; Davis et al., 2004; McRae et al., 2004; Reich et al., 2004; Akuchekian et al., 2004; Bartzokis et al., 2005; Davidson et al., 2006a; Davidson et al., 2006b; Neylan et al., 2006; Padala et al., 2006; Spivak et al., 2006; Becker et al., 2007; Davidson et al., 2007; Friedman et al., 2007; Lindley et al., 2007; Marshall et al., 2007; Martenyi et al., 2007; Raskind et al., 2007; Tucker et al., 2007; van der Kolk et al., 2007; Davis et al., 2008a; Davis et al., 2008b; Rothbaum et al., 2008; Hamner et al., 2009; Krystal et al., 2011; Mathew et al., 2011; Panahi et al., 2011; Yeh et al., 2011; Carey et al., 2012; Raskind et al., 2013; Ahmadpanah et al., 2014; Baniasadi et al., 2014; Batki et al., 2014; Naylor et al., 2015; Back et al., 2016; Mahabir et al., 2016; Petrakis et al., 2016; Ramaswamy et al., 2016; Villarreal et al., 2016; Dunlop et al., 2017; Li et al., 2017; Ramaswamy et al., 2017; Rezaei Ardani et al., 2017; Suris et al., 2017; Brunet et al., 2018; Hodgins et al., 2018; Raskind et al., 2018), six (Kosten et al., 1991; Marshall et al., 2001; Tucker et al., 2003; Davidson et al., 2006b; Martenyi et al., 2007; Ahmadpanah et al., 2014) conducted three separate trial arms (a comparison of two active drugs with placebo). Our research included 78 published double-blind, parallel RCTs. This meta- analysis involved 70 RCTs, which involved a comparison of 31 active drugs and placebo, and also extended to eight active- comparators trials (Kosten et al., 1991; Marshall et al., 2001; Tucker et al., 2003; McRae et al., 2004; Davidson et al., 2006b; Spivak et al., 2006; Martenyi et al., 2007; Ahmadpanah et al., 2014), comparing 12 active drugs, to explore the discovery of differences among different drugs. According to the pharmacological effects of drug classiﬁcation of pharmacological mechanisms and abbreviation, as shown in Supplemental Table 1, 8,083 patients diagnosed with PTSD were randomly assigned to active drug or placebo group and had at least one post baseline evaluation in the analysis. Supplemental Table 2 shows 44 of 66 studies or 66% obtained funding support from pharmaceutical companies. Quality Assessment The results of the quality assessment are shown in Supplemental Table 3. The following studies were judged to have a low risk of bias: 22 studies with sufﬁcient information on the generation of appropriate random sequences; 18 studies with a full description May 2020 \| Volume 11 \| Article 559 Frontiers in Pharmacology \| www.frontiersin.org Gender (Male) Race (White) Population Type of trauma Diagnostic criteria Baseline score Drug dose (mg/d) During of treatment (week) 50 (74.63%) NR Mixed Mixed DSM-IV TR (M.I.N.I.) 7 (0.35) Prazosin 1–15 mg/d versus hydroxyzine 10–100 8 47 (71.21%) NR Mixed Mixed DSM-IV TR (M.I.N.I.) 7 (0.28) Prazosin 1–15 mg/d versus placebo 8 47 (70.15%) NR Mixed Mixed DSM-IV TR (M.I.N.I.) 7 (0.45) Hydroxyzine 10–100 mg/d versus placebo 8 67 (100%) NR Veterans All combat-related DSM-IV CAPS: 49.81 (8.42) Topiramate (50–500 mg/d) versus placebo 12 26 (96.3%) 8 Veterans Military(combat5, non-combat 9), civilian-related events 13 DSM-IV CAPS: 63.88 (22.65) Fixed dose n-acetylcysteine 2400 versus placebo 8 92 (80.75%) NR Mixed Mixed DSM-III-R CAPS: 83.4 (17.95) Brofaromine, titrated up to 150 mg versus placebo 12 37 (100%) NR Veterans All combat-related DSM-IV-TR PCL-M 55.94 (7.65) Pregabalin 75–300 versus placebo 6 65 (100%) 44 veterans All combat-related DSM-IV CAPS: 100.43 (13.96) Risperidone 1 to 3 mg versus placebo 16 28 (93.3%) 16 Veterans 22 Combat-related DSM-IV-TR CAPS: 78.29 (16.55) Topiramate (25–300) versus placebo 12 22 (79%) 8 Mixed Mixed DSM-IV NR Bupropion sr 100–300 mg/d versus placebo 8 50 (26.74%) 158 Mixed Mixed DSM-III-R CAPS: 75.86 (17.56) Sertraline 133.3 (25–200) versus placebo 12 25 (41.7%) 42 Mixed Mixed DSM-IV-TR CAPS: 73.56 (15.94) 0.67 mg/kg of conventional (short-acting) propranolol, plus 1.0 mg/kg of long-acting propranolol versus placebo 6 1 (6.67%) 8 Mixed Mixed DSM-IV SI-PTSD 41.77 (9.43) Olanzapine 5–20 mg/d versus placebo 10 11 (39%) NR Civilians Civilians DSM-IV CAPS: 80.5 (13.64) olanzapine 5–15 mg/d versus placebo 8 5 (9%) 50 Civilians Mixed DSM-III-R DTS 76.55 (21.62) Fluoxetine 10–60 mg/d or placebo 12 NR NR Veterans All combat-related DSM-III IES 34.54 (7.59) Amitriptyline 160.7 (50 to 300) or placebo 8 46 (22.12%) 174 Mixed Mixed DSM-III-R CAPS: 73.69 (16.11) Sertraline 146.3 (25–200) versus placebo 12 11 (42.3%) NR Mixed Mixed DSM-IV SPRINT 22.84 (5.59) Mirtazapine (15–45) versus placebo 8 NR NR Mixed Mixed DSM-IV CAPS: 83.02 (15.24) Venlafaxine er (37.5–300 mg/d) versus sertraline (25–200 mg/d) 12 NR NR Mixed Mixed DSM-IV CAPS: 82.8 (14.84) Venlafaxine er (37.5–300 mg/d) versus placebo 12 NR NR Mixed Mixed DSM-IV CAPS: 81.8 (15.05) Sertraline (25–200 mg/d) versus placebo 12 151 (45.9%) NR Mixed Mixed DSM-IV CAPS: 81.97 (15.08) Venlafaxine er (37.5–300 mg/d) versus placebo 24 79 (34%) NR Mixed Mixed DSM-IV CAPS: 82.55 (15.08) Tiagabine (4–16) versus placebo 12 40 (97.6%) 22 Veterans (40) and Civilian(1) Combat-related, 40 (97.5%) DSM-IV CAPS: 81.8 (18.77) Sertraline (100–600) versus placebo 12 2 (98%) NR Veterans 78 Combat-related DSM-IV CAPS: 76.21 (17.3) Divalproex 2309 (1,000–3,000) versus placebo 8 (Continued) dy Sample (I/C) Mean Age (SD) Gender (Male) Race (White) Population Type of trauma Diagnostic criteria Baseline score Drug dose (mg/d) During of treatment (week) real et al., 2016 42/38 52.95 (11.07) 75 (93.75%) 42 Veterans All combat-related DSM-IV CAPS: 73.12 (14.24) Quetiapine (25–800) versus placebo 12 et al., 2011 17/14 40.45 (11.71) 10 (32.26%) NR Civilians Violence-related DSM-IV CAPS: 73.06 (18.65) Topiramate102.94 (25–200) versus placebo 12 ar et al., 2002 23/19 39.64 (7.56) 37 (88%) NR Veterans 32 combat-related DSM-III-R CAPS: 92.15 (12.5) Sertraline 50–200 mg/d versus placebo 10 S, Clinician-Administered PTSD Scale; DSM-IV-TR, the Diagnostic and Statistical Manual of Mental Disorders; ED 4, Text Rev; DTS, Davidson Trauma Scale; IES-R, the Impact of Event Scale-Revised; M.I.N.I., the Mini-International opsychiatric Interview; NR, not reported; PTSD, post-traumatic stress disorder; PCL-M, the Patient Checklist for PTSD-Military Version; SI-PTSD, Structured Interview for Post-traumatic Stress Disorder; SPRINT, Short Post Traumatic s Disorder Rating Interview. (Supplemental Table 4). For speciﬁc drug branches, amitriptyline (SMD = −0.75, 95% CI: −1.46 to −0.04), quetiapine (SMD = −0.53, 95% CI: −0.98 to −0.09), risperidone (SMD = −0.36, 95% CI: −0.56 to −0.15), ﬂuoxetine (SMD = −0.24, 95% CI: −0.41 to −0.08), paroxetine (SMD = −0.39, 95% CI: −0.51 to −0.27), sertraline (SMD = −0.26, 95% CI: −0.47 to −0.06), phenelzine (SMD = −1.07, 95% CI: −1.77 to −0.38), and venlafaxine (SMD = −0.20, 95% CI: −0.35 to −0.05) demonstrated greater efﬁcacy than placebo (Supplemental Table 4). Symptoms of Hyperarousal Compared with placebo, all active drugs (SMD = −0.34, 95% CI: −0.34 to −0.22) and different drug classiﬁcations, such as atypical antipsychotics (SMD = −0.37, 95% CI: −0.54 to −0.20), SNRIs (SMD = −0.25, 95% CI: −0.40 to −0.10), and SSRIs (SMD = −0.37, 95% CI: −0.49 to −0.25), signiﬁcantly reduced the severity of hyperarousal symptoms (Supplemental Table 4). For speciﬁc drug branches, quetiapine (SMD = −0.55, 95% CI: −1.00 to −0.10), ﬂuoxetine (SMD = −0.27, 95% CI: −0.43 to −0.10), paroxetine (SMD = −0.40, 95% CI: −0.52 to −0.27), sertraline (SMD = −0.45, 95% CI: −0.75 to −0.16), and venlafaxine (SMD = −0.25, 95% CI: −0.40 to −0.10), signiﬁcantly reduced the severity of hyperarousal (Supplemental Table 4). Symptoms of Avoidance Compared with placebo, all active drugs (SMD = −0.27, 95% CI: −0.33 to −0.21) and different drug classiﬁcations, such as MAOIs (SMD = −0.81, 95% CI: −1.49 to −0.14), SNRIs (SMD=−0.28, 95% CI: −0.43 to −0.13), and SSRIs (SMD = −0.34, 95% CI: −0.41 to −0.26), signiﬁcantly reduced the symptoms of avoidance (Supplemental Table 4). By contrast, in the speciﬁc drug branches, amitriptyline (SMD = −0.90, 95% CI:−1.62 to −0.18), phenelzine (SMD = −0.81, 95% CI: −1.49 to −0.14), venlafaxine (SMD = −0.28, 95% CI:−0.43 to −0.13), ﬂuoxetine (SMD = −0.20, 95% CI: −0.37 to −0.04), sertraline (SMD = −0.33, 95% CI: −0.46 to −0.20), and paroxetine (SMD = −0.42, 95% CI: −0.54 to −0.30) were associated with generally greater improvement for relieving avoidance symptoms (Supplemental Table 4). ) Gender (Male) Race (White) Population Type of trauma Diagnostic criteria Baseline score Drug dose (mg/d) During of treatment (week) 32 (91.43%) 25 Veterans All combat-related DSM-IV CAPS: 85.14 (17.38) Guanfacine (1–2 mg/d) versus placebo 8 0 (0%) 72 Mixed Mixed DSM-IV CAPS: 76.17 (16) Fixed dose 350 mg/d of gsk561679 versus placebo 6 135 (80%) 120 Veterans 120 Combat-related DSM-III-R CAPS: 72.93 (19.41) Sertraline (25–200 mg/day) or placebo 12 NR 17 Veterans All combat-related DSM-IV CAPS: 89.72 (18.31) Risperidone 20–40 versus placebo 5 28 (96.55%) 26 Veterans Combat (n = 28) and sexual assault (n = 1) DSM-IV CAPS: 77.1 (22.57) Divalproex 1196 mg (500–1,500) versus placebo 10 9 (64.29%) 4 Mixed Mixed DSM-IV SI-PTSD 44.29 (6.3) Lamotrigine 380 (25–500) versus placebo 12 12 (100%) 5 Veterans All combat-related DSM-IV DTS 108.5 (20.09) Fluoxetine 48 (10–60) versus placebo 12 0 (0%) 72 Civilians NR DSM-IV CAPS: 74.37 (16.2) gsk561679 versus placebo 6 34 (75.56%) NR Mixed Mixed DSM-III-R CAPS: 81.78 (18.11) Brofaromine 50–150 versus placebo 14 42 (100%) 39 Veterans All combat-related DSM III IES 33.83 (16.12) Imipramine 225 (50–300) versus phenelzine 68 mg (15–75) 8 37 (100%) 31 Veterans All combat-related DSM III IES 31.77 (14.21) Imipramine 225 (50–300) versus placebo 8 41 (100%) 34 Veterans all combat-related DSM III IES 34.96 (15.26) phenelzine 68 mg (15–75)versus placebo 8 258 (96.6%) 117 Veterans 209 combat-related events DSM-IV CAPS: 78.2 (14.82) Risperidone (1–4) versus placebo 24 63 (87.5%) 0 Mixed Mixed DSM-IV IES-R 64.35 (3.9) Fixed dose sertraline 135 mg daily versus placebo 12 40 (100%) 25 Veterans all combat-related NR CAPS: 61.55 (17.93) Topiramate (50–200) versus placebo 7 11 (26 83%) NR Mixed Mixed DSM IV CAPS: 79 4 (20 13) Fixed dose propranolol 1 mg/kg versus 1 (16.27) placebo 70 (28.2%) 192 Mixed Mixed DSM-IV CAPS: 77.22 (15.91) Fixed dose 40 mg/d ﬂuoxetine versus placebo 12 92 (28.5%) 242 Mixed Mixed DSM-IV CAPS: 78.57 (15.64) Fixed dose 20 mg/d ﬂuoxetine versus 40 mg/d ﬂuoxetine 12 16 (41%) 14 Mixed Mixed DSM-IV CAPS: 73 (13.7) GR205171 (5 mg/day) with placebo 8 (Continued) Gender (Male) Race (White) Population Type of trauma Diagnostic criteria Baseline score Drug dose (mg/d) During of treatment (week) 32 (91.43%) 25 Veterans All combat-related DSM-IV CAPS: 85.14 (17.38) Guanfacine (1–2 mg/d) versus placebo 8 0 (0%) 72 Mixed Mixed DSM-IV CAPS: 76.17 (16) Fixed dose 350 mg/d of gsk561679 versus placebo 6 135 (80%) 120 Veterans 120 Combat-related DSM-III-R CAPS: 72.93 (19.41) Sertraline (25–200 mg/day) or placebo 12 NR 17 Veterans All combat-related DSM-IV CAPS: 89.72 (18.31) Risperidone 20–40 versus placebo 5 28 (96.55%) 26 Veterans Combat (n = 28) and sexual assault (n = 1) DSM-IV CAPS: 77.1 (22.57) Divalproex 1196 mg (500–1,500) versus placebo 10 9 (64.29%) 4 Mixed Mixed DSM-IV SI-PTSD 44.29 (6.3) Lamotrigine 380 (25–500) versus placebo 12 12 (100%) 5 Veterans All combat-related DSM-IV DTS 108.5 (20.09) Fluoxetine 48 (10–60) versus placebo 12 0 (0%) 72 Civilians NR DSM-IV CAPS: 74.37 (16.2) gsk561679 versus placebo 6 34 (75.56%) NR Mixed Mixed DSM-III-R CAPS: 81.78 (18.11) Brofaromine 50–150 versus placebo 14 42 (100%) 39 Veterans All combat-related DSM III IES 33.83 (16.12) Imipramine 225 (50–300) versus phenelzine 68 mg (15–75) 8 37 (100%) 31 Veterans All combat-related DSM III IES 31.77 (14.21) Imipramine 225 (50–300) versus placebo 8 41 (100%) 34 Veterans all combat-related DSM III IES 34.96 (15.26) phenelzine 68 mg (15–75)versus placebo 8 258 (96.6%) 117 Veterans 209 combat-related events DSM-IV CAPS: 78.2 (14.82) Risperidone (1–4) versus placebo 24 63 (87.5%) 0 Mixed Mixed DSM-IV IES-R 64.35 (3.9) Fixed dose sertraline 135 mg daily versus placebo 12 40 (100%) 25 Veterans all combat-related NR CAPS: 61.55 (17.93) Topiramate (50–200) versus placebo 7 11 (26.83%) NR Mixed Mixed DSM-IV CAPS: 79.4 (20.13) Fixed dose propranolol 1 mg/kg versus placebo 1 119 (32.25%) NR Mixed Mixed DSM-IV CAPS: 74.85 (15.98) Fixed dose paroxetine (20 mg/d) versus placebo 12 117 (31.8%) NR Mixed Mixed DSM-IV CAPS: 74.35 (15.73) Fixed dose paroxetine (40 mg/d) versus placebo 12 112 (30.7%) NR Mixed Mixed DSM-IV CAPS: 74.8 (15.84) Fixed dose paroxetine (20 mg/d) versus paroxetine (40 mg/d) 12 17 (32.7%) NR Civilians Mixed DSM-IV CAPS: 83.53 (25.84) Paroxetine 10–60 mg/d versus placebo 10 245 (81.4%) 273 Mixed 301 combat-related DSM-IV CAPS: 80.7 (15.53) Fluoxetine 48 (10–60) versus placebo 12 72 (28.7%) 198 Mixed Mixed DSM-IV CAPS: 77.71 (16.27) Fixed dose 20 mg/d ﬂuoxetine versus placebo 12 70 (28.2%) 192 Mixed Mixed DSM-IV CAPS: 77.22 (15.91) Fixed dose 40 mg/d ﬂuoxetine versus placebo 12 92 (28.5%) 242 Mixed Mixed DSM-IV CAPS: 78.57 (15.64) Fixed dose 20 mg/d ﬂuoxetine versus 40 mg/d ﬂuoxetine 12 16 (41%) 14 Mixed Mixed DSM-IV CAPS: 73 (13.7) GR205171 (5 mg/day) with placebo 8 (Continued) ( %) ( ) p g ( ) p 258 (96.6%) 117 Veterans 209 combat-related events DSM-IV CAPS: 78.2 (14.82) Risperidone (1–4) versus placebo 24 63 (87.5%) 0 Mixed Mixed DSM-IV IES-R 64.35 (3.9) Fixed dose sertraline 135 mg daily versus placebo 12 40 (100%) 25 Veterans all combat-related NR CAPS: 61.55 (17.93) Topiramate (50–200) versus placebo 7 11 (26.83%) NR Mixed Mixed DSM-IV CAPS: 79.4 (20.13) Fixed dose propranolol 1 mg/kg versus placebo 1 119 (32.25%) NR Mixed Mixed DSM-IV CAPS: 74.85 (15.98) Fixed dose paroxetine (20 mg/d) versus placebo 12 117 (31.8%) NR Mixed Mixed DSM-IV CAPS: 74.35 (15.73) Fixed dose paroxetine (40 mg/d) versus placebo 12 112 (30.7%) NR Mixed Mixed DSM-IV CAPS: 74.8 (15.84) Fixed dose paroxetine (20 mg/d) versus paroxetine (40 mg/d) 12 17 (32.7%) NR Civilians Mixed DSM-IV CAPS: 83.53 (25.84) Paroxetine 10–60 mg/d versus placebo 10 245 (81.4%) 273 Mixed 301 combat-related DSM-IV CAPS: 80.7 (15.53) Fluoxetine 48 (10–60) versus placebo 12 72 (28.7%) 198 Mixed Mixed DSM-IV CAPS: 77.71 (16.27) Fixed dose 20 mg/d ﬂuoxetine versus placebo 12 70 (28.2%) 192 Mixed Mixed DSM-IV CAPS: 77.22 (15.91) Fixed dose 40 mg/d ﬂuoxetine versus placebo 12 92 (28.5%) 242 Mixed Mixed DSM-IV CAPS: 78.57 (15.64) Fixed dose 20 mg/d ﬂuoxetine versus 40 mg/d ﬂuoxetine 12 16 (41%) 14 Mixed Mixed DSM-IV CAPS: 73 (13.7) GR205171 (5 mg/day) with placebo 8 (Continued) 37 (100%) 31 Veterans All combat-related DSM III IES 31.77 (14.21) Imipramine 225 (50–300) versus placebo 8 41 (100%) 34 Veterans all combat-related DSM III IES 34.96 (15.26) phenelzine 68 mg (15–75)versus placebo 8 258 (96.6%) 117 Veterans 209 combat-related events DSM-IV CAPS: 78.2 (14.82) Risperidone (1–4) versus placebo 24 63 (87.5%) 0 Mixed Mixed DSM-IV IES-R 64.35 (3.9) Fixed dose sertraline 135 mg daily versus placebo 12 40 (100%) 25 Veterans all combat-related NR CAPS: 61.55 (17.93) Topiramate (50–200) versus placebo 7 11 (26.83%) NR Mixed Mixed DSM-IV CAPS: 79.4 (20.13) Fixed dose propranolol 1 mg/kg versus placebo 1 119 (32.25%) NR Mixed Mixed DSM-IV CAPS: 74.85 (15.98) Fixed dose paroxetine (20 mg/d) versus placebo 12 117 (31.8%) NR Mixed Mixed DSM-IV CAPS: 74.35 (15.73) Fixed dose paroxetine (40 mg/d) versus placebo 12 112 (30.7%) NR Mixed Mixed DSM-IV CAPS: 74.8 (15.84) Fixed dose paroxetine (20 mg/d) versus paroxetine (40 mg/d) 12 17 (32.7%) NR Civilians Mixed DSM-IV CAPS: 83.53 (25.84) Paroxetine 10–60 mg/d versus placebo 10 245 (81.4%) 273 Mixed 301 combat-related DSM-IV CAPS: 80.7 (15.53) Fluoxetine 48 (10–60) versus placebo 12 72 (28.7%) 198 Mixed Mixed DSM-IV CAPS: 77.71 (16.27) Fixed dose 20 mg/d ﬂuoxetine versus placebo 12 70 (28.2%) 192 Mixed Mixed DSM-IV CAPS: 77.22 (15.91) Fixed dose 40 mg/d ﬂuoxetine versus placebo 12 92 (28.5%) 242 Mixed Mixed DSM-IV CAPS: 78.57 (15.64) Fixed dose 20 mg/d ﬂuoxetine versus 40 mg/d ﬂuoxetine 12 16 (41%) 14 Mixed Mixed DSM-IV CAPS: 73 (13.7) GR205171 (5 mg/day) with placebo 8 (Continued) D) Gender (Male) Race (White) Population Type of trauma Diagnostic criteria Baseline score Drug dose (mg/d) During of treatment (week) 6 (23%) NR Civilians Mixed DSM-IV CAPS: 68.81 (13.53) Nafazodone 463 (100–600) versus sertraline 153 (50–200) 12 15 (100%) NR Veterans All combat-related DSM-IV PCL-M 72.47 Risperidone 0.5–2 mg versus placebo 6 9 (64.29%) 7 Veterans All combat-related DSM-IV CAPS: 86.45 (15.37) Aripiprazole 5–20 versus placebo 10 NR NR Veterans All combat-related DSM-IV CAPS: 68.34 (20.57) Guanfacine 0.5–3 versus placebo 8 0 (0%) 14 Civilians All related to sexual assault and domestic abuse DSM-IV CAPS: 79.89 Risperidone (1–6) versus placebo 12 70 (100%) NR Veterans Male Iranian veterans with combat-related PTSD DSM-IV-TR IES-R 65.25 (4.45) Sertraline 50–200 versus placebo 10 89 (92.71%) 78 Veterans All combat-related DSM-IV CAPS: 73.78 (17.77) Prazosin 14.5 (2–16) versus placebo 13 4 (13.33%) NR Civilians Mostly related to sexual or physical assault DSM-IV CAPS: 77.65 (17.66) Ziprasidone 40–160 versus placebo 9 57 (97%) 32 Veterans All Combat-Related DSM-IV CAPS: 75.45 (12.98) Vilazodone (10–40) versus placebo 12 2 (5%) 26 Veterans all Combat-Related DSM-IV CAPS: 77 (19.87) Prazosin 13 (2–15) versus placebo 8 57 (85.07%) 42 soldiers (65) and veterans (2) All Combat-Related DSM-IV CAPS: 81.69 (22.72) Prazosin 1–20 mg for men or 1–10 mg for women versus placebo 15 297 (97.7%) 203 Veterans All Combat-Related DSM-IV CAPS: 81.3 (16.3) Prazosin (1–20 mg for men or 1–12 mg for women) versus placebo 10 ) 21 (100%) 18 Civilians All childhood abuse related DSM-III-R CAPS: 69.1 (12.54) Risperidone (0.5–8) versus placebo 8 24 (100%) NR Veterans All combat-related DSM-IV-TR PCL-M 49.5 (5.85) Rivastigmin 3–6 mg/d versus placebo 12 4 (20%) 14 Civilians Mixed DSM-IV CAPS: 57.76 (20.8) Risperidone (0.5–3) versus placebo 8 15 (53.57%) NR Civilians All motor vehicle accident-related DSM-IV CAPS: 77.8 (16) Fixed dose Reboxetine (8 mg/d) versus ﬂuvoxamine (150 mg/d) 8 19 (100%) NR Veterans All combat-related DSM-IV CAPS: 85.11 (19.03) Olanzapine 15 (10–20) versus placebo 8 54 (100%) 36 Veterans all combat-related DSM-IV-TR PCL 55.85 (10.88) Dexamethasone 0.15 mg/kg versus placebo 2 105 (34.2%) 222 Mixed Mixed DSM-IV CAPS: 73.74 (16.7) Paroxetine (20–50) versus placebo 12 13 (27.08%) 41 Mixed Mixed DSM-IV CAPS: 87.6 (14.48) Citalopram 20–50 versus sertraline 50– 200 10 10 (28.57%) 29 Mixed Mixed DSM-IV CAPS: 91.91 (10.9) Citalopram 20–50 versus placebo 10 7 (21.21%) 31 Mixed Mixed DSM-IV CAPS: 87.03 (16.38) Sertraline 50–200 versus placebo 10 8 (21.05) 34 Civilians Mixed DSM-IV CAPS: 89.7 (13.64) Topiramate (25–400) versus placebo 12 42 (65.63%) NR Mixed Mixed DSM-III-R NR Fluoxetine 20–60 mg or placebo 5 8 (13.6%) 39 Mixed Mixed DSM-IV CAPS: 72.03 (13.2) Fluoxetine 30 (10–60) versus placebo 8 (Continued) Pharmaceutical Management for PTSD Huang et al. Frontiers in Pharmacology \| www.frontiersin.org Symptoms of Anxiety Compared with placebo, all active drugs (SMD = −0.23, 95% CI: −0.33 to −0.14) and different drug classiﬁcations, such as atypical antipsychotics (SMD = −0.32, 95% CI: −0.51 to −0.12), MAOI (SMD = −0.67, 95% CI: −1.34 to −0.01), TCAs (SMD = −0.68, 95% CI: −1.16 to −0.21), and TeCAs (SMD = −0.89, 95% CI: −1.74 to −0.04), signiﬁcantly reduced the symptoms of anxiety (Supplemental Table 4). For speciﬁc drug branches, amitriptyline (SMD = −0.99, 95% CI:−1.72 to −0.26), ﬂuoxetine (SMD = −0.28, 95% CI: −0.44 to −0.12, I2 = 0%), mirtazapine (SMD = −0.89, 95% CI: −1.74 to −0.04), and phenelzine (SMD = −0.67, 95% CI: −1.34 to −0.01) signiﬁcantly reduced the symptoms of anxiety (Supplemental Table 4). Symptoms of Depression Compared with placebo, depression symptoms were observed to have been noticeably reduced by using all active drugs (SMD = −0.28, 95% CI: −0.34 to −0.23) and different drug classiﬁcations, such as alpha blockers (SMD = −0.54, 95% CI: −0.95 to −0.13), atypical antipsychotics (SMD = −0.33, 95% CI: −0.51 to −0.15), SNRIs (SMD = −0.21, 95% CI: −0.36 to −0.06), SSRIs (SMD = −0.28, 95% CI: −0.41 to −0.16), and TeCAs (SMD = −0.91, 95% CI: −1.76 to −0.05), in a depression-related rating scale (Supplemental Table 4). For speciﬁc drug branches, prazosin (SMD = −0.54, 95% CI: −0.95 to −0.13), amitriptyline (SMD = −1.16, 95% CI: −1.90 to −0.41), olanzapine (SMD = −0.81, 95% CI: −1.41 to −0.20), quetiapine (SMD = −0.63, 95% CI: −1.08 to −0.18), mirtazapine (SMD = −0.91, 95% CI: −1.76 to −0.05), vilazodone (SMD = −0.21, 95% CI: −0.36 to −0.06), ﬂuoxetine (SMD = −0.25, 95% CI: −0.40 to −0.10), and paroxetine (SMD = TABLE 1 \| Continued May 2020 \| Volume 11 \| Article 559 8 Pharmaceutical Management for PTSD Huang et al. −0.49, 95% CI: −0.61 to −0.36) signiﬁcantly relieved the symptoms of depression (Supplemental Table 4). speciﬁc drug branches, which included only paroxetine (RR = 1.45, 95% CI: 1.02 to 2.07) and topiramate (RR = 2.61, 95% CI: 1.09 to 6.25), had signiﬁcantly higher discontinuation rate due to adverse effects than placebo (Supplemental Table 4). Outcomes for Active Drug vs. Active Comparators We also synthesised active-comparators studies separately to assess the differences between active drugs and active comparators (Supplemental Table 5). The analysis showed that sertraline relative to citalopram signiﬁcantly reduces total severity of PTSD based on a clinician-assessed scale (SMD = 0.65, 95% CI: 0.07 to 1.23) and avoidance symptoms (SMD = 0.93, 95% CI: 0.33 to 1.52). For symptoms of re-experiencing, the different drug classiﬁcations, including MAOI vs. TCAs (SMD = −0.64, 95% CI: −1.27 to −0.02), and speciﬁc drug branches, including phenelzine vs imipramine (SMD = −0.64, 95% CI: −1.27 to −0.02), had statistically signiﬁcant differences. Stratiﬁed Analyses All-Cause Discontinuation Rate Gender A pooling analysis of studies involving all-male population showed that all active drugs (SMD = −0.70, 95% CI: −1.24 to −0.16), risperidone (SMD = −0.57, 95% CI: −1.06 to −0.08), and topiramate (SMD = −1.14, 95% CI: −2.16 to −0.12) can signiﬁcantly reduce a clinician-assessed scale relative to placebo in Figures 4 and 5. However, the results of the combined analysis of female patients indicated that no drug has a better effect than placebo. total score relative to placebo in Figure 3. Severity of Trauma Based on CAPS Scores Severity of Trauma Based on CAPS Score Compared with placebo, all different drug classiﬁcations and speciﬁc drug branches, except for phenelzine (RR = 0.32, 95% CI: 0.12 to 0.80), did not show statistically signiﬁcant differences in the all-cause discontinuation rate (Supplemental Table 4). A stratiﬁed analysis of drug treatment in population with severe PTSD symptomatology (CAPS score of 60 to 79) showed that compared with placebo, all active drugs (MD = −4.93, 95% CI: −7.27 to −2.59) and different drug classiﬁcations, such as atypical antipsychotics (MD = −3.80, 95% CI: −7.01 to −0.58) and SSRIs (MD = −7.98, 95% CI: −11.18 to −4.79), can signiﬁcantly reduce the severity of PTSD total symptoms in Figure 2. Compared with placebo, in speciﬁc drug branches, ﬂuoxetine (MD = −5.23, 95% CI: −10.20 to −0.27), paroxetine (MD = −12.63, 95% CI: −15.78 Discontinuation Rate Due to Adverse Effects In terms of discontinuation rate due to adverse effects, all active drugs (RR = 1.47, 95% CI: 1.24 to 1.75), different drug classiﬁcations like atypical antipsychotics (RR = 2.06, 95% CI: 1.10 to 3.84) and SSRIs (RR = 1.37, 95% CI: 1.08 to 1.74), and FIGURE 2 \| Forest plot based on stratiﬁed analyses of severity of trauma based on CAPS scores of placebo-controlled comparisons. MAOIs, monoamine oxidase inhibitors; NK1, neurokinin-1 receptor antagonist; SARIs, serotonin antagonist and reuptake inhibitors; SNRIs, serotonin and norepinephrine reuptake inhibitors; SSRIs, selective serotonin reuptake inhibitors; NA, not applicable. FIGURE 2 \| Forest plot based on stratiﬁed analyses of severity of trauma based on CAPS scores of placebo-controlled comparisons. MAOIs, monoamine oxidase inhibitors; NK1, neurokinin-1 receptor antagonist; SARIs, serotonin antagonist and reuptake inhibitors; SNRIs, serotonin and norepinephrine reuptake inhibitors; SSRIs, selective serotonin reuptake inhibitors; NA, not applicable. FIGURE 2 \| Forest plot based on stratiﬁed analyses of severity of trauma based on CAPS scores of placebo-controlled comparisons. MAOIs, monoamine oxidase inhibitors; NK1, neurokinin-1 receptor antagonist; SARIs, serotonin antagonist and reuptake inhibitors; SNRIs, serotonin and norepinephrine reuptake inhibitors; SSRIs, selective serotonin reuptake inhibitors; NA, not applicable. FIGURE 2 \| Forest plot based on stratiﬁed analyses of severity of trauma based on CAPS scores of placebo-controlled comparisons. MAOIs, monoamine oxidase inhibitors; NK1, neurokinin-1 receptor antagonist; SARIs, serotonin antagonist and reuptake inhibitors; SNRIs, serotonin and norepinephrine reuptake inhibitors; SSRIs, selective serotonin reuptake inhibitors; NA, not applicable. May 2020 \| Volume 11 \| Article 559 9 Frontiers in Pharmacology \| www.frontiersin.org Huang et al. Severity of Trauma Based on CAPS Scores Pharmaceutical Management for PTSD FIGURE 3 \| Forest plot based on stratiﬁed analyses of severity of trauma based on CAPS scores of placebo-controlled comparisons in speciﬁc drug branches. FIGURE 3 \| Forest plot based on stratiﬁed analyses of severity of trauma based on CAPS scores of placebo-controlled comparisons in speciﬁc drug branches. to −9.48), and quetiapine (MD = −11.81, 95% CI: −22.18 to −1.44) can signiﬁcantly reduce the CAPS total score in patients with severe PTSD symptomatology (Figure 3). For patients with extreme PTSD symptomatology (CAPS ≥80), all active drugs (SMD = −5.91, 95% CI: −7.79 to −4.03) and different drug classiﬁcations, such as atypical antipsychotics (MD = −9.72, 95% CI: −15.55 to −3.89), SNRI (MD = −8.10, 95% CI: −12.27 to −3.92), and SSRIs (MD = −5.65, 95% CI: −8.58 to −2.72), can signiﬁcantly reduce the CAPS total score relative to placebo in Figure 2. In speciﬁc drug branches, ﬂuoxetine (MD = −7.80, 95% CI: −14.75 to −0.85), olanzapine (MD = −17.49, 95% CI: −32.68 to −2.30), sertraline (MD = −5.41, 95% CI: −8.70 to −2.11), and venlafaxine (MD = −8.10, 95% CI: −12.27 to −3.92) can signiﬁcantly reduce the CAPS total score relative to placebo in Figure 3. clinician-assessed scale, as can be observed in Figure 4. In speciﬁc drug branches, quetiapine (SMD = −0.49, 95% CI: −0.93 to −0.04), risperidone (SMD = −0.22, 95% CI: −0.44 to −0.01), and topiramate (SMD = −1.14, 95% CI: −2.16 to −0.12) can signiﬁcantly reduce the total PTSD severity based on a clinician-assessed scale in Figure 4. For civilian PTSD patients, compared with placebo, all active drugs (SMD = −0.27, 95% CI: −0.47 to −0.07), olanzapine (SMD = −0.93, 95% CI: −1.71 to −0.15), and ﬂuoxetine (SMD = −0.91, 95% CI: −1.52 to −0.31) had a signiﬁcant efﬁcacy in Figure 5. Therapeutic Regimens Compared active drugs with placebo, the monotherapy demonstrated a signiﬁcant reduction in CAPS scores (SMD = −0.31, 95% CI: −0.41 to −0.21, I < sp > 2 = 63%), meanwhile, the adjunctive therapy also indicated a positive effect on reducing CAPS scores (SMD = −0.49, 95% CI: −0.92 to −0.06, I2 = 63%). Different Populations A pooling analysis of the different drug classiﬁcations compared with placebo for veterans indicated that all active drugs (SMD = −0.27, 95% CI: −0.44 to −0.10) and atypical antipsychotics (SMD = −0.29, 95% CI: −0.48 to −0.10) can signiﬁcantly reduce the May 2020 \| Volume 11 \| Article 559 Frontiers in Pharmacology \| www.frontiersin.org 10 Pharmaceutical Management for PTSD Huang et al. FIGURE 4 \| Forest plot based on stratiﬁed analyses of different populations and gender of placebo-controlled comparisons. TCAs, tricyclic antidepressants. FIGURE 4 \| Forest plot based on stratiﬁed analyses of different populations and gender of placebo-controlled comparisons. TCAs, tricyclic antidepressants. based on stratiﬁed analyses of different populations and gender of placebo-controlled comparisons. TCAs, tricyclic antidepressants DISCUSSION Based on 78 RCTs from 66 studies, this study was the largest and latest meta-analysis of pharmacological treatments for PTSD in adults. We obtained a more comprehensive evidence for a detailed search of published literature; from the recent meta- analysis (Cipriani et al., 2018), this meta-analysis covered 15 additional studies. We investigated important results related to changes in PTSD total symptoms, reduction rate of core and other symptoms, all-cause discontinuation, discontinuation due to adverse effects, which were chosen to estimate pharmacological treatments efﬁcacy and acceptability. Necessary considerations to this study were the extrapolation of evidences based on speciﬁc treatment options for PTSD and exploration of the applicability of key evidences for the different Age and Races We did not ﬁnd any older populations (over 60 years of age) involved in our study, and there is no evidence of a comparison between older and non-older adults. For the races, only one study was concerned on a single race. A stratiﬁcation analysis based on racially diverse populations has not been performed in current studies. The results of the pooled analysis were not reversed by the trim- and-ﬁll method for the study of anticonvulsants and SSRIs. However, in terms of discontinuation rate due to adverse effects for atypical antipsychotics compared with placebo, the results of the pooled analysis were reversed (RR = 1.57, 95% CI: 0.86 to 2.87), suggesting that publication bias may affect the robustness of the ﬁndings of atypical antipsychotics (Supplemental Table 6). Meta-Regression For the change in overall PTSD symptoms, the meta-regression results for patients’ age resulted in a signiﬁcant coefﬁcient (coefﬁcient = 0.02, P = 0.046), implying that treatments, compared with placebo, in younger patients tend to be more effective than in older patients. A meta-regression model of the severity of trauma showed that active treatments, compared with placebo, tend to be less effective in studies with more severe patients (coefﬁcient = 0.03, P = 0.75). Meanwhile, the meta- regression of publication year (P = 0.26) and sponsorship (P = 0.68) indicated that it had not reached statistical difference. May 2020 \| Volume 11 \| Article 559 Frontiers in Pharmacology \| www.frontiersin.org 11 Pharmaceutical Management for PTSD Huang et al. FIGURE 5 \| Forest plot based on stratiﬁed analyses of different populations and gender of placebo-controlled comparisons in speciﬁc drug branch. FIGURE 5 \| Forest plot based on stratiﬁed analyses of different populations and gender of placebo-controlled comparisons in speciﬁc drug branch. FIGURE 5 \| Forest plot based on stratiﬁed analyses of different populations and gender of placebo-controlled comparisons especially for civilian-related trauma. For different doses, this study found that the discontinuation rate due to adverse effects of 40 mg/day ofﬂuoxetine is signiﬁcantly higher than 20 mg/day of ﬂuoxetine; however, there was no signiﬁcant difference in efﬁcacy, including reduction of various symptoms (Martenyi et al., 2007). By contrast, the use of different doses (20 mg/day vs. 40 mg/day) of paroxetine did not show signiﬁcant differences in efﬁcacy and acceptability (Marshall et al., 2001). Unfortunately, this study found no evidence in the comparisons of different doses within venlafaxine and sertraline. For the comparison between venlafaxine and sertraline, there was also no signiﬁcant difference, but no evidence was found for pair-wise comparisons of other two drugs. individual patient levels and sub-symptoms, such as basic characteristics of population, severity of trauma, special population, different classiﬁcations of pharmacological mechanisms, and speciﬁc drug branches. The APA and NICE guidelines (Association, 2017; Excellence NIfHaC, 2018) indicate that ﬂuoxetine, paroxetine, sertraline, and venlafaxine should be recommended for PTSD drug therapy. The results of our meta-analysis are consistent with this recommendation and provide more reliable evidence for these drugs. After conducting a stratiﬁcation analysis and considering fully the modiﬁcation factors, we clearly deﬁned the scope of application for these four drugs. Frontiers in Pharmacology \| www.frontiersin.org Meta-Regression Our study showed that paroxetine can be used for severe PTSD symptom levels, whereas sertraline and venlafaxine can be used for extremely severe PTSD symptom levels. Meanwhile, ﬂuoxetine can be used for the treatment of severe or extremely severe PTSD symptoms, The APA guidelines also indicate that there is currently insufﬁcient evidence to conﬁrm the efﬁcacy of risperidone and topiramate. However, the risperidone and topiramate showed a signiﬁcant effect when compared with placebo in our meta- May 2020 \| Volume 11 \| Article 559 Frontiers in Pharmacology \| www.frontiersin.org 12 Pharmaceutical Management for PTSD Huang et al. analysis. This study demonstrated a potential tendency that risperidone and topiramate could be considered in male patients with PTSD, whereas topiramate is more suitable for male veterans with PTSD. Risperidone was as an adjunctive drug in ﬁve of the six studies on risperidone (Hamner et al., 2003; Reich et al., 2004; Bartzokis et al., 2005; Padala et al., 2006; Rothbaum et al., 2008; Krystal et al., 2011); only one study (Padala et al., 2006) involved a monotherapy drug. It is common for PTSD patients to be combined with other psychiatric disorders that rely on medicine interventions. Therefore, we evaluated the effect of the monotherapy and adjunctive drugs. The results in this study suggested either monotherapy or adjunctive therapy could decrease CAPS scores, which were basically expected. Our subgroup analysis found that risperidone had a more signiﬁcant efﬁcacy as a monotherapy drug than as an adjunctive drug. Of the six studies on topiramate, only Akuchekian’s (Akuchekian et al., 2004) from 2004 results indicated higher effect values; the other studies found no signiﬁcant statistical signiﬁcance. The heterogeneity of the pooling analysis of the efﬁcacy of topiramate may be attributed to the difference in results. The maximum dose of 500 mg/day of titrated topiramate in Akuchekian’s study (Akuchekian et al., 2004) was higher than the 400 mg/day in other studies; the population in Akuchekian’s study (Akuchekian et al., 2004) also had a comorbid psychotic illness while having a high duration of illness (mean duration of 17 years). In the future, the study of topiramate must consider the difference in drug efﬁcacy caused by higher doses and different populations. showing a signiﬁcant advantage in reducing hyperarousal and re- experiencing symptoms. For patients diagnosed with military- related PTSD and had antidepressant-resistant symptoms, risperidone did not signiﬁcantly reduce the overall PTSD symptoms. Meta-Regression Further researches are required to conﬁrm or revise this result. Prazosin, paroxetine, ﬂuoxetine, and venlafaxine can be considered a priority in reducing symptoms of depression drug. Sertraline did not show a signiﬁcant advantage in reducing depression symptoms. Surprisingly, the pooling analysis of SSRIs did not show an advantage in decreasing the symptoms of anxiety. A further subgroup analysis showed that ﬂuoxetine signiﬁcantly reduces the symptoms of anxiety. Compared with previous meta-analyses, we conducted a speciﬁc stratiﬁed analysis of age, gender, patient type, and severity of PTSD for the maximized extrapolation of evidence based on the current guideline (Association, 2017). Unfortunately, only one RCT (Li et al., 2017) recruited a population of a single race (Asian). There was also no trial of PTSD for older adults (over 60 years of age) in these studies. Therefore, our conclusions may only be suitable for non-older adults (18–60 years old). Additionally, the meta-regression analysis found that the efﬁcacy of a drug becomes less with increasing age. Relevant studies (Olff, 2017) showed that compared with men, the risk of suffering from PTSD was twice or thrice higher for women, which may be related to psychosocial and biological characteristics. Female patients were only based on three RCTs that analysed GSK561679 and risperidone. The results showed that there were currently no effective drugs for female PTSD patients, which could be attributed to the small sample size. Consequently, more studies with a large sample are needed to verify this ﬁnding. The NICE guideline (Excellence NIfHaC, 2018) also pointed out the potential role of atypical antipsychotics for the treatment of PTSD. A study (Berger et al., 2007) showed that atypical antipsychotics may be an alternative when PTSD does not respond to SSRIs. Veterans with comorbidity of PTSD (McCauley et al., 2012) are more difﬁcult to treat than non- veterans patients; they have higher suicide attempts and worse treatment adherence (McCauley et al., 2012; Reisman, 2016). Therefore, compared with SSRIs, atypical antipsychotics may be more considered for veterans as a special group. Our research results showed that atypical antipsychotics are effective in the treatment of PTSD total symptoms, especially for veterans. This ﬁnding is different from the recent a meta-analysis (Hoskins et al., 2015) of pharmaceutical PTSD. We found that quetiapine and topiramate signiﬁcantly reduce the severity of PTSD for veterans compared with placebo in all active drugs. Moreover, olanzapine is suitable for civilian trauma and extremely severe PTSD symptoms. Frontiers in Pharmacology \| www.frontiersin.org Meta-Regression g y g Although these recommended drugs based on a comparative analysis of placebo-controlled trials showed signiﬁcant advantages over placebo in terms of efﬁcacy, the effect size of these drugs almost even did not reach the moderate effect (SMD range of 0.5–0.8) according to Cohen’s criteria (Cohen, 2013). On the contrary, those drugs (e.g., hydroxyzine, mirtazapine, and phenelzine) that achieved large effect values were derived from small sample studies. Most studies compare active drugs with placebo. We also focused on active-comparators studies to explore the differences among drugs; unfortunately, the information obtained was quite small. This ﬁnding is consistent with the conclusion of a previous meta-analysis (Cipriani et al., 2018). Phenelzine is the only drug that was found to be signiﬁcantly better than the placebo in terms of all- cause discontinuation rate. The effect value of the all-cause discontinuation for another drug group after subgroup analysis was around 1. Compared with placebo, atypical antipsychotics and SSRIs had signiﬁcantly higher discontinuation due to adverse effects. The discontinuation due to adverse drug reactions was signiﬁcantly higher than that of placebo by approximately 1.47 times. Speciﬁcally, when using paroxetine and topiramate clinically, the adverse reactions were signiﬁcantly higher than placebo. We found that the discontinuation due to adverse effects was attributed to CNS, including impaired cognition, paraesthesia, headache, dizziness or light- headedness, sedation, and sexual dysfunction, by analysing the Our study demonstrated that paroxetine, sertraline, venlafaxine, and ﬂuoxetine can also be used as the ﬁrst choice of treatment for the three main core symptoms. A pooling analysis of atypical antipsychotics drugs involved a moderate heterogeneity (40%) for avoidance symptoms, and subgroup analysis showed heterogeneity in two studies (Butterﬁeld et al., 2001; Carey et al., 2012) of olanzapine; excluding these latter two studies, atypical antipsychotics drugs did not show signiﬁcant efﬁcacy. Risperidone can also reduce re-experiencing symptoms and achieve signiﬁcant efﬁcacy. Of the studies on risperidone, only Krystal’s study (Krystal et al., 2011) used a large sample, May 2020 \| Volume 11 \| Article 559 Frontiers in Pharmacology \| www.frontiersin.org 13 Pharmaceutical Management for PTSD Huang et al. controlled trials rather than on active-comparators trials, making it difﬁcult to obtain direct evidence of beneﬁt. After the subgroup analysis to eliminate clinical heterogeneity as much as possible was strictly implemented, we used I2 to select ﬁxed or random effect models based on the Cochrane’s Handbook (Higgins and Green, 2011) for deal with statistical heterogeneity. Meta-Regression It is worth noting that the uncertain pooled results of small sample studies, such as phenelzine, may affect our estimation of the true effect size no matter which effect model is implemented (Borenstein et al., 2010; Riley et al., 2011). We look forward to more large samples and high-quality studies to conﬁrm our ﬁndings in the future. Second, up to two thirds of the research included in the original study was supported by funds from pharmaceutical companies, which may have some impact on our conclusions. Third, most RCTs included in our analysis did not report adequate information on random sequence generation and allocation concealment. The insufﬁcient description of study design may lead to bias in the results. original study on topiramate. The main cause of adverse reactions during a headache was the reason for discontinuation of paroxetine. Higher discontinuation due to adverse effects was observed in sertraline compared with placebo but was not signiﬁcant. A network meta-analysis that compares multiple treatments is clinically more useful than that an analysis using pairwise comparisons alone because, in the right circumstances, the former enables competing interventions to be ranked (Cipriani et al., 2013). However, considering the small amount of available data, the results of a published network meta-analysis (Cipriani et al., 2018) of pharmacotherapy for PTSD in 2018 are probably not robust enough to suggest that it is the best option as a drug of choice. Most importantly, this network meta-analysis did not consider modiﬁers for effect of results at the individual patient level (e.g., age, sex, severity of symptoms, special population, or type of trauma). Cipriani’s study (Cipriani et al., 2018) showed that phenelzine has a huge advantage compared with other drugs based on the indirect comparisons of active-comparators experiments. However, the efﬁcacy of phenelzine was only derived from one small RCT (Kosten et al., 1991) with 60 participants in 1991. Therefore, the accuracy of phenelzine requires more research to conﬁrm. Pharmacological interventions can be effective in PTSD, which may reduce its core symptoms (re-experiencing, avoidance, and hyperarousal) and thus should be considered in improving the symptoms of PTSD. This systematic review, meta- analysis supports the use of SSRIs, SNRIs, and atypical antipsychotics as pharmacological interventions for overall symptoms of PTSD and three core symptoms, except for atypical antipsychotics for the improvement of avoidance symptoms. Different drugs should be selected for the treatment of patients with varying clinical features of PTSD. Meta-Regression The atypical antipsychotics should be prioritized rather than SSRIs for veterans and males. SSRIs, SNRIs, and atypical antipsychotics are the medication of choice for patients with severe or extremely severe PTSD symptoms. There is insufﬁcient evidence for drug selection for PTSD treatment in civilians and females patients. Based on current guidelines and evidence, we can make the following recommendations for clinicians and future research. The core symptoms of PTSD should be treated preferentially (Excellence NIfHaC, 2018) because the symptoms of comorbidity usually improve with the success of treatment of PTSD. However, comorbidity symptoms must be treated ﬁrst if they seriously impact a patient’s life or are sufﬁcient to make the treatment of PTSD difﬁcult. An example is the comorbidity of suicide and depression of veterans (Reisman, 2016). The evidence for the drugs recommended in the current guidelines (Association, 2017; Excellence NIfHaC, 2018) has been updated in our study, but the efﬁcacy of these drugs is still quite small (around 0.3). These drugs may be more suitable to relieve certain emergency symptoms in cases where psychotherapy does not yield rapid efﬁcacy. The clinician should consider fully the individual factors for the choice of drugs rather than ﬁxed use of a drug to treat, such as the severity of symptom, different populations, or some critical sub-symptoms. Clinicians may use our results to ﬁnd a preliminary drug selection strategy and should consider a patient’s own preferences (A. McHugh et al., 2013; Zoellner et al., 2019). Especially for veterans, common drugs may not be suitable for patients who are resistant to common psychotropic substances, such as SSRIs. Atypical antipsychotics can be used as a strategy for clinical application (Berger et al., 2007). In the future, making great progress in pharmacological treatments of PTSD may need to be researched for some special drugs that have strong effect values from small sample studies. Future research should continue to conduct rigorous stratiﬁcation analysis for different populations or baseline symptoms to determine individual differences among drugs. Differences in efﬁcacy due to dose variances are worthy of further investigation in the same drug. Our research has some limitations. First, most studies have focused on placebo- The magnitude of pharmacological interventions for PTSD is small and evidence regarding the efﬁcacy for PTSD is inadequate, thereby highlighting the need for more research in this area to conﬁrm the use of pharmacological treatments for this disorder. Meta-Regression Despite these objective limitations, our ﬁndings represented the best currently available evidence to guide the initial choice of pharmacological treatments of PTSD in adults and inform future guidelines in deciding what drugs to use to treat PTSD. AUTHOR CONTRIBUTIONS Substantial contributions to conception and design or analysis and interpretation of data: Z-DH, JL, Y-FZ, SL, H-YG, L-LL, Z-YY, Y- MN, and CZ. Substantial contributions to drafting the article or revising it critically for important intellectual content: Z-DH, JL, and Y-FZ. Final approval of the version to be published: Z-DH, JL, Y-FZ, SL, H-YG, L-LL, Z-YY, Y-MN, and CZ. 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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. Spivak, B., Strous, R. D., Shaked, G., Shabash, E., Kotler, M., and Weizman, A. (2006). Reboxetine versus ﬂuvoxamine in the treatment of motor vehicle accident-related posttraumatic stress disorder: a double-blind, ﬁxed-dosage, controlled trial. J. Clin. Psychopharmacol. 26, 152–156. doi: 10.1097/ 01.jcp.0000203195.65710.f0 May 2020 \| Volume 11 \| Article 559 Frontiers in Pharmacology \| www.frontiersin.org
https://openalex.org/W2561221491	https://revistas.ufg.br/fen/article/download/36763/22109	Portuguese	null	Nível de evidência das tecnologias de cuidado de enfermagem na angioplastia de membros inferiores	Revista Eletrônica de Enfermagem	2,016	cc-by	7,746	RESUMO 4 Enfermeira, Doutora em Enfermagem. Professor Associado da Universidade Federal de Santa Catarina. Florianópolis, SC, Brasil. E-mail: luciamante@gmail.com. 5 Enfermeira, Mestre em Enfermagem. Discente do Programa de Pós-Graduação em Enfermagem, nível Doutorado, da Universidade Federal de Santa Catarina. Professor Substituto da Universidade Federal de Santa Catarina. Florianópolis, SC, Brasil. E-mail: darla.fernandez@ufsc.br. 6 Enfermeira, Doutora em Enfermagem. Professor Adjunto da Universidade Federal de Santa Catarina. Florianópolis, SC, Brasil. E-mail: fabiane.sebold@ufsc.br. Descritores: Hemodinâmica; Assistência Perioperatória; Enfermagem Baseada em Evidências. Recebido: 18/07/2015. Aceito: 30/06/2016. Publicado: 21/12/2016. Publicado: 21/12/2016. Como citar esse artigo: Soares V, Girondi JBR, Hammerschmidt KSA, Amante LN, Fernandez DLR, Sebold LF. Nível de evidência das tecnologias de cuidado de enfermagem na angioplastia de membros inferiores. Rev. Eletr. Enf. [Internet]. 2016 [acesso em: __/__/__];18:e1187. Disponível em: http://dx.doi.org/10.5216/ree.v18.36793. Artigo de Revisão Nível de evidência das tecnologias de cuidado de enfermagem na angioplastia de membros inferiores Viviane Soares1, Juliana Balbinot Reis Girondi2, Karina Silveira de Almeida Hammerschmidt3, Lúcia Nazareth Amante4, Darla Lusia Ropelato Fernandez5, Luciara Fabiane Sebold6 Viviane Soares1, Juliana Balbinot Reis Girondi2, Karina Silveira de Almeida Hammerschmidt3, Lúcia Nazareth Amante4, Darla Lusia Ropelato Fernandez5, Luciara Fabiane Sebold6 1 Enfermeira, Mestre em Enfermagem. Florianópolis, SC, Brasil. E-mail: vivi_zimba@hotmail.com. 2 Enfermeira, Doutora em Enfermagem. Professor Titular da Universidade Federal de Santa Catarina. Florianópolis, SC, Brasil. E-mail: juliana.balbinot@ufsc.br. 3 Enfermeira, Doutora em Enfermagem. Professor Adjunto da Universidade Federal de Santa Catarina. Florianópolis, SC, Brasil. E-mail: karina.h@ufsc.br. 4 Enfermeira, Doutora em Enfermagem. Professor Associado da Universidade Federal de Santa Catarina. Florianópolis, SC, Brasil. E-mail: luciamante@gmail.com. 5 Enfermeira, Mestre em Enfermagem. Discente do Programa de Pós-Graduação em Enfermagem, nível Doutorado, da Universidade Federal de Santa Catarina. Professor Substituto da Universidade Federal de Santa Catarina. Florianópolis, SC, Brasil. E-mail: darla.fernandez@ufsc.br. 6 Enfermeira, Doutora em Enfermagem. Professor Adjunto da Universidade Federal de Santa Catarina. Florianópolis, SC, Brasil. E-mail: fabiane.sebold@ufsc.br. RESUMO Objetivou-se descrever as produções científicas sobre tecnologias de cuidado de enfermagem ao paciente adulto submetido à angioplastia de membros inferiores. Revisão Sistemática da Literatura com busca nas bases de dados eletrônicas: LILACS, PUBMED, Web of Science, SciELO, além de Google Acadêmico. Foram analisados e classificados 12 artigos conforme os níveis de evidência. Todos os estudos qualitativos foram classificados com baixo grau de recomendação e dos estudos quantitativos apenas um apresentou alto grau de recomendação. A pesquisa apresentou estudos com o mais baixo nível de evidência científica, além da escassez de pesquisas e o frágil embasamento científico no qual os cuidados perioperatórios de enfermagem ao paciente submetido à angioplastia de membros inferiores vêm sendo desenvolvidos. Por conseguinte, há falta de informações e formação deficiente culminando no despreparo para atender os pacientes, compreender e liderar esse serviço de alta complexidade consoante aos princípios de segurança do paciente. Descritores: Hemodinâmica; Assistência Perioperatória; Enfermagem Baseada em Evidências. INTRODUÇÃO No setor de hemodinâmica são realizados procedimentos terapêuticos do aparelho circulatório servindo aos propósitos de especialidades médicas como cirurgia vascular, cardiologia e gastroenterologia. Nesse serviço é comum o tratamento de doenças Rev. Eletr. Enf. [Internet]. 2016 [acesso em: __/__/__];18:e1187. Disponível em: http://dx.doi.org/10.5216/ree.v18.36793. Rev. Eletr. Enf. [Internet]. 2016 [acesso em: __/__/__];18:e1187. Disponível em: http://dx.doi.org/10.5216/ree.v18.367 2 Soares V, Girondi JBR, Hammerschmidt KSA, Amante LN, Fernandez DLR, Sebold LF. vasculares periféricas, afecções caracterizadas pela redução do fluxo sanguíneo proveniente de obstrução por placas ateroscleróticas, trombos, êmbolos, válvulas venosas incompetentes ou por redução na ação de bombeamento dos músculos circunvizinhos(1). Doenças vasculares são tratadas especialmente por procedimento de angioplastia transluminal percutânea, que possui algumas vantagens sobre a cirurgia convencional, quais sejam: tempo de internação reduzido, menor risco de complicações, dor de pequena intensidade e menor custo(2). Estudos têm evidenciado eficácia no tratamento e vantagens para a recuperação dos pacientes submetidos e esse tipo de procedimento(3). A isquemia crítica, por exemplo, é uma doença vascular que pode levar à amputação de membros inferiores e é importante causa de mortalidade(4-5). Nesses casos, um número crescente de centros está adotando primeiramente o tratamento por abordagem endovascular, por angioplastia, obtendo sucesso significativo, como o estudo realizado pela Universidade da Califórnia no Davis Medical Center, que analisou pacientes que se submeteram a procedimentos endovasculares para tratamento de isquemia crítica, entre 2006 e 2012 e obteve taxas favoráveis de salvamento do membro afetado(6). O setor de hemodinâmica é considerado serviço de alta complexidade e requer uma equipe de enfermagem atuante, capacitada, competente, eficiente, ágil e atualizada. Além de atividades assistenciais, o enfermeiro no serviço de hemodinâmica desenvolve funções de liderança, gerenciamento de materiais e de pessoal, o que exige agilidade e tomada de decisões rápidas. Em meio a esse processo, compete ao enfermeiro o dimensionamento de pessoal, supervisão e treinamento da equipe, controle dos materiais médico-hospitalares utilizados em cada procedimento, bem como o conhecimento de condutas em relação ao reprocessamento dos materiais utilizados(7). Em meio à alta tecnologia que envolve o procedimento de angioplastia, a enfermagem deve se fazer valer de instrumentos metodológicos para embasar a assistência prestada. Para tanto, o cuidado de enfermagem baseado em evidências científicas relevantes, propicia eficácia e segurança, já que é obtido por meio de estudos e pesquisas em bases de dados confiáveis(8). Rev. Eletr. Enf. [Internet]. 2016 [acesso em: __/__/__];18:e1195. Disponível em: http://dx.doi.org/10.5216/ree.v18.38214. INTRODUÇÃO A identificação destas tecnologias de cuidado embasadas em evidências científicas se faz necessária para implementação de cuidados de enfermagem condizentes com as necessidades da população. As doenças cardiovasculares são um tema amplamente debatido atualmente devido ao aumento das taxas de morbidade e mortalidade e pelo fato de serem doenças evitáveis e tratáveis. Além disso, são foco de políticas públicas específicas no território brasileiro, como a Política Nacional de Atenção Cardiovascular de Alta Complexidade e outras relacionadas à prevenção destas como, a Política Nacional de Promoção da Saúde, Política Nacional de Atenção Básica, Política Nacional de Atenção Integral à Hipertensão Arterial Sistêmica e ao Diabetes Mellitus. Consoante, as tecnologias de cuidados devem estar articuladas a esta rede de cuidados para que os portadores de doenças cardiovasculares possam ser melhor assistidos, tendo garantia de acesso, cobertura e atendimento em uma rede de cuidados organizada em todos os níveis de atenção à saúde. Nesse contexto, cabe averiguar quais níveis de evidência têm embasado as tecnologias de cuidados Nesse contexto, cabe averiguar quais níveis de evidência têm embasado as tecnologia Rev. Eletr. Enf. [Internet]. 2016 [acesso em: __/__/__];18:e1195. Disponível em: http://dx.doi.org/10.5216/ree.v18.38214. Soares V, Girondi JBR, Hammerschmidt KSA, Amante LN, Fernandez DLR, Sebold LF. 3 empregadas pela enfermagem a pacientes submetidos à angioplastia de membros inferiores, no período perioperatório. A necessidade de investigação sobre esse nível de atenção, especificamente, se faz necessária uma vez que estes cuidados ainda são baseados na experiência clínica e empírica dos profissionais que prestam assistência a este tipo de paciente. Estes cuidados poucas vezes são baseados em evidências científicas e foi justamente esta lacuna que motivou a buscar argumentos científicos que justifiquem os cuidados prestados com foco neste procedimento. Assim, os objetivos do presente estudo foram: descrever as produções científicas disponíveis na literatura que englobem as tecnologias de cuidado de enfermagem ao paciente adulto submetido à angioplastia de membros inferiores. Rev. Eletr. Enf. [Internet]. 2016 [acesso em: __/__/__];18:e1195. Disponível em: http://dx.doi.org/10.5216/ree.v18.38214. MÉTODO Trata-se de uma Revisão Sistemática de Literatura de natureza descritiva(9). Previamente a este estudo se verificou a existência de Revisões Sistemáticas sobre o tema proposto na Biblioteca Cochrane (The Cochrane Database of Systematic Reviews), mas não foram encontradas revisões nesta área. A pergunta de pesquisa que originou esta Revisão foi: “Quais são as produções científicas existentes sobre as tecnologias de cuidado de Enfermagem ao paciente submetido à angioplastia de membros inferiores”? A busca dos estudos primários foi realizada nas bases de dados, PubMed/MEDLINE (Public MEDLINE), Web of Science, Scientific Eletronic Library Online (SciELO) e Literatura Latino-Americana e do Caribe em Ciências da Saúde (LILACS). Além das bases pesquisou-se no Google Acadêmico®. Para cada base de dados foi estabelecida uma estratégia de busca baseando-se na pergunta de pesquisa, conforme Quadro 1. Para a composição da estratégia de busca, utilizou-se os termos do MeSH (Medical Subject Headings Terms), que foram aplicados na busca PUBMED/MEDLINE. Para a LILACS empregou-se o DECs (descritores em ciências da saúde). Utilizou-se também os operadores booleanos (delimitadores) representados pelos termos AND, OR e NOT. As palavras-chave foram pesquisadas em português e inglês. O período da busca foi de agosto de 2014 a maio de 2016, por dois autores de forma independente. Os critérios de inclusão foram estudos: a) quantitativos, qualitativos ou quali-quantitativos que apresentassem em seu título ou no resumo a temática relacionada às tecnologias de cuidado de enfermagem no período perioperatório de angioplastia de membros inferiores; b) com seres humanos na faixa etária maior ou igual a 18 anos, submetidos à angioplastia de membros inferiores; c) disponíveis nas línguas inglesa, portuguesa ou espanhola. Os estudos incluídos não tiveram como filtro o ano de publicação, visto que é uma revisão sistemática e o tema ainda pouco estudado na área da Enfermagem. Foram critérios de exclusão: normas técnicas, manuais, leis, resoluções, editoriais, cartas, comentários, resumo de anais e publicações duplicadas. Rev. Eletr. Enf. [Internet]. 2016 [acesso em: __/__/__];18:e1195. Disponível em: http://dx.doi.org/10.5216/ree.v18.38214. 4 Soares V, Girondi JBR, Hammerschmidt KSA, Amante LN, Fernandez DLR, Sebold LF. Soares V, Girondi JBR, Hammerschmidt KSA, Amante LN, Fernandez DLR, Sebold LF. , , , , , Quadro 1: Estratégias de busca por bases de dados. Rev. Eletr. Enf. [Internet]. 2016 [acesso em: __/__/__];18:e1195. Disponível em: http://dx.doi.org/10.5216/ree.v18.38214. Fluxo de Seleção dos Estudos na Revisão Si t áti Figura 1: Fluxograma de seleção de estudos, de acordo com os critérios pré-estabelecidos. Fluxo de Seleção dos Estudos na Revisão Sistemática Referências excluídas (n=3.492) Referências completas analisadas (n= 12) Referências completas excluídas da análise (n= 28) Referências selecionadas (n=42) Estudos identificados através de busca nas bases eletrônicas (n=2.752) Referências identificadas por busca em outras fontes (n=1072) Referências após remoção dos estudos duplicados (n=3.571) Identificação Seleção Elegibilidade Inclusão Estudos incluídos na síntese qualitativa (n=06) Estudos incluídos na síntese quantitativa (n=05) Estudos incluídos na síntese quali- quanti (n=01) Referências identificadas por busca em outras fontes (n=1072) Estudos identificados através de busca nas bases eletrônicas (n=2.752) Referências após remoção dos estudos duplicados (n=3.571) Seleção Referências selecionadas (n=42) Referências excluídas (n=3.492) Elegibilidade Referências completas excluídas da análise (n= 28) Referências completas analisadas (n= 12) Estudos incluídos na síntese quali- quanti (n=01) Estudos incluídos na síntese qualitativa (n=06) Estudos incluídos na síntese quantitativa (n=05) Figura 1: Fluxograma de seleção de estudos, de acordo com os critérios pré-estabelecidos. Para a extração dos dados foi elaborada uma tabela com as características dos estudos incluídos, que permitiu a comparabilidade das variáveis destes, o país/ano e origem de publicação, o objetivo e os métodos empregados. Os resultados foram apresentados na forma descritiva, sendo os estudos classificados de acordo com o nível de evidência(11). MÉTODO Base de Dados Estratégia de busca PUBMED ("angioplasty"[MeSH Terms] OR "angioplasty"[All Fields] OR "Endovascular treatment"[All Fields] OR "endovascular procedures"[MeSH Terms] OR "endovascular procedures"[All Fields] OR "endovascular procedure"[All Fields) AND ("nursing"[Subheading] OR "nursing"[Title/Abstract] OR "nursing"[MeSH Terms] OR "nursing care"[All Fields] OR "nursing care"[MeSH Terms] OR "nurses"[MeSH Terms] OR "nurses"[All Fields] OR "nurse"[All Fields]) AND (English[lang] OR Portuguese[lang] OR Spanish[lang]) Web of Science Tópico: (angioplast* OR Endovascular treatment OR endovascular procedure*) AND Tópico: (nursing OR nursing care OR nurse OR nurses) SciELO (angioplast$ OR endovascular$ OR "tratamento endovascular" OR "tratamentos endovasculares" OR "procedimento endovascular" OR"procedimentos endovasculares" OR "Endovascular treatment" OR "endovascular procedures" OR "endovascular procedure") AND (nursing OR enfermagem OR enfermeir$ OR nurse$) LILACS (mh:("Angioplastia" OR "Procedimentos Endovasculares") OR tw:("Angioplastia" OR "Procedimentos Endovasculares" OR "Procedimento Endovascular" OR "tratamento endovascular" OR "tratamentos endovasculares")) AND (mh:("enfermagem" OR "enfermeiras" OR "enfermeiros" OR "cuidados de enfermagem" OR "Avaliação em Enfermagem" OR "Diagnóstico de Enfermagem" OR "Enfermagem Baseada em Evidências" OR "Enfermagem Cardiovascular" OR "Enfermagem Perioperatória") OR tw:("enfermagem" OR enfermeir$ OR "cuidados de enfermagem" OR "Avaliação em Enfermagem" OR "Diagnóstico de Enfermagem" OR "Enfermagem Baseada em Evidências" OR "Enfermagem Cardiovascular" OR "Enfermagem Perioperatória")) Estratégia de busca A seleção dos estudos baseou-se nos critérios de inclusão e na pergunta de pesquisa no formato acrônimo PICO(10) onde: onde P= População/Participantes: Pacientes adultos submetidos à angioplastia de membros inferiores; I= Intervenções: Cuidados de Enfermagem na angioplastia de membros inferiores e o uso de Tecnologias de Cuidados de Enfermagem aos pacientes submetidos à angioplastia de membros inferiores; C= Comparações: não há; O= Outcomes/Desfecho: Cuidados e Tecnologias de cuidado de Enfermagem eficazes no período perioperatório de angioplastia de membros inferiores. Nesta Revisão Sistemática (RS) foram identificados inicialmente 3.824 estudos. A Figura 1 representa o fluxograma do processo de seleção e inclusão das produções científicas. 5 Soares V, Girondi JBR, Hammerschmidt KSA, Amante LN, Fernandez DLR, Sebold LF. Fluxo de Seleção dos Estudos na Revisão Sistemática Rev. Eletr. Enf. [Internet]. 2016 [acesso em: __/__/__];18:e1195. Disponível em: http://dx.doi.org/10.5216/ree.v18.38214. R, Hammerschmidt KSA, Amante LN, Fernandez DLR, Sebold LF. 6 RESULTADOS Nesta RS foram analisados 12 estudos que atenderam aos critérios de inclusão e exclusão do protocolo de pesquisa. O Quadro 2 apresenta a caracterização destes e a classificação dos níveis de evidência, onde se Rev. Eletr. Enf. [Internet]. 2016 [acesso em: __/__/__];18:e1195. Disponível em: http://dx.doi.org/10.5216/ree.v18.38214. Soares V, Girondi JBR, Hammerschmidt KSA, Amante LN, Fernandez DLR, Sebold LF. 6 Soares V, Girondi JBR, Hammerschmidt KSA, Amante LN, Fernandez DLR, Sebold LF. 6 Soares V, Girondi JBR, Hammerschmidt KSA, Amante LN, Fernandez DLR, Sebold LF. 6 pode perceber que a maioria das publicações ocorreu entre o ano de 2006 a 2016, apresentando um número maior a partir de 2011. A publicação mais antiga identificada sobre o tema é do ano de 2006 e a mais recente é do ano de 2016. 7 Soares V, Girondi JBR, Hammerschmidt KSA, Amante LN, Fernandez DLR, Sebold LF. Quadro 2: Síntese dos artigos quanto a identificação, base de dados, país de origem de publicação, objetivo, resultados, método, resultados e nível de evidência. Autor/Ano Base de Dados País de origem da publicação Objetivo Resultados Nível de Evidência ESTUDOS COM MÉTODO QUALITATIVO (12)Kasthuri R, Karunaratne D, Andrew H, Sumner J, Chalmers N. / 2007. PubMed/MEDLINE Reino Unido Auditar a segurança da intervenção arterial periférica, dos casos de Angioplastia Periférica, por enfermeiros para aconselhamento, cuidados e acompanhamento desses pacientes pós alta hospitalar. Em pacientes encaminhados para a intervenção vascular periférica eletiva, a hemostasia pós-procedimento foi realizada com compressão manual. Após 3 horas no leito (deitado na cama), os pacientes foram mobilizados e tiveram alta em 5 horas. Os pacientes foram contatados por telefone no próximo dia útil para auditar as complicações. Das 183 cirurgias eletivas realizadas em 2 anos, em cinco paciente (2%) houve necessidade de retorno pois complicações locais na região de virilha, sendo que num dos casos o paciente tinha falso aneurisma. Nos demais quatro casos não houve necessidade de tratamentos adicionais. VI (13)Vieira LC, Contrin LM, Rol JL, Conte HD, Lima ARS, Castro EDR, et al./ 2009. Google Acadêmico Brasil Identificar as dificuldades e necessidades da equipe de Enfermagem em serviços de hemodinâmica e angiografia no país. O estudo identificou a dificuldade do enfermeiro no processo de gerenciamento e liderança no setor de Hemodinâmica. Da mesma forma esta dificuldade foi percebida durante a execução de diversas atividades, desde as mais simples até as mais complexas. RESULTADOS A responsabilidade de supervisão do enfermeiro de hemodinâmica é ampla, como gerenciar materiais de alto custo e liderança da equipe. VI (14)Krauzer IM, Brocardo D, Scarsi T./2011. Google Acadêmico Brasil Identificar o perfil de saúde dos clientes assistidos no Centro de Hemodinâmica e aplicar o Processo de Enfermagem, de acordo com Callista Roy. O estudo identificou o perfil de 52 clientes atendidos no Centro de Hemodinâmica de um hospital privado brasileiro. Os resultados obtidos foram o perfil de saúde dos clientes atendidos, de modo a oferecer características sobre o tipo de clientela, por meio da identificação da faixa etária; do local de moradia; estado civil; da escolaridade; ocupação; dos fatores de risco para doenças cardiovasculares; Índice de Massa Corporal e dos tipos mais frequentes de intervenções cirúrgicas. Depois foi delineado o Processo de Enfermagem, onde foram elencados os diagnósticos de Enfermagem no período pós-operatório e o percentual de incidência nos clientes: Ansiedade relacionada à morte (100%), Risco de dor aguda (85%), Integridade da pele prejudicada (75%), Perfusão tissular ineficaz, Perfusão tissular ineficaz, Risco de infecção (24%) e Risco de inibição da cascata de coagulação (16%). VI Rev. Eletr. Enf. [Internet]. 2016 [acesso em: __/__/__];18:e1195. Disponível em: http://dx.doi.org/10.5216/ree.v18.38214. 8 Soares V, Girondi JBR, Hammerschmidt KSA, Amante LN, Fernandez DLR, Sebold LF. Autor/Ano Base de Dados País de origem da publicação Objetivo Resultados Nível de Evidência (15)Bomfim FMTS, Lima SG, Victor EG./ 2013. Google Acadêmico Brasil Avaliar a prevalência e as condições do reprocessamento de cateteres de hemodinâmica na cidade do Recife, PE – Brasil. Foram avaliados quatro hospitais da rede pública e seis da rede privada em uma capital brasileira. Todas as instituições reprocessam cateteres. Apenas um deles realiza todas as etapas do reprocessamento na própria instituição. Na maioria dos hospitais, os cateteres são reutilizados, em média, quatro vezes. Observou-se significativa divergência nas respostas fornecidas pelos participantes do estudo, o que refletiu na falta de padronização das técnicas utilizadas no reprocessamento. A maioria dos profissionais já detectou fraturas e tortuosidades nos cateteres reprocessados. A preparação enzimática é o produto mais empregado para a limpeza dos cateteres. Em relação à limpeza do lúmen destes dispositivos não houve uniformidade entre as respostas fornecidas pelos investigados. Embora a maioria dos profissionais utilize a água da torneira para o enxágue dos cateteres, as técnicas utilizadas são bastante diversificadas. Testes para verificação de materiais pirogênicos não são executados em todos os materiais reprocessados. VI (16)Maher P. Rev. Eletr. Enf. [Internet]. 2016 [acesso em: __/__/__];18:e1195. Disponível em: http://dx.doi.org/10.5216/ree.v18.38214. RESULTADOS J Vasc Nurs./2014. PubMed/MEDLINE Irlanda Examinar a viabilidade de alta no mesmo dia após angioplastia, com ênfase na obtenção de resultados seguros paciente. O estudo explora como os avanços nas tecnologias e técnicas endovasculares contribuíram para tornar a alta no mesmo dia uma opção cada vez mais viável. A pré-admissão clínica dos pacientes desenvolvidas por enfermeiros especializados facilita a seleção do paciente de forma apropriada e segura, onde os pacientes em risco de problemas pós-procedimento podem ser identificados de forma eficaz com base em critérios clínicos pré-definidos. Com base nestas condutas, alta hospitalar no mesmo dia após angioplastia para doença vascular periférica é uma opção segura e viável. VI Resultados etr. Enf. [Internet]. 2016 [acesso em: __/__/__];18:e1195. Disponível em: http://dx.doi.org/10.5216/ree.v18.38214. 9 Soares V, Girondi JBR, Hammerschmidt KSA, Amante LN, Fernandez DLR, Sebold LF. Autor/Ano Base de Dados País de origem da publicação Objetivo Resultados Nível de Evidência (17)Wann-Hansson C, Wennick A. BMC Nurs./2016. PubMed/MEDLINE Suécia Elucidar como os pacientes com doença arterial periférica comunicam o seu conhecimento sobre a sua doença e tratamentos. A pesquisa identificou que o curto espaço de tempo gasto com os pacientes com doença arterial periférica requer orientação inovadora na prática clínica para atender às necessidades dos indivíduos, independentemente de o paciente entender sua doença arterial periférica. Durante as discussões sobre fatores de risco, observou-se o tabagismo como um importante fator de risco, que desencadeou sentimento de culpa. É necessário para os pacientes consultar fontes de informação para gerir as suas vidas diárias. Nesse processo o enfermeiro é essencial para o desenvolvimento de ações de educação em saúde. VI ESTUDOS COM MÉTODO QUANTITATIVO (18)Akopian G, Katz SG./ 2006. PubMed/MEDLINE Estados Unidos Avaliar a possibilidade de alta hospitalar no mesmo dia após angioplastia periférica Nos 97 pacientes submetidos à angioplastia periférica, a artéria femoral superficial foi o local mais frequente de intervenção (47%) e estes pacientes receberam alta hospitalar no mesmo dia de realização do procedimento. Nessa população estudada, um paciente foi internado por um grande hematoma no local da punção necessitando de transfusão de sangue, dois pacientes para a observação de um hematoma menor no local da punção, um por dor no peito, e um para observação de bradicardia transitória. O tempo médio para a mobilização pós procedimento foi de 1,4 horas, e o tempo médio de alta foi de 2,8 horas pós intervenção. RESULTADOS Nenhuma morte ou internação não planejada ocorreu em tempo igual ou superior a 30 dias de intervenção no hospital. VI (19)Gioppato S, Castello HJ Jr, Conforti TB, Gonçalves SLP, Morais FGS, Cantarelli MJC. / 2011. Google Acadêmico Brasil Avaliar a relação custo-efetividade da retirada imediata da bainha femoral e oclusão com dispositivo de oclusão vascular AngioSeal TM comparativamente à hemostasia por compressão manual e/ou mecânica após intervenções endovasculares. Comparado à compressão manual e/ou mecânica como técnicas de hemostasia em procedimentos terapêuticos endovasculares o dispositivo AngioSeal TM não houve diferença específicas em relação ao sucesso da hemostasia sem complicações. O custo individual do tratamento das complicações relacionadas à hemostasia por compressão foi maior que o custo do dispositivo de oclusão. VI Resultados de educação em saúde. ESTUDOS COM MÉTODO QUANTITATIVO (18)Akopian G, Katz SG./ 2006. PubMed/MEDLINE Estados Unidos Avaliar a possibilidade de alta hospitalar no mesmo dia após angioplastia periférica Nos 97 pacientes submetidos à angioplastia periférica, a artéria femoral superficial foi o local mais frequente de intervenção (47%) e estes pacientes receberam alta hospitalar no mesmo dia de realização do procedimento. Nessa população estudada, um paciente foi internado por um grande hematoma no local da punção necessitando de transfusão de sangue, dois pacientes para a observação de um hematoma menor no local da punção, um por dor no peito, e um para observação de bradicardia transitória. O tempo médio para a mobilização pós procedimento foi de 1,4 horas, e o tempo médio de alta foi de 2,8 horas pós intervenção. Nenhuma morte ou internação não planejada ocorreu em tempo igual ou superior a 30 dias de intervenção no hospital. VI (19)Gioppato S, Castello HJ Jr, Conforti TB, Gonçalves SLP, Morais FGS, Cantarelli MJC. / 2011. Google Acadêmico Brasil Avaliar a relação custo-efetividade da retirada imediata da bainha femoral e oclusão com dispositivo de oclusão vascular AngioSeal TM comparativamente à hemostasia por compressão manual e/ou mecânica após intervenções endovasculares. Comparado à compressão manual e/ou mecânica como técnicas de hemostasia em procedimentos terapêuticos endovasculares o dispositivo AngioSeal TM não houve diferença específicas em relação ao sucesso da hemostasia sem complicações. O custo individual do tratamento das complicações relacionadas à hemostasia por compressão foi maior que o custo do dispositivo de oclusão. VI Rev. Eletr. Enf. [Internet]. 2016 [acesso em: __/__/__];18:e1195. Disponível em: http://dx.doi.org/10.5216/ree.v18.38214. 10 10 Soares V, Girondi JBR, Hammerschmidt KSA, Amante LN, Fernandez DLR, Sebold LF. RESULTADOS Autor/Ano Base de Dados País de origem da publicação Objetivo Resultados Nível de Evidência (20)Covello CM. / 2011. Google Acadêmico Brasil Comparar as taxas de complicações vasculares no sítio de acesso femoral em idosos submetidos à hemostasia, mecânica ou clássica da artéria femoral após procedimentos percutâneos. Foram randomizados 110 pacientes, destes 54 submetidos a compressão manual e 56 a compressão mecânica. As taxas de complicações vasculares maiores foram na fase hospitalar e no seguimento de sete dias. No seguimento clínico de sete dias foi observado que a taxa total de complicações vasculares maior no grupo da compressão manual (64%) bem como a taxa de pacientes com complicações vasculares nesta técnica (48%). A taxa de complicações vasculares menores não foi diferente para os grupos nos dois períodos. II (21)Albayati MA, Gohel MS, Patel SR, Riga CV, Cheshire NJW, Bicknell CD./ 2011. Web of Science Reino Unido Investigar falhas na segurança do paciente submetidos à procedimentos vasculares e endovasculares para orientar a qualidade e a segurança nas futuras intervenções. Em 66 procedimentos vasculares observados foram identificadas 1145 falhas que comprometerem a segurança do paciente durante os procedimentos. Destas 23,5% das falhas foram relacionadas a equipamentos, 21% falhas relacionadas à comunicação da equipe, 3,6% falhas técnicas e 5,4% falhas psicomotoras. VI (22)Eufrásio S, Sousa P, Oliveira N, Gonçalves F, Alves G, Rodrigues H, et al. / 2013. Scielo Portugal Registrar, avaliar e quantificar a dose de radiação distribuída pela equipe cirúrgica pela exposição à radiação dispersa, emitida por um aparelho portátil de radioscopia com arco cirúrgico (arco em C), durante a utilização em bloco operatório. O estudo constatou como nível máximo de exposição a radiação dispersa pela equipe assistente à altura ao solo de 120 cm. Os níveis de radiação foram inferiores quando utilizados os meios de proteção radiológica. VI ESTUDOS COM MÉTODO MISTO (23)Lundén M, Lundgren SM, Persson LO, Lepp M. / 2013. PubMed/MEDLINE Estados Unidos Identificar pacientes que são predominantemente ansiosos ou calmos antes do tratamento com angioplastia transluminal percutânea e explorar as razões para estes sentimentos. O estudo mensurou o estado emocional dos pacientes pré- angioplasta percutnea evidenciando que a maioria (69%) deles sentiu bastante calma antes da angioplastia. Este sentimento se deu em virtude da confiança na equipe de saúde cuidadora, bem como na capacidade desta de prever os riscos cirúrgicos. RESULTADOS Os pacientes que se sentiram ansiosos antes do procedimento tiveram essa causa relacionada ao medo de um resultado negativo e se sentiram inseguros em relação as opções de tratamento. VI Resultados Rev. Eletr. Enf. [Internet]. 2016 [acesso em: __/__/__];18:e1195. Disponível em: http://dx.doi.org/10.5216/ree.v18.38214. Soares V, Girondi JBR, Hammerschmidt KSA, Amante LN, Fernandez DLR, Sebold LF. 11 Soares V, Girondi JBR, Hammerschmidt KSA, Amante LN, Fernandez DLR, Sebold LF. 11 Das 12 produções conclui-se que estas concentram-se no Brasil (cinco), seguido pelos Estados Unidos (duas), Reino Unido (duas), Portugal (uma), Portugal, Irlanda (uma), Suécia (uma). Em relação à fonte pesquisada, um artigo foi encontrado na base de dados SciELO, um na base de dados Web of Science, cinco artigos na PUBMED e cinco produções na ferramenta Google Acadêmico®. A base de dados LILACS não trouxe nenhuma produção pertinente. Dessa forma, o idioma mais publicado foi o português (seis), seguido do inglês (cinco). Não foram encontradas produções no idioma espanhol. DISCUSSÃO Os 12 estudos selecionados para essa revisão sistemática foram explorados na sua totalidade de acordo com a temática proposta. A partir dos achados encontrados nos estudos, optou-se por criar categorias para uma melhor discussão. Estas categorias baseiam-se na metodologia dos estudos e delas emergiram três grupos: a) estudos qualitativos; b) estudos quantitativos; c) estudo quali-quantitativo (método misto). Estudos qualitativos Rev. Eletr. Enf. [Internet]. 2016 [acesso em: __/__/__];18:e1195. Disponível em: http://dx.doi.org/10.5216/ree.v18.38214. Estudos qualitativos Ao estudar 52 pacientes de um hospital particular de Chapecó/SC identificou-se como fatores de risco: hipertensão arterial sistêmica, idade acima de 60 anos, obesidade e sedentarismo. Dos fatores de risco identificados nesta amostra elencaram-se os diagnósticos de enfermagem: ansiedade relacionada à morte (100%), risco de dor aguda (85%), integridade da pele prejudicada (75%), risco de perfusão tissular ineficaz tipo renal (42%), risco de infecção (24%) e risco de inibição da cascata de coagulação (16%)(14). Portanto, a implementação do Processo de Enfermagem no cuidado destes pacientes é premissa essencial para a efetivação de um cuidado seguro e de qualidade. Em relação ao reprocessamento de cateteres de hemodinâmica foram entrevistados 92 profissionais de enfermagem atuantes em 10 serviços na cidade de Recife/PE. Verificou-se que todos estes serviços reprocessam os cateteres utilizados nos procedimentos hemodinâmicos em média há cinco anos, 70% das instituições reprocessam aproximadamente 20 dispositivos ao dia e estes cateteres são reutilizados em média quatro vezes. O estudo destaca que não há uma padronização das técnicas de reprocessamento de cateteres de hemodinâmica e que nenhum dos serviços pesquisados segue um protocolo validado que oriente as etapas no reprocessamento. Indica ainda, que a vigilância na qualidade do reprocessamento não deve ser restrita aos dispositivos utilizados, mas ao registro das ocorrências adversas ao paciente(15). As evidências científicas dos estudos qualitativos, de forma geral, discorrem sobre as atividades desenvolvidas por enfermeiros, que vão desde atividades assistenciais, perpassando as gerenciais, de educação em saúde e supervisão. Enfatizam essa gama de atividades, mas destacando enquanto principais atribuições do enfermeiro: uma pré-avaliação do paciente planejada e sistematizada(16), o monitoramento do paciente no pós-procedimento e na alta hospitalar, o fornecimento de informações/orientações no período perioperatório; além da detecção precoce de complicações pós-procedimento, mediante identificação prévia dos riscos cirúrgicos e da implementação do Processo de Enfermagem. Para o desempenho de tais atribuições as tecnologias de cuidado emergentes são: acompanhamento e monitoramento do paciente por contato telefônico(12); elaboração e aplicação do processo de enfermagem durante o cuidado prestado no período perioperatório de angioplastia(13); avaliação clínica pré-admissional feita pelo enfermeiro para detecção precoce de riscos cirúrgicos e alta precoce(16); educação em saúde com base nas necessidades do paciente(17); vigilância na qualidade do reprocessamento de cateteres utilizados na hemodinâmica e acompanhamento das ocorrências adversas(12-15). Estudos qualitativos Foram encontrados quatro produções científicas nesta categoria(12-17). Primeiramente destaca-se um estudo descritivo realizado em 2007 com 183 pacientes que realizaram procedimento de intervenção arterial periférica e objetivou averiguar a segurança no procedimento realizado. Foi feito contato telefônico para avaliar o acompanhamento dos enfermeiros durante o período perioperatório, onde a maioria dos pacientes considerou a estratégia importante. Esse acompanhamento pelo enfermeiro é realizado no período pré-operatório via telefone, onde estes fornecem informações sobre o procedimento que será realizado. Além disso, o paciente recebe via correio um folheto com informações relativas à intervenção. Na ocasião da alta, os pacientes recebem do enfermeiro presencialmente as informações pertinentes, além do telefone de contato do serviço e do enfermeiro de plantão para os contatos necessários. No pós-operatório, via contato telefônico, dos 183 casos, apenas dois pacientes (1%) tiveram complicações e cinco tiveram no dia seguinte a alta hospitalar edema em região inguinal e retornaram para avaliação. O estudo evidencia que o acompanhamento dos casos por enfermeiros é importante, pois além de satisfazer o paciente, reduz custos hospitalares e evita problemas de falta de leitos para a internação no caso de procedimentos eletivos(12). Outra pesquisa qualitativa foi o estudo exploratório-descritivo realizado em 2009 em 30 cidades brasileiras, o que contempla 17 estados, para identificar as dificuldades e necessidades da equipe de enfermagem que atua nos serviços de hemodinâmica e angiografia destes locais. Dentre as dificuldades encontradas estão: a falta de experiência na área, a escassez de literatura na área voltada para a enfermagem, o gerenciamento da unidade, a fragilidade de programas de educação permanente e a dificuldade de gerenciamento de tempo para verificação do reprocessamento de materiais. Referencia ainda, as atribuições assistenciais desenvolvidas por enfermeiros. Evidencia que as dificuldades de troca de informações e a formação deficiente levam ao despreparo para atender os pacientes, de compreender e Rev. Eletr. Enf. [Internet]. 2016 [acesso em: __/__/__];18:e1195. Disponível em: http://dx.doi.org/10.5216/ree.v18.38214. Soares V, Girondi JBR, Hammerschmidt KSA, Amante LN, Fernandez DLR, Sebold LF. 12 12 liderar esse serviço de alta complexidade(13). liderar esse serviço de alta complexidade(13). liderar esse serviço de alta complexidade(13). Corroborando esse achado, outro estudo destaca que a Enfermagem deve embasar o cuidado no Processo de Enfermagem a fim de identificar fatores de risco e atuar na prevenção das complicações decorrentes de procedimentos endovasculares. Rev. Eletr. Enf. [Internet]. 2016 [acesso em: __/__/__];18:e1195. Disponível em: http://dx.doi.org/10.5216/ree.v18.38214. Estudos quantitativos Essa categoria foi composta por cinco estudos(18-22), sendo que quatro deles se enquadram na classificação VI, ou seja, estudos com baixo grau de recomendação. Apenas um estudo se destacou enquanto nível de evidência II por se tratar de ensaio clínico randomizado-controlado, ou seja, estudo experimental desenvolvido com seres humanos que visou o conhecimento do efeito das técnicas de hemostasia pós- angioplastia, configurando-se como uma das ferramentas mais poderosas para obtenção de evidências clínicas para a prática. Um estudo de 2013, feito por meio de uma simulação registrou, avaliou e quantificou a dose de radiação reproduzindo as condições habituais em que ocorrem os procedimentos endovasculares, na equipe cirúrgica e no paciente, que estão expostos à radiação dispersa. Identificou que nos doentes, procedimentos prolongados com a utilização de magnificação eletrônica podem causar queimaduras na pele e aumentam consideravelmente a taxa de dose de radiação incidente no doente. Como meio para diminuir a quantidade de radiação incidente no doente e consequente radiação dispersa, preconiza a aproximação do intensificador de imagem ao paciente, além do controle eficaz do tempo de procedimento, modo de fluoroscopia e magnificação. Destaca ainda a importância da monitoração dos valores de radiação e que o uso adequado dos equipamentos de proteção radiológica pode permitir condições de segurança para a equipe de saúde e para o paciente(16). Já um estudo realizado em 2011 investigou 66 procedimentos endovasculares por nove meses e encontrou 1.847 eventos adversos e 1.145 falhas identificadas, mediante aplicação de check list de verificação de segurança cirúrgica da Organização Mundial da Saúde. As falhas mais comuns identificadas foram: 23,5% em equipamentos (indisponibilidade, falhas na configuração, gestão do espaço e falhas do aparelho); 21% na comunicação entre cirurgião e circulante, anestesista e cirurgião; 9% em técnicas. A pesquisa descreve que poucas falhas graves foram identificadas, mas houve o acúmulo de falhas menores que aconteceram em momentos mais críticos do procedimento. Destaca que a sala de cirurgia vascular é um ambiente complexo, dinâmico e abriga uma equipe multidisciplinar envolvendo a troca de grande quantidade de informações e tarefas cirúrgicas de alta complexidade. Logo, a prevenção do erro cirúrgico representa um grande desafio, mas deste problema(17). Uma publicação de 2006 descreve sobre o uso de protocolo para alta hospitalar no mesmo dia pós- angioplastia eletiva em 97 pacientes por um período de 27 meses. Estudos qualitativos Apesar de importantes tecnologias de cuidados para a Enfermagem, todos os estudos qualitativos que compuseram a amostra desta RS foram classificados como nível de evidência VI, demonstrando que são estudos com baixo grau de recomendação. As várias tecnologias de cuidados que subsidiam o fazer do enfermeiro em hemodinâmica, enfatizando a responsabilidade deste pela assistência integral ao paciente e no desenvolvimento de atividades gerenciais, de ensino e pesquisa(22-26). Em relação à educação de pacientes submetidos à angioplastia transluminal Rev. Eletr. Enf. [Internet]. 2016 [acesso em: __/__/__];18:e1195. Disponível em: http://dx.doi.org/10.5216/ree.v18.38214. Soares V, Girondi JBR, Hammerschmidt KSA, Amante LN, Fernandez DLR, Sebold LF. 13 percutânea os enfermeiros devem se tornar conhecedores do estilo de vida do paciente para implementar um plano de cuidados individualizado no pré e pós intervenção(27). percutânea os enfermeiros devem se tornar conhecedores do estilo de vida do paciente para implementar um plano de cuidados individualizado no pré e pós intervenção(27). Rev. Eletr. Enf. [Internet]. 2016 [acesso em: __/__/__];18:e1195. Disponível em: http://dx.doi.org/10.5216/ree.v18.38214. Estudos quantitativos A pesquisa aponta que a angioplastia periférica em pacientes com claudicação pode ser realizada com segurança em ambulatório, com significativa redução de custos. A conclusão aponta que a alta no mesmo dia pós-angioplastia periférica é segura e eficaz, portanto é considerada padrão outro de atendimento. Para ter este desfecho, apesar de ser resultados de aplicação de um protocolo médico, o artigo aponta alguns aspectos inerentes ao cuidado de enfermagem, Rev. Eletr. Enf. [Internet]. 2016 [acesso em: __/__/__];18:e1195. Disponível em: http://dx.doi.org/10.5216/ree.v18.38214. Soares V, Girondi JBR, Hammerschmidt KSA, Amante LN, Fernandez DLR, Sebold LF. 14 como: uso de medicamentos pré e trans-procedimento, tempo de mobilização pós-procedimento e tempo de compressão manual no local de realização da intervenção(18). No entanto, na realidade brasileira, grande parte dos procedimentos endovasculares exige permanência hospitalar até o dia seguinte à realização do procedimento, pois a maioria destes envolve situações clínicas e/ou anatômicas complexas(19). Dois estudos dessa categoria avaliaram a questão das técnicas de hemostasia pós-angioplastia. Em um deles foi estudado os resultados, complicações e custos envolvidos no uso de um dispositivo de oclusão vascular comparado à compressão manual/mecânica pós-intervenção endovascular. Foram analisados 407 pacientes submetidos à intervenções endovasculares de 2006 a 2009, num hospital de Campinas/SP. A conclusão é que a técnica de hemostasia por compressão manual e/ou mecânica quando bem realizada é tão efetiva quanto à hemostasia com uso de oclusão vascular, porém com um custo significativamente menor. Ou seja, o dispositivo de oclusão vascular não se mostrou mais seguro ou com melhor relação custo- efetividade quando comparado à compressão manual ou mecânica(19). O segundo estudo comparou as taxas de complicações vasculares no sítio de acesso femoral em 110 idosos, em virtude da hemostasia mecânica (com dispositivo grampo tipo “C”) ou clássica após procedimentos percutâneos. A pesquisa concluiu que não houve diferença entre estas taxas de complicações vasculares entre as técnicas hemostáticas(20). Nessa categoria as evidência científicas apontam para tecnologias de cuidados emergentes, quais sejam: adotar estratégias para redução da quantidade de radiação incidente no paciente durante o procedimento de angioplastia(16); monitorar os valores de radiação e adequação dos meios de proteção radiológica(16); implantar um protocolo de segurança pré-procedimentos endovasculares(17); implantar a lista de verificação de segurança cirúrgica(17); padronizar a técnica de compressão hemostática pós-procedimento de angioplastia(19-20). Estudo quali-quantitativo (método misto) Rev. Eletr. Enf. [Internet]. 2016 [acesso em: __/__/__];18:e1195. Disponível em: http://dx.doi.org/10.5216/ree.v18.38214. Estudo quali-quantitativo (método misto) Nessa categoria foi contemplado apenas um estudo americano, onde foram entrevistados 42 pacientes submetidos a procedimentos endovasculares e aplicada a escala de Likert a fim de identificar os pacientes ansiosos ou calmos antes do procedimento e explorar as razões para estes sentimentos. As razões listadas para estar calmo foram estar seguros, ter boas expectativas quanto ao procedimento e segurança nos cuidadores (médicos e enfermeiros). Em contrapartida, a ansiedade esteve relacionada ao desespero e apreensão sobre o procedimento, medo de um resultado negativo e insegurança quanto às opções de tratamento. A pesquisa aponta que estudos sobre os sentimentos dos pacientes pré-angioplastia são escassos. O estudo apesar de ser classificado quanto ao nível de evidência em VI traz algumas importantes tecnologias de cuidado a serem empregadas nesse contexto, quais sejam: conhecer as percepções do paciente pré-procedimento; estar aberto ao diálogo e escuta qualificada; utilizar terminologia adequada durante orientações; fornecer orientações pré-procedimento (escritas, orais e por vídeo) sobre o procedimento, fatores de risco, local de realização da intervenção e tratamentos; utilizar musicoterapia antes Rev. Eletr. Enf. [Internet]. 2016 [acesso em: __/__/__];18:e1195. Disponível em: http://dx.doi.org/10.5216/ree.v18.38214. Soares V, Girondi JBR, Hammerschmidt KSA, Amante LN, Fernandez DLR, Sebold LF. 15 do procedimento; incentivar a criação de grupos de pacientes; utilizar abordagem individualizada e acolhedora antes da angioplastia(21). do procedimento; incentivar a criação de grupos de pacientes; utilizar abordagem individualizada e acolhedora antes da angioplastia(21). 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W2120318578.txt	https://downloads.hindawi.com/journals/ijmms/2006/024549.pdf	fr		Inégalité de Markov en plusieurs variables	International journal of mathematics and mathematical sciences	2,006	cc-by	5,414	INÉGALITÉ DE MARKOV EN PLUSIEURS VARIABLES WIESŁAW PLEŚNIAK Reçu le 16 mai 2005 ; Révisé le 13 mars 2006 ; Accepté le 26 avril 2006 L’inégalité de Markov joue un rôle important en théorie constructive de fonctions. Dans les deux dernières décennies, on a développé sa théorie multidimensionnelle. Le but de cet article est de présenter le récent progrès de cette belle théorie. Copyright © 2006 Hindawi Publishing Corporation. All rights reserved. 1. Inégalité de Markov 1.1. Introduction. En 1889, Andrei Andreievitch Markov a répondu à une question posée deux ans plus tôt par Mendeleev en montrant que, pour tout polynôme p ∈ C[x], on a p (x) ≤ (deg p)2 p[−1,1] pour x ∈ [−1,1], (1.1) où pI = sup \| p\|(I). Ce résultat est optimal puisque, pour les polynômes de Tchebyschev Tk (x) = cosk arccos x x ∈ [−1,1] , k = 1,2,..., (1.2) on a Tk [−1,1] = 1 et \|Tn (±1)\| = n2 . L’inégalité de Markov a été le point de départ d’une énorme littérature. En eﬀet, elle a de nombreuses applications en analyse et en physique. Pour des détails on peut se reporter à l’ouvrage de Rahman et Schmeisser ([44]). La théorie correspondante en plusieurs variables est relativement nouvelle : avant 1980, toutes les extensions connues de l’inégalité de Markov, dans la pratique, concernaient uniquement des ensembles compacts, convexes et d’intérieur non vide de Rn (voir, e.g., Baouendi-Goulaouic [2]). Le principal obstacle à l’étude des cas plus généraux provenait de l’existence, dans Rn , d’ensembles à pointes n’admettant aucune extension multidimensionnelle de l’inégalité de Markov. Cette remarque est due à Zerner ([55]). Exemple 1.1. On pose E = {(x, y) ∈ R2 : 0 < y ≤ exp(−1/x), 0 < x ≤ 1} ∪ {(0,0)} et Pk (x, y) = y(1 − x)k pour k = 1,2,.... On a donc degPk = k + 1, ∂Pk /∂y E = 1, tandis Hindawi Publishing Corporation International Journal of Mathematics and Mathematical Sciences Volume 2006, Article ID 24549, Pages 1–12 DOI 10.1155/IJMMS/2006/24549 2 Inégalité de Markov en plusieurs variables √ que Pk E < exp(− k) pour k = 1,2,.... Par conséquent, il n’existe pas de constantes M > 0 et r > 0 telles qu’on ait, pour tout k, ∂Pk /∂y ≤ M(k + 1)r Pk . E E (1.3) Il est alors naturel de chercher les parties de Rn pour lequelles il est possible d’établir des majorations de l’exemple 1.1. On est ainsi ramené à la définition suivante. Définition 1.2. On dira qu’une partie compacte de Rn admet (ou presérve) l’inégalité de Markov, ou bien qu’elle est markovienne, s’il existe des constantes M > 0 et r > 0 telles que, pour tout polynôme P ∈ R[x1 ,...,xn ], on ait grad P E ≤ M(degP)r P E . (M) Une théorie satisfaisante de l’inégalité de Markov en plusieurs variables, abordant en particulier le cas d’ensembles à pointes, a été développée dans les dernières deux décennies par Pawłucki, Pleśniak, Goetgheluck, Baran, Białas-Cież, Jonsson, Bos, Levenberg, Milman, Taylor, Goncharov, Totik et autres. Donnons ici un exemple d’un compact markovien de Rn à pointes, dû à Goetgheluck ([24]). Théorème 1.3. Soit, pour k ≥ 1, Ek = (x, y) ∈ R2 : 0 ≤ y ≤ xk , 0 ≤ x ≤ 1 . (1.4) Alors l’ensemble Ek est markovien, pour l’exposant r = 2k. De plus, la valeur 2k est optimale. L’ensemble Ek , défini ci-dessus, est un sous-ensemble semi-analytique de R2 . On peut alors se demander si toute partie semi-analytique (plus général, sous-analytique) de Rn admet une inégalité de Markov. La réponse à cette question est fournie par la théorie de Pawłucki-Pleśniak présentée ci-dessous, dont le point de départ était l’exemple de Goetgheluck. 1.2. Ensembles semi-analytiques, sous-analytiques et à pointes polynomiales Définition 1.4. Une partie E de Rn est dite semi-analytique si, pour tout x ∈ Rn , on peut trouver un voisinage U de x et un nombre fini de fonctions réel analytiques fi j et gi j définies dans U, telles que E∩U = i fi j > 0, gi j = 0 . (1.5) j La projection d’un semi-analytique n’est pas nécessairement semi-analytique (Łojasiewicz ([34])). On appelle classe d’ensembles sous-analytiques, la famille obtenue en ajoutant à la famille des semi-analytiques, toutes les projections d’ensembles semianalytiques. Plus précisement, Définition 1.5. Un sous-ensemble E de Rn est dit sous-analytique si, pour tout x ∈ Rn , il existe un voisinage U de x tel que E ∩ U soit la projection d’un semi-analytique borné A de Rn+m , où m est un entier non négatif. Wiesław Pleśniak 3 L’ensemble Ek de Goetgheluck est, de façon évidente, semi-analytique, et à fortiori sous-analytique, tandis que l’ensemble de Zerner est trop plat à l’origine pour être semianalytique. Il se trouve que les sous-analytiques n’admettent que des pointes polynomiales. Définition 1.6. On dit qu’une partie E de Rn est à pointes polynomiales (en bref, UPC = uniformly polynomially cuspidal) s’il existe des constantes positives M et m, un entier positif d et une application h : E × [0,1] → E, tels que, pour tout x ∈ E, on ait h(x,1) = x, la fonction h(x, ·) soit un polynôme de degré ≤ d et dist h(x,t), Rn \ E ≥ M(1 − t)m pour (x,t) ∈ E × [0,1]. (1.6) En utilisant le théorème de rectilinéarisation de Hironaka (voir [17]) et la séparation régulière de Łojasiewicz ([34]), on prouve (Pawłucki-Pleśniak [37]) Théorème 1.7. Tout ensemble sous-analytique borné de Rn , à intérieur dense dans E, est UPC. La famille des ensembles UPC est visiblement plus large que celle des sous-analytiques. Un exemple simple d’ensemble UPC qui n’est pas sous-analytique est fourni par [0,1] × [−1,1] \ E, où E est l’ensemble de Zerner. Les ensembles UPC admettent une fonction de Green pluricomplexe continue de façon hölderienne. Soit E une partie compacte de Cn , on pose, pour tout z ∈ Cn , VE (z) = sup u(z) : u ∈ ᏸ Cn , u\|E ≤ 0 , (1.7) où ᏸ(Cn ) = {u ∈ PSH(Cn ) : supz∈Cn [u(z) − log(1 + \|z\|)] < ∞} est la classe de Lelong des fonctions plurisous-harmoniques à croissance logarithmique. D’après la théorie du pluripotentiel due à Bedford-Taylor (voir [31]), si E est non pluripolaire (c’est-à-dire, si pour toute fonction u ∈ PSH(Cn ), l’inclusion E ⊂ {u(z) = −∞} implique u(z) ≡ −∞), alors la régularisée supérieure VE∗ (z) = limsup VE (w) w→z (1.8) de VE est un équivalent multidimensionnel de la fonction de Green classique pour C \ E, où E désigne l’enveloppe polynomiale de E. L’importance de la classe UPC se traduit par le résultat suivant dû à Pawłucki-Pleśniak [37]. Théorème 1.8. Soit E une partie UPC compacte de Rn , à paramètre m. On a s VE (z) ≤ M dist(z,E) , où s = 1/2[m] et [m] := k pour k − 1 < m ≤ k avec k ∈ Z. (HCP) 4 Inégalité de Markov en plusieurs variables Si VE est continue de façon HCP (abrév. de Hölder Continuity Property), en appliquant la formule intégrale de Cauchy, on montre que E est markovien. Ceci entraı̂ne (PawłuckiPleśniak [37]) Corollaire 1.9. Toute partie compacte E de Rn de type UPC, en particulier tout sousanalytique compact E ⊂ Rn , et d’intérieur dense dans E, est markovien. Le résultat ci-dessus reste vrai dans le cadre, beaucoup plus général, des structures o-minimales polynomialement bornées. En particulier, si on considère une classe de fonctions quasi-analytiques de Carleman liée à une suite strictement logarithmiquement convexe, les ensembles définissables dans la structure de Rolin-Speissegger-Wilkie engendrée par cette classe sont UPC (voir Pierzchała [39]). 1.3. Dynamique complexe. La géométrie sous-analytique n’est pas seule à fournir des exemples d’ensembles markoviens. On peut en trouver plusieurs en dynamique complexe. Soit P : Cn → Cn une application polynomiale, on définit l’ensemble de Julia rempli, associé à P, par JP := z ∈ Cn : P j (z) j ∈N0 est borné . (1.9) Si l’exposant de Łojasiewicz de P (à l’infini) ł(P) := sup δ : liminf \|z\|→∞ P(z) \|z\|δ >0 (1.10) est strictement plus grand que 1, alors JP est HCP, d’où markovien (voir Kosek [32, 33]). Les ensembles markoviens décrits ci-dessus sont tous de type UPC, et donc HCP. Cependant, il y a des ensembles HCP qui ne sont pas UPC. Les premiers exemples de tels ensembles (de type Cantor) ont été construits par Jonsson ([29]). Il s’est avéré diﬃcile de déterminer si le Cantor triadique classique est markovien ou non. Une réponse aﬃrmative à ce problème a été fournie par Białas et Volberg ([13]) qui ont montré que cet ensemble est même de type HCP. Il convient de noter qu’il existe également des ensembles de type Cantor qui ne sont pas markoviens et pour lequels la fonction classique de Green est continue (voir Pleśniak [40], Goetgheluck-Pleśniak [26], Totik [52]). Quant à savoir si la propriété de Markov de E entraı̂ne la propriété HCP de E, ce problème reste toujours ouvert sauf pour des cas particuliers de C (voir Białas-Cież [14], Totik [52]). En général, on ne sait même pas si la propriété de Markov de E entraı̂ne la continuité de la fonction de Green VE ou la non-pluripolarité de E. Néanmoins, Białas-Cież ([15]) a montré que toute partie compacte de Markov de C est de capacité logarithmique positive, et par conséquent, qu’elle n’est pas polaire. 1.4. Relations avec les polynômes orthogonaux. On termine cette section par la mise en évidence des relations entre l’inégalité classique de Markov et les polynômes orthogonaux. Soit E une partie compacte de R et soit μ une mesure de probabilité, à support E, telle que, pour tout disque Δ(x,r) centré en x ∈ E de rayon 0 < r ≤ 1, on ait Wiesław Pleśniak 5 μ(Δ(x,r)) ≥ c0 r s avec des constantes c0 > 0 et s > 0 ne dépendant pas de x et r. Soit {Pn : n = 0,1,2,...} une suite de polynômes orthogonaux associée à μ. On suppose que le degré de Pn est n. On sait que, pour tout n, les zéros {x1 ,x2 ,...,xn } du polynôme Pn sont tous simples, réels et contenus dans le plus petit intervalle couvrant E. On a le résultat suivant dû à Jonsson (voir [30]). Théorème 1.10. Le compact E est markovien si et seulement si l’on peut trouver des constantes γ ≥ 1, L > 0 et β > 0 telles que (i) il existe d, 0 < d ≤ 1, tel que, pour tout x0 ∈ E et tout r > 0, r ≤ 1, x ∈ R : dr γ ≤ \|x − x0 \| ≤ r ∩ E = ∅; (1.11) (ii) si xi et xi−1 sont des zéros consécutifs de Pn , avec n ≥ 2, alors xi − xi−1 ≥ L/nβ . (1.12) 2. Applications Nous allons présenter quelques applications importantes de l’inégalité de Markov. Nous commençons par 2.1. Approximation polynomiale de fonctions Ꮿ∞ . Rappelons le théorème classique de Bernstein [12]. Théorème 2.1. Une fonction f : I = [a,b] ⊂ R → C se prolonge en une fonction Ꮿ∞ sur R si et seulement si, pour tout s > 0, on a lim ks distI f ,ᏼk = 0. k→∞ (2.1) La démonstration classique du théorème de Bernstein ne s’applique pas, en général, au cas d’un compact quelconque de Rn . En eﬀet, contrairement au cas d’un intervalle de R, il existe un compact E de Rn et une fonction f : E → R de classe Ꮿ∞ dans int E, tels que f et toutes ses dérivées partielles se prolongent continûment à E et, qu’en même temps, f n’admette aucun prolongement Ꮿ∞ sur un voisinage de E. En voici un exemple. Exemple 2.2. Soit E = E1 ∪ E2 ⊂ R2 , où E1 = {(x, y) ∈ R2 : 0 ≤ x ≤ 1, g(x) ≤ y ≤ 1} avec g(x) = exp(−1/x) si 0 < x ≤ 1, g(0) = 0, et E2 = [0,1] × [−1,0]. Soit ⎧ ⎪ ⎨g(x) f (x, y) = ⎪ ⎩0 si (x, y) ∈ E1 ; si (x, y) ∈ E2 . (2.2) Alors f n’admet aucun prolongement Ꮿ∞ sur un voisinage ouvert de E. Le problème de caractérisation de type Bernstein des fonctions Ꮿ∞ en plusieurs variables a été résolu par Pawłucki-Pleśniak ([37]). Dans ce qui suit, on dira que E est déterminant pour les fonctions Ꮿ∞ dans Rn si, pour tout f ∈ Ꮿ∞ (Rn ), f = 0 sur E entraı̂ne Dα f = 0 sur E pour tout α ∈ Zn+ . On montre (Pleśniak [41]) que tout compact 6 Inégalité de Markov en plusieurs variables markovien de Rn est déterminant. Le résultat de Pawłucki-Pleśniak (voir aussi Pleśniak [41]) s’écrit comme suit. Théorème 2.3. Si un compact E de Rn est déterminant pour les fonctions Ꮿ∞ , alors les conditions suivantes sont équivalentes : (i) E est markovien ; (ii) (théorème de type Bernstein) pour toute fonction f :E → R, si la suite {distE ( f ,ᏼk )} est à décroissance rapide, c’est pour tout s > 0, ks distE ( f ,ᏼk ) → 0 si k → ∞, alors f s’étend en une fonction Ꮿ∞ dans Rn . Ici ᏼk désigne l’espace des polynômes de degré ≤ k et distE ( f ,ᏼk ) := inf { f − pE : p ∈ ᏼk }. L’ensemble E de l’exemple 2.2 est markovien puisqu’il est UPC. Compte tenu du théorème 2.3, la suite {distE ( f ,ᏼk )} n’est pas à décroissance rapide lorsque f est la fonction de cet exemple. 2.2. Applications en analyse diﬀérentielle. L’inégalité de Markov permet de construire de façon relativement simple un opérateur linéaire continu d’extension pour les jets de Whitney de classe C ∞ (voir Whitney [53]), définis sur un compact markovien de Rn . Le problème d’existence d’un tel opérateur a une longue histoire. Les premiers résultats sont dûs à Mityagin [35] (cas d’un intervalle de R) et à Seeley [48] (cas d’un demi-espace de Rn ). Stein [50] a montré qu’un tel opérateur existe si E est l’adhérence d’un domaine borné de Rn dont le bord est localement de classe Lip1. Bierstone [16] a étendu ce résultat au cas où le bord de E est Lipα, avec 0 < α ≤ 1. Dans [16], Bierstone a également montré qu’un tel opérateur existe quand E est un sous-analytique fermé de Rn tel que int E ⊃ E. Sa méthode a été essentiellement basée sur le célèbre théorème de désingularisation de Hironaka. Une autre méthode, basée sur des résultats de Vogt et Wagner concernant des suites exactes des espaces nucléaires, a été appliquée par Tidten [51] pour montrer l’existence d’un opérateur d’extension pour des fermés de Rn admettant certaines pointes polynomiales. Tous les ensembles mentionnés ci-dessus sont UPC, d’où de Markov. Le théorème suivant de Pawłucki-Pleśniak [38] est une généralisation essentielle des résultats précédents. De plus, il est de caractère constructif. Théorème 2.4. Soit E un compact markovien de Rn . Si, pour tout k ∈ N, Lk f désigne un polynôme d’interpolation de Lagrange de f de degré k, ayant pour nœuds des points extrémaux de Fekete-Leja, et (uk ; k ∈ N) désigne une famille appropriée de fonctions de troncature de classe Ꮿ∞ , alors la formule L f = u1 L 1 f + ∞ uk Lk+1 f − Lk f (2.3) k =1 définit un opérateur linéaire continu étendant les jets de Whitney de classe C ∞ sur E en fonctions de classe C ∞ sur l’espace Rn tout entier. En utilisant des techniques similaires, Zeriahi [54] a montré que si E est un compact de Rn de Markov, alors Ꮿ∞ (E) (équipé de la topologie de Whitney) est isomorphe à l’espace Wiesław Pleśniak 7 ᏿ des suites de réels positifs à décroissance rapide, ce qui est une généralisation non triviale du résultat de Mityagin [35] mentionné ci-dessus. Le théorème de Pawłucki-Pleśniak admet un complément (voir Pleśniak [41]) : la propriété de Markov de E est équivalente à l’existence d’un opérateur d’extension linéaire continu par rapport à la topologie de Jackson de l’espace Ꮿ∞ (E). Cette équivalence n’est plus valable lorsque l’espace Ꮿ∞ (E) est muni de la topologie de Whitney. En eﬀet, Goncharov [27] a construit un compact non markovien de R admettant l’existence d’un opérateur d’extension linéaire continu (par rapport à la topologie de Whitney). Pour d’autres résultats concernant l’opérateur d’extension de Pawłucki-Pleśniak voir AltunGoncharov ([1]). Notons encore que pour qu’un opérateur d’extension linéaire continu existe, il faut quelques restrictions sur les pointes de E. En eﬀet, Tidten ([51]) a montré que si E est le compact de l’exemple 1.1, alors il n’existe aucun opérateur d’extension (linéaire continu) de Ꮿ∞ (E) dans Ꮿ∞ (R2 ). 2.3. Exposant de Markov. Soit E un compact de Rn , on pose μ(E) := inf r > 0 : E satisfait à (M) avec exposant r (2.4) et on l’appelle exposant de Markov de E. La connaissance de la valeur exacte de μ(E) permet en particulier de minimiser la perte de régularité dans des problèmes liés à l’extension linéaire des classes de fonctions ultradiﬀérentiables (voir Pleśniak [42], Beaugendre [11]). Si E est un compact connexe de C contenant au moins deux points diﬀérents, alors par Pommerenke [43], 1 ≤ μ(E) ≤ 2. Si E est un compact de Rn quelconque, en utilisant des propriétés extrémales des polynômes de Tchebyschev, on montre que μ(E) ≥ 2. Si E est un compact convexe de Rn d’intérieur non-vide, alors μ(E) = 2. Rappellons encore l’exemple de Goetgheluck de Ek ⊂ R2 , où μ(E) = 2k. Si E est un compact UPC de Rn à paramètre m ≥ 1, alors μ(E) ≤ 2m (voir Baran [3]). Notons finalement que si E est l’ensemble de Zerner (voir exemple 1.1), alors μ(E) = ∞. Il se trouve que l’exposant de Markov est un invariant de « bonnes » applications analytiques. On a (voir Baran-Pleśniak [7]) Théorème 2.5. Soient E un compact de Rn satisfaisant à (M) pour l’exposant r et f une application analytique d’un voisinage U de E dans Rn , telle que f (E) soit non pluripolaire (dans Cn ) et, pour tout x ∈ E, detdx f = 0. Alors f (E) satisfait à (M) pour le même exposant r que celui de E. 2.4. Inégalité de Markov sur les ensembles algébriques de Cn . Dans les années récentes, l’inégalité de Markov a été intensivement étudiée sur des sous-variétés algébriques de Rn (travaux de Feﬀerman-Narasimhan, Roytwarf-Yomdin, Bos-Levenberg-Milman-Taylor, Brudnyı̆, Baran-Pleśniak, Gendre, Kosek, Comte-Yomdin, Erdélyi-Kroó, Kroó-Szabados). Notons ici un résultat important de Sadullaev [46]. Théorème 2.6. Un ensemble analytique A de Cn est algébrique si et seulement si la fonction de Green pluricomplexe VE∗ est localement bornée sur A pour tout compact non-pluripolaire E de A. 8 Inégalité de Markov en plusieurs variables Le critère de Sadullaev est crucial pour l’étude des inégalités de type Bernstein-Walsh, Markov ou de van der Corput-Schaake sur les ensembles algébriques (voir Bos-LevenbergTaylor [21], Bos et al. [19, 20], Baran-Pleśniak [8–10]). L’inégalité tangentielle de Markov avec l’exposant égal à 1 caractérise la propriété d’un compact K de Rn d’être morceau d’une variété algébrique. Plus précisement, on a (voir Baran-Pleśniak [9]) Théorème 2.7. Soit K un compact de Rn admettant une paramétrisation analytique φ : Bm → K, où Bm est la boule unitée de Rm (1 ≤ m ≤ n). Alors la dimension de Zariski de K est égale à m si et seulement si on peut trouver une constante C > 0 telle que, pour tout x ∈ K et tout polynôme P ∈ C[x1 ,... ,xn ], DT(t,v) P(x) ≤ C(degP)P K , (2.5) où t ∈ φ−1 (x) ∩ Bm , v ∈ Sm−1 et T(t,v) = Dv φ(t) est la dérivée de φ en direction v. Si K est une sous-variété compacte lisse de Rn de dimension m, 1 ≤ m ≤ n, le théorème ci-dessus a été montré auparavant par Bos et al. [19]. Soient E un compact polynomialement convexe de Cm de type HCP, avec s pour exposant, et f une application analytique non-dégénerée définie sur un voisinage ouvert de E, à valeurs dans un sous-ensemble algébrique M de Cn de dimension k, 1 ≤ k ≤ n. On a alors (Baran-Pleśniak [10]) Théorème 2.8. Il existe une constante C > 0 telle que, pour tout polynôme Q ∈ C[z1 ,... ,zn ] et tout t ∈ E, DT(t,v) Q f (t) ≤ C1 d 1/s Q f (E) . (2.6) Corollaire 2.9. Soit f une application polynomiale de R dans Rn avec f (t) = (1 − t)s1 (1 + t)s2 Q(t), (2.7) où Q est une application polynomiale de R dans Rn telle que Q(t) = 0 sur [−1,1]. Soit α = max(s1 ,s2 ). Alors il existe une constante A>0 telle que, pour tout polynôme P ∈ R[x1 ,...,xn ] de degré d, on ait DT P(x) ≤ Ad 2+2α P N , x pour x ∈ N = f [−1,1] , (2.8) avec DTx P(x) = D(Q(t)/Q(t)) P(x), où x = f (t). Exemple 2.10. Soient m et l deux entiers positifs premiers entre eux, m > l ≥ 2. Soit f (t) = (((1 + t)/2)l ,((1 + t)/2)m ). On a alors N = f [−1,1] = (x, y) ∈ R2 : 0 ≤ x, y ≤ 1, xm = y l . (2.9) Wiesław Pleśniak 9 En eﬀet, comme α = l − 1, le corollaire précédent donne l’inégalité DT (x,y) P(x, y) ≤ Ad2l P N , (2.10) pour tout polynôme P ∈ R[x, y] de degré d. (Voir aussi Bos et al. [20], Gendre [23].) Dans [19], on peut trouver un exemple d’une fonction analytique f : [0,1] → R telle que f ([0,1]) n’admette aucune inégalité de Markov tangentielle dont l’exposant est fini. Ainsi on pourrait penser qu’une telle inégalité n’a lieu que si f ([0,1]) est morceau d’une variété algébrique. Mais, un contre-exemple a récemment été donné par Bos et al. ([18]). Théorème 2.11. Soit T ∈ R[x] un polynôme quelconque. Pour tout intervalle [a,b] ⊂ R, on peut trouver une constante C > 0 telle que, pour tout polynôme P ∈ C[x, y], on ait d max x∈[a,b] dx 4 T(x) , P x,eT(x) ≤ C(degP) max P x,e x∈[a,b] (2.11) l’exposant 4 étant optimal. 2.5. Remarques finales. La théorie présentée ici témoigne de la force ainsi que de la beauté de l’inégalité classique de Markov. C’est pourquoi je l’appelle « une vénérable dame qui n’a pas perdu de son attrait. » Il est diﬃcile de traiter toutes les ramifications possibles de l’inégalité de Markov en plusieurs variables. Pour cette raison, je me suis restreint à sa forme en norme uniforme. Pour les versions L p voir des travaux de Bos-Milman ([22]), Goetgheluck ([25]) et Baran ([4, 5]). Notons encore que l’inégalité de Markov a été également étudiée en dimension infinie ; nous renvoyons le lecteur aux travaux de Harris ([28]), Sarantopoulos ([47]), MuñozSarantopoulos ([36]), Révész-Sarantopoulos ([45]), Skalyga ([49]) et Baran ([6]). Bibliographie [1] M. Altun and A. Goncharov, A local version of the Pawłucki-Pleśniak extension operator, Journal of Approximation Theory 132 (2005), no. 1, 34–41. [2] M. S. Baouendi and C. Goulaouic, Approximation of analytic functions on compact sets and Bernstein’s inequality, Transactions of the American Mathematical Society 189 (1974), 251–261. [3] M. Baran, Markov inequality on sets with polynomial parametrization, Annales Polonici Mathematici 60 (1994), no. 1, 69–79. , New approach to Markov inequality in L p norms, Approximation Theory, Monogr. Text[4] books Pure and Applied Mathematics, vol. 212, Marcel Dekker, New York, 1998, pp. 75–85, (in memory of A. K. 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Sarantopoulos, Bernstein and Markov-type inequalities for polynomials on real Banach spaces, Mathematical Proceedings of the Cambridge Philosophical Society 133 (2002), no. 3, 515–530. [37] W. Pawłucki and W. Pleśniak, Markov’s inequality and Ꮿ∞ -functions on sets with polynomial cusps, Mathematische Annalen 275 (1986), no. 3, 467–480. [38] , Extension of Ꮿ∞ -functions from sets with polynomial cusps, Studia Mathematica 88 (1988), no. 3, 279–287. [39] R. Pierzchała, UPC condition in polynomially bounded o-minimal structures, Journal of Approximation Theory 132 (2005), no. 1, 25–33. [40] W. Pleśniak, A Cantor regular set which does not have Markov’s property, Annales Polonici Mathematici 51 (1990), 269–274. [41] , Markov’s inequality and the existence of an extension operator for Ꮿ∞ -functions, Journal of Approximation Theory 61 (1990), no. 1, 106–117. [42] , Extension and polynomial approximation of ultradiﬀerentiable functions in Rn , Bulletin de la Société Royale des Sciences de Liège 63 (1994), no. 5, 393–402. [43] Ch. Pommerenke, On the derivative of a polynomial, The Michigan Mathematical Journal 6 (1959), 373–375. [44] Q. I. Rahman and G. Schmeisser, Analytic Theory of Polynomials, London Mathematical Society Monographs. New Series, vol. 26, The Clarendon Press, Oxford University Press, Oxford, 2002. [45] S. G. Révész and Y. Sarantopoulos, On Markov constants of homogeneous polynomials over real normed spaces, East Journal on Approximations 9 (2003), no. 3, 277–304. [46] A. Sadullaev, An estimate for polynomials on analytic sets, Mathematics of the USSR - Izvestiya 20 (1983), no. 3, 493–502. [47] Y. Sarantopoulos, Bounds on the derivatives of polynomials on Banach spaces, Mathematical Proceedings of the Cambridge Philosophical Society 110 (1991), no. 2, 307–312. [48] R. T. Seeley, Extension of Ꮿ∞ functions defined in a half space, Proceedings of the American Mathematical Society 15 (1964), 625–626. [49] V. I. Skalyga, Analogues of Markov’s inequality in normed spaces, Mathematical Notes 75 (2004), no. 5-6, 739–743. [50] E. M. Stein, Singular Integrals and Diﬀerentiability Properties of Functions, Princeton Mathematical Series, no. 30, Princeton University Press, New Jersey, 1970. [51] M. Tidten, Fortsetzungen von Ꮿ∞ -Funktionen, welche auf einer abgeschlossenen Menge in Rn definiert sind, Manuscripta Mathematica 27 (1979), no. 3, 291–312. [52] V. Totik, Markoﬀ constants for Cantor sets, Acta Scientiarum Mathematicarum (Szeged) 60 (1995), no. 3-4, 715–734. [53] H. Whitney, Analytic extensions of diﬀerentiable functions defined in closed sets, Transactions of the American Mathematical Society 36 (1934), no. 1, 63–89. 12 Inégalité de Markov en plusieurs variables [54] A. Zeriahi, Inegalités de Markov et développement en série de polynômes orthogonaux des fonctions Ꮿ∞ et Ꮽ∞ , Several Complex Variables (Stockholm, 1987/1988) (J. F. Fornaess, ed.), Math. Notes, vol. 38, Princeton University Press, New Jersey, 1993, pp. 683–701. [55] M. Zerner, Développement en séries de polynômes orthonormaux des fonctions indéfiniment diﬀérentiables, Comptes Rendus de l’Académie des Sciences. Série I. Mathématique 268 (1969), 218–220. Wiesław Pleśniak : Institut de mathématiques, Université Jagellonne de Cracovie, UL. 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https://openalex.org/W4372347577	https://www.medrxiv.org/content/medrxiv/early/2023/05/05/2023.05.04.23289538.full.pdf	English	null	COMMON ADOLESCENT MENTAL HEALTH DISORDERS SEEN IN FAMILY MEDICINE CLINICS IN GHANA AND NIGERIA	medRxiv (Cold Spring Harbor Laboratory)	2,023	cc-by	6,001	. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 5, 2023. ; https://doi.org/10.1101/2023.05.04.23289538 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 5, 2023. ; https://doi.org/10.1101/2023.05.04.23289538 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 5, 2023. ; https://doi.org/10.1101/2023.05.04.23289538 doi: medRxiv preprint COMMON ADOLESCENT MENTAL HEALTH DISORDERS SEEN IN FAMILY MEDICINE CLINICS IN GHANA AND NIGERIA 1Kumbet JS, 2Oseni TIA, 3Mensah-Bonsu M, 4Damagum FM, 5Opare-Lokko EA, 6Namisango E 7Olawumi AL, 8Ephraim OC, 9Aweh B, 1Department of Family Medicine, Geriatric Medicine Sub Division, Federal Medical Centre, Keffi, Nigeria. 2Department of Family Medicine, Ambrose Alli University, Ekpoma, Nigeria/Lifestyle and Behavioural Medicine Unit, Department of Family Medicine, Irrua Specialist Teaching Hospital, Irrua, Nigeria. 3Department of Family Medicine, Komfo Anokye Teaching Hospital, Kumasi, Ghana. 4Department of Family Medicine, Aminu Kano Teaching Hospital, Kano, Nigeria. 5Department of Family Medicine, Greater Accra Regional Hospital, Accra/Faculty of Family Medicine, Ghana College of Physicians and Surgeons, Accra, Ghana. 6African Palliative Care Association/College of Health Sciences, Makerere University, Kampala, Uganda 7 Department of Family Medicine, Aminu Kano Teaching Hospital, Kano, Nigeria. 8Department of Family Medicine, Alex Ekwueme Federal University Teaching Hospital, Abakaliki, Nigeria 9Department of Mental Health and Behavioural Medicine, Irrua Specialist Teaching Hospital, Irrua, Nigeria. Corresponding Author: Dr Tijani I. A. Oseni, Department of Family Medicine, Ambrose Alli University, Ekpoma, Nigeria/Lifestyle and Behavioural Medicine Unit, Department of Family Medicine, Irrua Specialist Teaching Hospital, Irrua, Nigeria. tijanioseni@aauekpoma.edu.ng +2348036281897 NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice. COMMON ADOLESCENT MENTAL HEALTH DISORDERS SEEN IN FAMILY MEDICINE CLINICS IN GHANA AND NIGERIA 1Kumbet JS, 2Oseni TIA, 3Mensah-Bonsu M, 4Damagum FM, 5Opare-Lokko EA, 6Namisango E 7Olawumi AL, 8Ephraim OC, 9Aweh B, 1Department of Family Medicine, Geriatric Medicine Sub Division, Federal Medical Centre, Keffi, Nigeria. Centre, Keffi, Nigeria. 2Department of Family Medicine, Ambrose Alli University, Ekpoma, Nigeria/Lifestyle and Behavioural Medicine Unit, Department of Family Medicine, Irrua Specialist Teaching Hospital, Irrua, Nigeria. 7 Department of Family Medicine, Aminu Kano Teaching Hospital, Kano, Nigeria. 8Department of Family Medicine, Alex Ekwueme Federal University Teaching Hospital, Abakaliki, Nigeria 9Department of Mental Health and Behavioural Medicine, Irrua Specialist Teaching Hospital, Irrua, Nigeria. Corresponding Author: Dr Tijani I. A. Oseni, Department of Family Medicine, Ambrose Alli University, Ekpoma, Nigeria/Lifestyle and Behavioural Medicine Unit, Department of Family Medicine, Irrua Specialist Teaching Hospital, Irrua, Nigeria. tijanioseni@aauekpoma.edu.ng +2348036281897 NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice. . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 5, 2023. ; https://doi.org/10.1101/2023.05.04.23289538 doi: medRxiv preprint Methodology A descriptive cross-sectional study involving 302 Physicians practicing in Family Medicine Clinics in Nigeria and Ghana who were randomly selected for the study. Data were collected using self-administered semi-structured questionnaire, and were entered into excel spreadsheet before analysing with IBM-SPSS version 22. Descriptive statistics using frequencies and percentages was used to describe variables. Ethical approval was obtained prior to commencement of the study. Background Mental health disorders among adolescents is on the rise globally. For fear of stigmatization, patients seldom present to mental health physicians. They are mostly picked during consultations with Family Physicians. This study seeks to evaluate the common mental health disorders seen by family Physicians in Family Medicine Clinics in Nigeria and Ghana. Conclusion Family Physicians in Nigeria and Ghana attend to a good number of adolescents with mental health disorders in their Family Medicine clinics. There is the need for Family Physicians to have specialized training and retraining to be able to recognize and treat adolescent mental health disorders. This will help reduce stigmatization and improve the management of the disease thus reducing the burden. Key Words: adolescent, mental health, disorder, family physicians, Ghana, Nigeria. Results Of the 302 Physicians recruited for the study, only 233 completed the study, in which 168 (72.1%) practiced in Nigeria and 65 (27.9%) in Ghana. They were mostly in urban communities (77.3%) and tertiary health facilities (65.2%). Over 90% of Family Medicine practitioners attended to adolescents with mental health issues with over 70% of them seeing at least 2 adolescents with mental health issues every year. The burden of mental health disorder was 16% and the common mental health disorders seen were depression (59.2%), Bipolar Affective Disorder (55.8%), Epilepsy (51.9%) and Substance Abuse Disorder (44.2%). . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 5, 2023. ; https://doi.org/10.1101/2023.05.04.23289538 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 5, 2023. ; https://doi.org/10.1101/2023.05.04.23289538 doi: medRxiv preprint INTRODUCTION The burden of mental health disorders is on the rise globally. In 2019, 1 in every 8 people, or 970 million people around the world were living with a mental disorder, with anxiety and depressive disorders the most common.1 In 2020, the amount of people living with anxiety and depressive disorders rose considerably because of the COVID-19 pandemic.1,2 Adolescents are group of persons with chronological age 10-19 years of age. Adolescence is a critically important stage of life for mental health and well-being of individuals, not only for the reason that this is when young people acquire autonomy, social interaction, self-control, and rapid learning, but also because the abilities and potentials formed in this period have a direct bearing on their mental health for the rest of their lives.3 Although adolescents generally are highly susceptible to mental health challenges, they receive very little attention, especially in developing countries.4 Globally, one in seven of adolescents experiences a mental disorder, accounting for 13% of the global burden of disease in this age group with depression, anxiety and behavioural disorders are among the leading causes of illness and disability among them.5 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 5, 2023. ; https://doi.org/10.1101/2023.05.04.23289538 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 5, 2023. ; https://doi.org/10.1101/2023.05.04.23289538 doi: medRxiv preprint According to the World Health Organization, common mental disorders such as depression and anxiety account for the largest proportion of mental, developmental and substance use disorders. Behavioural disorders including attention-deficit and hyperactivity disorder plus conduct disorder are more prevalent among 10-14-year olds, while alcohol and drug use disorders become more common in older adolescence (15-19-year olds). Statistical analysis Data were analysed using the Statistical Package for Social SciencesTM (IBM Corp, Armonk, NY, USA) version 22.0. They were presented in tables and were described using frequencies and percentages. Study Design and Setting The study was a descriptive cross-sectional study conducted among Family Physicians practising in Nigeria and Ghana from May to September, 2022. It is part of a larger study with some of the findings already published.12 The sample size was calculated to be 302 using fisher’s formula and finite correction was made based on the total population of Family Physicians in each country: 1200 and 125 for Nigeria and Ghana respectively. The sample size (302) was proportionately distributed to each country based on their population size at the time of data collection: Nigeria had 254 while Ghana had 48 Family Physicians. The study sites included General Outpatient Clinics of Teaching Hospitals, Specialist, General and District Hospitals and other Primary Healthcare Centers, where Family Physicians practice in both countries. The consenting Physicians were recruited into the study using multi-stage sampling methods. The validated semi- structured self-administered questionnaire was administered to study participants. Other details of the study design and methodology are contained in the published article.12 Authors did not have access to information that could identify individual participants during or after data collection as the study was anonymised. INTRODUCTION The total number in both age groups combined is 17 million young people, equivalent to almost 20% or one in five of the population in this age group.3 A systematic review of studies from Sub-Saharan African countries revealed that 40.8% of adolescents have emotional and behavioral problems, 29.8% have anxiety disorders, 26.9% have depression, 21.5% have PTSD, and 20.8% have suicidal thoughts.6 One out of every six young Nigerian aged 15- 24 is suffering from poor mental health, according to a report released by the United Nations Children Fund (UNICEF).7 A study in Nigeria found that the prevalence of depression among adolescents ranged from 4.2% to 13.6%.8 In Ghana, WHO estimated that 650,000 are suffering from a severe mental disorder and a further 2,166,000 are suffering from a moderate to mild mental disorder and the treatment gap is 98% of the total population.9 However, the prevalence of mental illness and its burden among adolescents is not known on a national level.10 There is a dearth of mental health experts in West Africa. This is worsened by the stigma associated with mental health disorders in the region. The WHO and World Organization of Family Doctors (WONCA) advocates for the integration of mental health services into primary care as the most viable way of closing the treatment gap and ensuring people get the mental health care they need.11 The aim of this study was to evaluate the common mental health disorders among adolescents as seen by family Physicians in Family Medicine Clinics in Nigeria and Ghana. . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 5, 2023. ; https://doi.org/10.1101/2023.05.04.23289538 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 5, 2023. ; https://doi.org/10.1101/2023.05.04.23289538 doi: medRxiv preprint Ethical Consideration Ethical approval for the study was obtained from the Health Research Ethics Committee of Irrua Specialist Teaching Hospital, Irrua, Nigeria (ISTH/HREC/20221403/275). . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 5, 2023. ; https://doi.org/10.1101/2023.05.04.23289538 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 5, 2023. ; https://doi.org/10.1101/2023.05.04.23289538 doi: medRxiv preprint RESULTS A total of 233 Family Physicians completed the study (77.2% response rate), in which 65 (27.90%) in Ghana and 168 (72.10%) in Nigeria participated in the study. They worked in facilities that were mainly in the urban setting 180 (77.25%). Majority of the facilities were Tertiary institutions 152 (65.24%), which was either Teaching Hospitals or Federal Medical Centres. The socio-demographic characteristics are shown in Table 1. Table 1: Socio-demographic characteristics of respondents (N=233) VARIABLE FREQUENCY PERCENTAGE Country Ghana 65 27.90 Nigeria 168 72.10 Location of Health Facility Rural 53 22.75 Urban 180 77.25 Type of Facility General/District Hospital 55 23.61 Teaching Hospital/Federal Medical Centre 152 65.24 Private/Mission Hospital 26 11.15 Table 1: Socio-demographic characteristics of respondents (N=233) VARIABLE FREQUENCY PERCENTAGE Table 2 shows the adolescent mental health disorders seen in Family Medicine Clinics in Ghana and Nigeria. Over 90% of Family Medicine practitioners attend to adolescents with mental health issues with over 70% of them seeing at least 2 adolescents with mental health issues every year. . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 5, 2023. ; https://doi.org/10.1101/2023.05.04.23289538 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 5, 2023. ; https://doi.org/10.1101/2023.05.04.23289538 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 5, 2023. ; https://doi.org/10.1101/2023.05.04.23289538 doi: medRxiv preprint Table 2: Adolescent mental health disorders seen in primary care clinics (N=233) VARIABLE FREQUENCY PERCENTAGE Do you attend to adolescents with mental health issues? (n=233) Yes 212 90.99 No 21 9.01 How often do you attend to adolescents with mental health issues? RESULTS (n=212) Often (> 3 patients a year) 33 15.57 Sometimes (2 – 3 patients a year) 117 55.19 Rarely (≤ 1 patient a year) 62 29.24 ble 2: Adolescent mental health disorders seen in primary care clinics (N=233) FREQUENCY PERCENTAGE The burden of mental health disorder in this study was 16% and the distribution of the common adolescent mental health disorders seen in Family Medicine Clinics in Ghana and Nigeria are as shown in Table 3. Depression 138 (59.23%) was the most commonly seen disorder followed by Bipolar Disorders 130 (55.79%), Epilepsy 121 (51.93%), and Substance Use Disorders 103 (44.21%) in that order. . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 5, 2023. ; https://doi.org/10.1101/2023.05.04.23289538 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 5, 2023. ; https://doi.org/10.1101/2023.05.04.23289538 doi: medRxiv preprint Table 3: Common Adolescent Mental Health Disorders Seen in Primary Care Clinics (N=233) DISORDER FREQUENCY PERCENTAGE Depression 138 59.23 Substance Use Disorders 103 44.21 Bipolar Disorders 130 55.79 Psychosis 52 22.32 Suicide or Self Harm 71 30.47 Schizoaffective Disorder 58 24.89 ADHD 39 16.74 Epilepsy 121 51.93 Enuresis 29 12.45 ommon Adolescent Mental Health Disorders Seen in Primary Care Clinics DISCUSSION The study participants and their distribution have already been described in another publication from the study.12 Our study showed that 91% of respondents attend to adolescents with mental health issues with over half of them attending to about two to three adolescents with mental health disorders yearly. This is worrisome, it shows that the burden of adolescent mental health in primary care is enormous. If not attended to, it usually leads to major mental problems including suicide and self-harm which have been on the rise among adolescents.13,14 It calls for improved capacity in the diagnosis and management of mental health disorders among Family Physicians. This is particularly so as patients hardly present for care as they are generally stigmatized, shunned and denied access to care by their families, caregivers and the society.15 The burden of mental health disorders among adolescents in primary care in west Africa in this study (16%) is similar to the Global findings of 14% burden of mental health disorders among adolescents5 and 16% burden found by Robert et al in England.16 The high burden found in this study can be attributed to the . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 5, 2023. ; https://doi.org/10.1101/2023.05.04.23289538 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 5, 2023. ; https://doi.org/10.1101/2023.05.04.23289538 doi: medRxiv preprint large number of presentation of adolescent to primary care centres as compared to specialist clinics, the fear of stigmatization of mental health disorders and the recent development of subspecialty clinics such as adolescent clinics, and geriatric clinics in General outpatient clinics.3,10 Respondents reported a variety of mental health disorders seen. The most common disorder was depression. This was followed by bipolar disorder, epilepsy and substance use disorders with the least common disorders as enuresis, Attention Deficit Hyperactivity Disorder (ADHD), Psychosis and Schizoaffective Disorders. DISCUSSION WHO report that 12 billion work hours and 1 trillion US dollars are lost annually to depression and anxiety alone.17 On a global scale WHO stated in 2021 that “Depression, anxiety and behavioural disorders are among the leading causes of illness and disability among adolescents”,18 which is in tandem with this study having 59.23% of the respondents treating depression. However, a study done in Nigeria and Ghana 8,19 had Schizophrenia and Epilepsy as the commonest mental health disorders in Nigeria and Ghana respectively. The above two studies were not done in primary care setting and Providers and stakeholders had limited or no training in adolescent mental health and that could be the reason for the slight difference. The high number of depressions among adolescent can be explained by the high level of poverty and few numbers of specialist to attend to the huge burden of mental health disorder among adolescents.3 Suicidal attempt, Suicide and Self Harm may be a complications or an end spectrum of adolescent mental health disorders13, in this study 30.47% have suicidal attempt and self-harm which is close to findings of a study done in SSA6 which slight difference may be attributable to smaller number of countries involved in our own study. Bipolar disorder was the second leading mental health disorder identified in this study. This could be due to the fact that the most frequent range of onset of bipolar disorder is between the ages of large number of presentation of adolescent to primary care centres as compared to specialist clinics, the fear of stigmatization of mental health disorders and the recent development of subspecialty clinics such as adolescent clinics, and geriatric clinics in General outpatient clinics.3,10 large number of presentation of adolescent to primary care centres as compared to specialist clinics, the fear of stigmatization of mental health disorders and the recent development of subspecialty clinics such as adolescent clinics, and geriatric clinics in General outpatient clinics.3,10 Respondents reported a variety of mental health disorders seen. The most common disorder was depression. This was followed by bipolar disorder, epilepsy and substance use disorders with the least common disorders as enuresis, Attention Deficit Hyperactivity Disorder (ADHD), Psychosis and Schizoaffective Disorders. DISCUSSION WHO report that 12 billion work hours and 1 trillion US dollars are lost annually to depression and anxiety alone.17 On a global scale WHO stated in 2021 that “Depression, anxiety and behavioural disorders are among the leading causes of illness and disability among adolescents”,18 which is in tandem with this study having 59.23% of the respondents treating depression. However, a study done in Nigeria and Ghana 8,19 had Schizophrenia and Epilepsy as the commonest mental health disorders in Nigeria and Ghana respectively. The above two studies were not done in primary care setting and Providers and stakeholders had limited or no training in adolescent mental health and that could be the reason for the slight difference. The high number of depressions among adolescent can be explained by the high level of poverty and few numbers of specialist to attend to the huge burden of mental health disorder among adolescents.3 Suicidal attempt, Suicide and Self Harm may be a complications or an end spectrum of adolescent mental health disorders13, in this study 30.47% have suicidal attempt and self-harm which is close to findings of a study done in SSA6 which slight difference may be attributable to smaller number of countries involved in our own study. Bipolar disorder was the second leading mental health disorder identified in this study. This could be due to the fact that the most frequent range of onset of bipolar disorder is between the ages of Bipolar disorder was the second leading mental health disorder identified in this study. This could be due to the fact that the most frequent range of onset of bipolar disorder is between the ages of 14-21 years; which falls with the adolescent and early adult age group.20 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 5, 2023. ; https://doi.org/10.1101/2023.05.04.23289538 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 5, 2023. ; https://doi.org/10.1101/2023.05.04.23289538 doi: medRxiv preprint In this study, epilepsy was placed third among adolescent mental illnesses. DISCUSSION This is not surprising given that, compared to all other continents, Africa has the highest prevalence of epilepsy in children and adolescents.21 According to a meta-analysis, there are 13–15 new cases of epilepsy for every 1000 people in West Africa.22 This was attributed to the high prevalence of parasitic and infectious diseases in Africa.21 The influence of epileptogenic recreational drugs and substances; which are commonly used in this age group could also explain the high burden of seizure disorder in them.21 The frequent exposure to computer screens, phone screens and flashing lights may also predispose the adolescents to photosensitive epilepsies; which had been reported to be high among them.23 Substance use disorder and suicide or self-harm were also prevalent among adolescent in this study. This is similar to the findings of Birhanu et al in Ethiopia,24 Mavura et al in northern Tanzania,25 and Volkow et al in the US.26 The reasons may not be unconnected to the high level of peer influence, risk taking behavior and experimentation with substances due to developmental changes and challenges in adolescence.24,25 The high burden of self-harm or suicide in this study could be due to the strong relationship between substance abuse and suicide or self-harm especially, among adolescents and young adults.27,28 There is an urgent need for Family Physicians to look out for adolescent mental health issues and address them at the early stage before they progress to more complicated forms. There is also the need for policy makers to increase awareness on the burden of mental health disorders among adolescents and put measures in place to mitigate them. Limitations The study was conducted in two countries in West Africa. Though most Family Medicine Clinics in the region are in these two countries, the results still may not be a true representation of the entire region. Also, the study was conducted among doctors. However, relatively most primary health care centres in the region are run by primary care nurses, community health officers and community health extension workers. These categories of primary care providers were not included in the study even though they attend to most of the patients presenting to primary care facilities in these regions. Conclusion The prevalence of mental disorders among adolescents seen by Family Physicians in Family Medicine clinics are high in Nigeria and Ghana. There is the need for Family Physicians to have . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 5, 2023. ; https://doi.org/10.1101/2023.05.04.23289538 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 5, 2023. ; https://doi.org/10.1101/2023.05.04.23289538 doi: medRxiv preprint specialized training and retraining on mental health issues concerning adolescents. There is also a need to have more subspecialty adolescent clinics in Family Medicine Clinics to be able to handle adolescents’ challenges including recognising and treating adolescent mental health disorders easily. There is also the need for high index of suspicion for these disorders in adolescents when they present. Policy makers should also put measures in place to improve awareness and care for patients. specialized training and retraining on mental health issues concerning adolescents. There is also a need to have more subspecialty adolescent clinics in Family Medicine Clinics to be able to handle adolescents’ challenges including recognising and treating adolescent mental health disorders easily. There is also the need for high index of suspicion for these disorders in adolescents when they present. Policy makers should also put measures in place to improve awareness and care for patients. Funding Financing of the project was exclusively at the expense of the researchers. Financing of the project was exclusively at the expense of the researchers. Author Contribution SK, OTIA, MBM, DF, OLE and EO conceived the idea, designed the study and conducted the study. NE analysed the data. All authors wrote the manuscript, edited and revised the manuscript as well as approved the final copy of the manuscript. Conflicts of Interests The researchers declare that there is no conflict of interest whatsoever. The researchers declare that there is no conflict of interest whatsoever. . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 5, 2023. ; https://doi.org/10.1101/2023.05.04.23289538 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 5, 2023. ; https://doi.org/10.1101/2023.05.04.23289538 doi: medRxiv preprint Acknowledgement We thank Afriwon Research Group ARG of Afriwon Renaissance for bringing young researchers in Africa together and providing the platform from which the research was conducted. REFERENCES 1. Institute of Health Metrics and Evaluation. Global Health Data Exchange (GHDx). Global burden of disease 2019. Available from: https://www.vizhub.healthdata.org/gbd-results/. [Accessed 14th May 2022]. 1. Institute of Health Metrics and Evaluation. Global Health Data Exchange (GHDx). Global burden of disease 2019. Available from: https://www.vizhub.healthdata.org/gbd-results/. [Accessed 14th May 2022]. 2. World Health Organization. Mental Health and COVID-19: Early evidence of the pandemic’s impact. Geneva: World Health Organization; 2022. 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Acknowledgement JAMA Pediatr. 2021;175(6):640–643. doi:10.1001/jamapediatrics.2020.6981. . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 5, 2023. ; https://doi.org/10.1101/2023.05.04.23289538 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 5, 2023. ; https://doi.org/10.1101/2023.05.04.23289538 doi: medRxiv preprint 27. Breet E, Bantjes J, Lewis I. Substance use and self-harm: a cross-sectional study of the prevalence, correlates and patterns of medical service utilisation among patients admitted to a South African hospital. 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https://openalex.org/W2141252253	https://escholarship.org/content/qt85j204j4/qt85j204j4.pdf?t=ppuhcn	English	null	Cellular retinoic acid-binding proteins are essential for hindbrain patterning and signal robustness in zebrafish	Development	2,012	cc-by	5,791	Peer reviewed Peer reviewed Authors Copyright Information This work is made available under the terms of a Creative Commons Attribution License, availalbe at https://creativecommons.org/licenses/by/4.0/ Permalink https://escholarship.org/uc/item/85j204j4 Permalink https://escholarship.org/uc/item/85j204j4 UC Irvine Cellular Retinoic Acid-Binding Proteins Are Essential For Hindbrain Patterning and Signal Robustness in Zebrafish UC Irvine UC Irvine Previously Published Works Title Cellular Retinoic Acid-Binding Proteins Are Essential For Hindbrain Patterning and Signal Robustness in Zebrafish Permalink https://escholarship.org/uc/item/85j204j4 Authors Nie, Q Cai, A Radtke, K et al. Publication Date 2012 Copyright Information This work is made available under the terms of a Creative Commons Attribution License, availalbe at https://creativecommons.org/licenses/by/4.0/ UC Irvine UC Irvine Previously Published Works Title Cellular Retinoic Acid-Binding Proteins Are Essential For Hindbrain Patterning and Signal Robustness in Zebrafish Permalink https://escholarship.org/uc/item/85j204j4 Authors Nie, Q Cai, A Radtke, K et al. Publication Date 2012 Copyright Information This work is made available under the terms of a Creative Commons Attribution License, availalbe at https://creativecommons.org/licenses/by/4.0/ INTRODUCTION facilitating its degradation by Cyp26s (Boylan and Gudas, 1992; Fiorella and Napoli, 1994; Won et al., 2004). Deletion of both Crabp1 and Crabp2 in mice causes supernumerary digits on the forelimb at low penetrance, but adults are otherwise viable (Lampron et al., 1995), suggesting that other proteins can compensate for Crabps in RA signaling (Romand et al., 2000). Morphogen gradients induce different cell fates depending on concentration. Gradient shape is determined by the source and rate of ligand production, as well as its transport properties and stability (Ben-Zvi and Barkai, 2010; Sample and Shvartsman, 2010; Umulis et al., 2010). Feedback mechanisms such as self-enhanced receptor- mediated degradation also shape morphogen gradients and make them robust (i.e. able to compensate for changes in morphogen availability), as has been demonstrated for growth factors of the TGF, Wingless (Wg) and Hedgehog (Hh) families (Eldar et al., 2003; Lander, 2007; Meinhardt, 2009; Wartlick et al., 2009). The vitamin A derivative retinoic acid (RA) determines the identities of hindbrain rhombomeres along the anterior-posterior (A-P) axis (Dupé and Lumsden, 2001; Maves and Kimmel, 2005; White et al., 2007). However, unlike peptide morphogens, RA is hydrophobic and is typically bound to soluble proteins. How these binding proteins function in modulating RA signaling remains unclear. RA signaling is also remarkably robust, as might be expected for a signal derived from a dietary precursor. Hindbrain development occurs normally over a ~20-fold range of RA concentrations (Hernandez et al., 2007; White and Schilling, 2008). We previously developed a model explaining how robustness critically depends upon negative feedback, and showed that RA induces expression of the RA-degrading enzyme Cyp26a1 (White et al., 2007). However, our models suggest that self-enhanced degradation alone can only account for a small fraction of the robustness. Crabps might provide additional negative feedback by preventing ligand access to receptors and facilitating degradation (Häcker et al., 2005; Lander et al., 2007). Cellular RA-binding proteins (Crabps) bind RA with high affinity and solubilize it intracellularly. Vertebrates have two highly conserved Crabps, both of which transport RA to its receptors (RARs) in vitro (Aström et al., 1991; Delva et al., 1999; Budhu et al., 2001; Budhu and Noy, 2002). In addition, Crabp1 protects cells from excess RA by binding it in the cytosol, away from RARs, and Here we show that, in contrast to mice, zebrafish Crabp2a and Crabp2b are essential for expression of hoxb4 and hoxb5 in the hindbrain. Powered by the California Digital Library University of California eScholarship.org eScholarship.org RESEARCH REPORT 2150 Development 139, 2150-2155 (2012) doi:10.1242/dev.077065 © 2012. Published by The Company of Biologists Ltd Development 139, 2150-2155 (2012) doi:10.1242/dev.077065 © 2012. Published by The Company of Biologists Ltd SUMMARY The vitamin A derivative retinoic acid (RA) is a morphogen that patterns the anterior-posterior axis of the vertebrate hindbrain. Cellular retinoic acid-binding proteins (Crabps) transport RA within cells to both its nuclear receptors (RARs) and degrading enzymes (Cyp26s). However, mice lacking Crabps are viable, suggesting that Crabp functions are redundant with those of other fatty acid-binding proteins. Here we show that Crabps in zebrafish are essential for posterior patterning of the hindbrain and that they provide a key feedback mechanism that makes signaling robust as they are able to compensate for changes in RA production. Of the four zebrafish Crabps, Crabp2a is uniquely RA inducible and depletion or overexpression of Crabp2a makes embryos hypersensitive to exogenous RA. Computational models confirm that Crabp2a improves robustness within a narrow concentration range that optimizes a ‘robustness index’, integrating spatial information along the RA morphogen gradient. Exploration of signaling parameters in our models suggests that the ability of Crabp2a to transport RA to Cyp26 enzymes for degradation is a major factor in promoting robustness. These results demonstrate a previously unrecognized requirement for Crabps in RA signaling and hindbrain development, as well as a novel mechanism for stabilizing morphogen gradients despite genetic or environmental fluctuations in morphogen availability. KEY WORDS: Danio rerio, Hindbrain, Morphogen KEY WORDS: Danio rerio, Hindbrain, Morphogen Anna Q. Cai1,2,3,, Kelly Radtke2,4,5,, Angela Linville4,5, Arthur D. Lander2,4,5, Qing Nie1,2,‡ and Thomas F Schilling2,4,5,‡ Anna Q. Cai1,2,3,, Kelly Radtke2,4,5,, Angela Linville4,5, Arthur D. Lander2,4,5, Qing Nie1,2,‡ and Thomas F. Schilling2,4,5,‡ 1Department of Mathematics, University of California Irvine, Irvine, CA 92697, USA. 2Center for Complex Biological Systems, University of California Irvine, Irvine, CA 92697, USA. 3School of Mathematics and Statistics, University of New South Wales, Sydney NSW 2052, Australia. 4Department of Developmental and Cell Biology, University of California Irvine, Irvine, CA 92697, USA. 5Developmental Biology Center, University of California Irvine, Irvine, CA 92697, USA. INTRODUCTION In addition, Crabp2a is uniquely RA inducible and required for robustness. When Crabp2a is depleted or overexpressed, hindbrain patterning becomes hypersensitive to exogenous RA. Computational models with which we simulate the effects of Crabps on gradient robustness indicate that Crabps must be present within a narrow concentration range and must deliver RA both to its receptors and to Cyp26s for degradation. DEVELOPMENT *These authors contributed equally to this work ‡Authors for correspondence (qnie@math.uci.edu; tschilli@uci.edu) Morpholino (MO) and mRNA injections One-cell stage embryos were injected with 10 ng Crabp2a-MO1 (CGGG - AATTTTACGATCCATCTTCCG) or 5 ng Crabp2b-MO1 (TGTTTTCT - CCGTGTTAGATTCCATG). Two independent MOs for each gene – Crabp2a-MO2 (TTCTGTCGTTCCTCTGAAAGTAACC) and Crabp2b- MO2 (GTTTCTGCGTGTCTTTCTTTCACTC) – gave similar phenotypes, confirming MO specificity. All MOs were designed to block translation. crabp2a:mCherry and crabp2b:mCherry constructs were generated by fusion PCR of the crabp2a cDNA templates and mCherry from a Gateway vector (Invitrogen) (for primers see supplementary material Table S1). Injections were performed with 200 pg crabp2a:mCherry or crabp2b:mCherry mRNA per embryo, alone or with each MO to confirm MO efficiency. For rescue and overexpression experiments, two full-length crabp2a-pCS2 clones, one with and one without the crabp2a-MO binding sites, were transcribed using SP6 mMessage (Ambion). Modeling h d l The model was solved with our newly developed numerical solver (supplementary material Appendices S1, S2), using a scaled domain x[0,1] containing the RA gradient between 0≤x≤0.9. A domain of Cyp26a1 posterior to x>0.9 creates a discontinuity in the gradient. Therefore, xref0.85 was used and the gradient was modeled between x0 and x0.9. Established biological ranges of parameter values were used or estimated within a realistic range (supplementary material Appendix S3, Tables S2, S3). Exploration of parameter space was performed on a logarithmic scale to include large orders of magnitude. We measured robustness using a probability density distribution N(E), interpreted as the probability of the system producing robustness values in the range [E1,E2] with the following formula: ∫ < < = E E E N e de Pr{ } ( ) . (1) E E 1 2 1 2 To determine the requirements for Crabps in signal robustness, we treated embryos injected with Crabp2a-MO or Crabp2b-MO, or both, during gastrulation with 1-10 nM RA (Fig. 2G-L). Surprisingly, Crabp2a-deficient embryos were ~10-fold more sensitive to exogenous RA than wild-type RA-treated embryos; Crabp2b-MO did not affect RA sensitivity alone or when combined with Crabp2a-MO. Treatment of controls with 1 nM RA caused no hindbrain defects (100%, n22; Fig. 2H), whereas treatment of Crabp2a-MO-injected embryos virtually eliminated the R3 domain of krox20 and pax2a expression at the midbrain- hindbrain boundary (70%, n10; Fig. 2K). Defects resembled those of controls treated with 10-fold higher amounts of RA (100%, n18; Fig. 2I). By contrast, treatments of Crabp2a- deficient embryos with 10 nM RA caused complete loss of krox20 and anterior expansion of hoxb4 expression throughout the hindbrain (100%, n8; Fig. 2L). These patterning defects correlate with an expansion of Tg(RARE-gata2:NTD-eYFP)Id1 transgene expression in Crabp2a-deficient embryos treated with 1 nM exogenous RA (supplementary material Fig. S6) (Linville et al., 2009). Thus, embryos lacking Crabp2a are hypersensitive to small changes in exogenous RA and hindbrain patterning is much less robust. (1) To ensure that N(E) was representative of the system, we performed consistency checks using increasing numbers of simulations to find the representative distribution. N(E) typically becomes invariant at ~10,000 simulations, but we included many more for higher resolution. RT-PCR and cloning To test functional requirements for Crabps we compared hindbrain patterning in zebrafish embryos injected with antisense morpholino oligonucleotides (MOs) targeting crabp2a and crabp2b. MOs depleted each target effectively (supplementary material Fig. S3) and specifically, and microinjection of crabp2a mRNAs not recognized by MOs targeting their 5UTRs partially rescued the morphant phenotypes (supplementary material Fig. S4). Embryos injected with either MO alone showed normal patterns of krox20 (egr2 – Zebrafish Information Network) (R3, R5) and reduced hoxb4 (71%, n14) and hoxb5 (100%, n25) expression in R7 and spinal cord (Fig. 1A-C,I-K). By contrast, embryos co-injected with Crabp2a-MO and Crabp2b-MO completely lacked hoxb4 (73%, n15; Fig. 1D) and hoxb5a (90%, n20; Fig. 1L) expression, suggesting a strong reduction in RA signaling similar to that caused by the loss of aldh1a2 (Begemann et al., 2001). hoxb4 expression was not rescued by treatment with 5-20 nM exogenous RA, despite a strong posteriorization of the hindbrain (n14; Fig. 1E-H). Thus, Crabps are required for RA-dependent Hox gene expression in the posterior hindbrain. RT-PCR was performed with whole-embryo RNA preparations (for primers see supplementary material Table S1). crabp2a and crabp2b were amplified and cloned into pCS2 (Rupp et al., 1994) for mRNA synthesis. Embryo treatments Wild-type or Tg(RARE-gata2:NTD-eYFP)Id1 transgenic embryos (Perz- Edwards et al., 2001) were treated with all-trans RA (Sigma) or diethylaminobenzaldehyde (DEAB) as described previously (White et al., 2007). Treatments began at 6 hours postfertilization (hpf) and continued until embryos were fixed in 4% paraformaldehyde (PFA). Accepted 4 April 2012 2151 RESEARCH REPORT Crabps and retinoid signaling Crabp2a is RA inducible and required for signal robustness Mammalian Crabp2 contains a retinoic acid response element (RARE) and is RA inducible in vitro (Aström et al., 1994). We examined crabp2a and crabp2b expression after treatment with RA or an Aldh1a2 inhibitor (DEAB) during gastrulation. Whereas 1 nM RA had no effect on the expression of either gene (data not shown), 10 nM RA induced crabp2a expression throughout the CNS (Fig. 2A,B). Conversely, 5 mM DEAB treatments, which phenocopy mutations in aldh1a2 (Begemann et al., 2001), virtually eliminated crabp2a expression (Fig. 2C) but had no effect on crabp2b (Fig. 2D-F; supplementary material Fig. S5). Thus, RA is both necessary and sufficient to induce crabp2a expression. In situ hybridization Antisense crabp2a and crabp2b mRNA probes were synthesized from linearized pCS2 clones (Thisse et al., 2004). Bright-field in situ hybridization and fluorescent images were acquired on a Zeiss Axioplan II compound microscope with Improvision software. RESULTS AND DISCUSSION A novel requirement for Crabps in Hox gene expression Crabps facilitate interactions between RA and RARs in vitro (Aström et al., 1991; Delva et al., 1999; Budhu and Noy, 2002), but also bind RA in the cytosol preventing its entry into the nucleus and promoting degradation (Boylan and Gudas, 1991; Boylan and Gudas, 1992; Fiorella and Napoli, 1994; Chen et al., 2003). Consistent with previous studies (Sharma et al., 2005) of the four zebrafish Crabps (crabp1a, crabp1b, crabp2a, crabp2b), all but crabp1a were detected during gastrulation (6-9 hpf; supplementary material Figs S1, S2). crabp2a and crabp2b remained expressed in the presumptive hindbrain at 10-24 hpf. crabp2a was expressed in the posterior hindbrain up to rhombomere (R) 4 (supplementary material Fig. S1) and in the anterior spinal cord. crabp2b was initially expressed throughout the ectoderm during gastrulation and became progressively restricted to the anterior hindbrain (particularly R2) and somites (supplementary material Fig. S1). These expression patterns suggest overlapping but spatially and temporally distinct roles for each Crabp. DEVELOPMENT We also overexpressed Crabp2a and treated embryos with RA or DEAB (Fig. 2M-R). Injection of 50-400 pg crabp2a mRNA caused no hindbrain defects (92%, n65; Fig. 2M,P). However, treatment of these embryos with 10 nM RA eliminated krox20 expression in R3 and both krox20 and hoxb4 expression in R5 expanded 2152 RESEARCH REPORT Development 139 (12) Fig. 1. crabp2a and crabp2b are required for zebrafish hindbrain patterning. (A-L)Whole- mount in situ hybridization (ISH) for pax2a at the midbrain-hindbrain boundary (MHB), krox20 in rhombomere (R) 3 and R5, and hoxb4 (A-H) or hoxb5 (I-L) in R7 and spinal cord at 18 hpf; dorsal views, anterior left. Compared with uninjected controls (A), morpholino (MO) depletion of Crabp2a (B,J) or Crabp2b (C,K) slightly reduces and co-injection (D,L) eliminates hoxb4 and hoxb5 expression (arrows). Exogenous RA treatments (5-20 nM) that posteriorize the hindbrain do not rescue hoxb4 expression (E-H). wt, wild type. anteriorly (64%, n11; Fig. 2N,Q). Conversely, Crabp2a- overexpressing embryos were less sensitive to 10 mM DEAB (73%, n15; Fig. 2R), which in uninjected embryos eliminated krox20 expression in R5 and all hoxb4 expression (89%, n18; Fig. 2O). Thus, excess Crabp2a also reduces robustness. varied up to 10-fold in each case (Fig. 3F-I; supplementary material Figs S7, S8). For both increases (2-, 5-, 10-fold; Fig. 3F) and decreases (2-, 5-, 10-fold; supplementary material Fig. RESULTS AND DISCUSSION A novel requirement for Crabps in Hox gene expression S9) in RA synthesis, E values were consistently lower in models that included Crabps and showed more simulations with extremely low E values (E0-0.25) (Fig. 3F-I; supplementary material Fig. S9B-D). A computational model predicts crucial roles for Crabps in robustness We modeled the effects of varying Crabp levels using randomly generated parameter sets with 5-fold increases or decreases in RA synthesis rate and simulated for 20 different Crabp synthesis rates Our previous computational model for RA signaling in the zebrafish hindbrain was based on evidence that RA induces (and Fgf inhibits) Cyp26a1 expression, thereby creating a feedback system of RA degradation (White et al., 2007). This system cannot explain the robustness over a 20-fold concentration range observed experimentally (Hernandez et al., 2007; White and Schilling, 2008), suggesting that reversible binding to receptors and binding proteins is crucial. To test this hypothesis, we expanded our model to include equations that model RA interactions with RARs ([R] in models) and Crabps ([BP]) (supplementary material Appendix S1; Fig. 3A). Our model: (1) represents these in a reaction-diffusion system in one spatial dimension (the A-P axis); (2) assumes that extracellular RA diffuses; and (3) is solved at the steady state. The new model captures receptor (ron/off) and/or binding protein (mon/off) saturation by RA. RA-BP and RA-BP-RAR interactions (jA,B) have been demonstrated (Dong et al., 1999), but there is no direct evidence that RA-BP forms a complex with Cyp26, so we used RAbpdeg to represent Crabp-assisted RA degradation. Fig. 2. crabp2a is RA inducible and required for signal robustness. (A-F)Whole-mount ISH for crabp2a/b expression at 15 hpf; dorsal views, anterior left. crabp2a (A-C) is expressed posterior to the R5/6 boundary, whereas crabp2b (D-F) expression is restricted to somites. Treatment with 10 nM RA expands crabp2a expression anteriorly throughout the CNS (B) but has no effect on crabp2b (E). Treatment with 5mM DEAB eliminates crabp2a expression (C) but does not disrupt crabp2b (F). (G-R)pax2a, krox20 and hoxb4 expression at 18 hpf in uninjected controls (G-I), Crabp2a-MO-injected (J-L) and crabp2a mRNA-injected embryos (M-R) treated with 1 nM RA (H,K), 10 nM RA (I,L,N,Q) or 10mM DEAB (O,R). so, somites. To study robustness characteristics, we sampled randomly generated parameter sets (supplementary material Appendix S1), comparing two values for the RA synthesis rate. We quantified the change in the RA gradient with respect to a fold-change in the RA synthesis of a ‘reference’ gradient, to define a robustness index designated ‘E’ (Fig. 3B). E is the normalized mean horizontal shift in two gradients between two thresholds y1 and y2 (representing 20% and 80% of the maximum reference value, respectively) when RA synthesis (or any other parameter) is varied. A computational model predicts crucial roles for Crabps in robustness (A)Schematic of RA signaling in one cell illustrating model components: RA (red), Crabps (purple crescents), Cyp26s associated with endoplasmic reticulum (ER, blue), RA-degradation products ([deg], red dots). Green arrows indicate components induced by RA and red inhibitory symbols indicate components repressed by RA. (B)A robustness index (E) utilized: ∫ = Δ = + = + E x y y y x dy y y y y y y y y 1 ( ) 1 – , 0.2( – ) , 0.8( – ) , y y ref ref 1 2 1 2 1 min min 2 min min 1 2 ∫ = Δ = + = + E x y y y x dy y y y y y y y y 1 ( ) 1 – , 0.2( – ) , 0.8( – ) , y y ref ref 1 2 1 2 1 min min 2 min min 1 2 ∫ = Δ = + = + E x y y y x dy y y y y y y y y 1 ( ) 1 – , 0.2( – ) , 0.8( – ) , (2 y y ref ref 1 2 1 2 1 min min 2 min min 1 2 (2) where ymin is the minimum value and yref(xref) is an imposed maximum value of the reference gradient. (C-E)Three examples of calculated gradients very similar (C) or different (D,E) from the reference gradient (red line) across the hindbrain primordium (x-axis) and corresponding E values. (F-I)Probability density distributions (percentages, y-axis) of E values (x-axis) for models that either include binding proteins (solid lines) or do not (dashed lines) for models with a 2-fold (black lines, G), 5-fold (blue lines, H) or 10-fold (red lines, I) increase in [RA]out synthesis rate. [Vbp] (supplementary material Fig. S10). Synthesis rates were obtained by logarithmically dividing its parameter range (supplementary material Appendix S3). The lowest E values corresponded to an intermediate level of Vbp in both cases. This suggests an optimal range of Crabp concentration, above or below which robustness is compromised. To examine this in more detail, E value distributions were generated for simulations with a 5-fold increase in RA synthesis rate coupled with either a 5- or 10-fold increase or a 2-fold decrease in the Crabp synthesis rate Vbp (Fig. 4A). A computational model predicts crucial roles for Crabps in robustness Smaller E values denote higher similarity between the two gradients and therefore a more robust system (Fig. 3C-E). This integrates spatial information across the entire signaling gradient as opposed to quantifying robustness with respect to a threshold value at one location. Parameter space sampling combined with a robustness index dramatically improves comparisons between systems because a representative distribution of E values is produced for each one. T tif i fl f C b RA i l b t Fig. 2. crabp2a is RA inducible and required for signal Fig. 2. crabp2a is RA inducible and required for signal robustness. (A-F)Whole-mount ISH for crabp2a/b expression at 15 hpf; dorsal views, anterior left. crabp2a (A-C) is expressed posterior to the R5/6 boundary, whereas crabp2b (D-F) expression is restricted to somites. Treatment with 10 nM RA expands crabp2a expression anteriorly throughout the CNS (B) but has no effect on crabp2b (E). Treatment with 5mM DEAB eliminates crabp2a expression (C) but does not disrupt crabp2b (F). (G-R)pax2a, krox20 and hoxb4 expression at 18 hpf in uninjected controls (G-I), Crabp2a-MO-injected (J-L) and crabp2a mRNA-injected embryos (M-R) treated with 1 nM RA (H,K), 10 nM RA (I,L,N,Q) or 10mM DEAB (O,R). so, somites. Fig. 2. crabp2a is RA inducible and required for signal robustness. (A-F)Whole-mount ISH for crabp2a/b expression at 15 hpf; dorsal views, anterior left. crabp2a (A-C) is expressed posterior to the R5/6 boundary, whereas crabp2b (D-F) expression is restricted to somites. Treatment with 10 nM RA expands crabp2a expression anteriorly throughout the CNS (B) but has no effect on crabp2b (E). Treatment with 5mM DEAB eliminates crabp2a expression (C) but does not disrupt crabp2b (F). (G-R)pax2a, krox20 and hoxb4 expression at 18 hpf in uninjected controls (G-I), Crabp2a-MO-injected (J-L) and crabp2a mRNA-injected embryos (M-R) treated with 1 nM RA (H,K), 10 nM RA (I,L,N,Q) or 10mM DEAB (O,R). so, somites. DEVELOPMENT 2153 RESEARCH REPORT Crabps and retinoid signaling Fig. 3. Computational models indicate essential roles for Crabps in robustness. (A)Schematic of RA signaling in one cell illustrating model components: RA (red), Crabps (purple crescents), Cyp26s associated with endoplasmic reticulum (ER, blue), RA-degradation products ([deg], red dots). Green arrows indicate components induced by RA and red inhibitory symbols indicate components repressed by RA. (B)A robustness index (E) utilized: Fig. 3. Computational models indicate essential roles for Crabps in robustness. A computational model predicts crucial roles for Crabps in robustness E value distributions were lowest with a 5-fold increase in Vbp, with many E values close to zero (Fig. 4A). Similarly, E value distributions for simulations with a 5-fold decrease in RA synthesis rate were lower when Vbp was increased (Fig. 4B; supplementary material Fig. S11). These simulations show that proportional changes in Crabp and RA synthesis produce the best robustness. of crabp2a ‘rescue’ mRNA and treating the embryos with 5 nM exogenous RA. This relatively low amount of RA reduces krox20 expression in R3 in wild types (mild), whereas in Crabp2a-depleted embryos R3 expression is lost and R5 is reduced (severe). Co- injection of 100-200 pg/embryo crabp2a mRNA partially rescued these phenotypes, whereas 25-50 pg had little effect and higher amounts (>300 pg) increased the severity of the phenotype (Fig. 4C; supplementary material Fig. S4). DEVELOPMENT References Allenby, G., Janocha, R., Kazmer, S., Speck, J., Grippo, J. F. and Levin, A. A. (1994). Binding of 9-cis-retinoic acid and all-trans-retinoic acid to retinoic acid receptors alpha, beta, and gamma. Retinoic acid receptor gamma binds all- trans-retinoic acid preferentially over 9-cis-retinoic acid. J. Biol. Chem. 269, 16689-16695. Fig. 4. Robustness gains require Crabp-mediated RA degradation. Fig. 4. Robustness gains require Crabp-mediated RA degradation. (A,B)E value distributions for models with either a 5-fold increase (A) or decrease (B) of RA synthesis rate with respect to 10- (green), 5- (magenta), or 2-fold (red) increases in Vbp. (C)Percentages (x-axis) of wild-type (blue bars), mild (red bars), moderate (green bars) and severe (black bars) phenotypes resulting from injection of different amounts of Crabp2a mRNA per embryo (y-axis) treated with 5 nM RA. (D,E)Simulations of 5-fold increases in RA synthesis rate in which binding proteins: (1) have all three mechanisms; (2) do not transport RA to receptors (jB0); (3) do not sequester RA (jA0); (4) do not transport RA to Cyp26 degradation (rabpdeg0); (5) must bind RA to allow degradation (radeg0); and (6) are absent. Fig. 4. Robustness gains require Crabp-mediated RA degradation. (A,B)E value distributions for models with either a 5-fold increase (A) or decrease (B) of RA synthesis rate with respect to 10- (green), 5- (magenta), or 2-fold (red) increases in Vbp. (C)Percentages (x-axis) of wild-type (blue bars), mild (red bars), moderate (green bars) and severe (black bars) phenotypes resulting from injection of different amounts of Crabp2a mRNA per embryo (y-axis) treated with 5 nM RA. (D,E)Simulations of 5-fold increases in RA synthesis rate in which binding proteins: (1) have all three mechanisms; (2) do not transport RA to receptors (jB0); (3) do not sequester RA (jA0); (4) do not transport RA to Cyp26 degradation (rabpdeg0); (5) must bind RA to allow degradation (radeg0); and (6) are absent. Aström, A., Tavakkol, A., Pettersson, U., Cromie, M., Elder, J. T. and Voorhees, J. J. (1991). Molecular cloning of two human cellular retinoic acid- binding proteins (CRABP). Retinoic acid-induced expression of CRABP-II but not CRABP-I in adult human skin in vivo and in skin fibroblasts in vitro. J. Biol. Chem. 266, 17662-17666. Aström, A., Pettersson, U., Chambon, P. and Voorhees, J. J. (1994). References Retinoic acid induction of human cellular retinoic acid-binding protein-II gene transcription is mediated by retinoic acid receptor-retinoid X receptor heterodimers bound to one far upstream retinoic acid-responsive element with 5-base pair spacing. J. Biol. Chem. 269, 22334-22339. g Begemann, G., Schilling, T. F., Rauch, G. J., Geisler, R. and Ingham, P. W. (2001). The zebrafish neckless mutation reveals a requirement for raldh2 in mesodermal signals that pattern the hindbrain. Development 128, 3081-3094. Ben-Zvi, D. and Barkai, N. (2010). Scaling of morphogen gradients by an expansion-repression integral feedback control. Proc. Natl. Acad. Sci. USA 107, 6924-6929. Crabps bind RA and prevent interactions with receptors, jA0 sequestering RA as [RA-BP] and allowing less [RA-R] (supplementary material Figs S12, S13); and (3) Crabps transport RA for degradation by Cyp26s. These roles are not mutually exclusive. Boylan, J. F. and Gudas, L. J. (1991). Overexpression of the cellular retinoic acid binding protein-I (CRABP-I) results in a reduction in differentiation-specific gene expression in F9 teratocarcinoma cells. J. Cell Biol. 112, 965-979. Boylan, J. F. and Gudas, L. J. (1992). The level of CRABP-I expression influences the amounts and types of all-trans-retinoic acid metabolites in F9 teratocarcinoma stem cells. J. Biol. Chem. 267, 21486-21491. To test each role computationally, we calculated E values for models incorporating different combinations (Fig. 4D,E): (1) all three functions; (2) only sequestration and degradation; (3) transport to receptors and degradation; (4) transport to receptors and sequestration; (5) degradation only when bound to RA (RAdeg0); and (6) no binding proteins present. Simulations with either 5-fold increases (Fig. 4D,E) or decreases (supplementary material Fig. S14) in RA synthesis rate revealed that combined positive and negative roles were most robust, particularly a model incorporating all three roles. By contrast, a system without sequestration was least robust. The receptor/sequestration model (Fig. 4E) was less robust than the dual sequestration/degradation model, suggesting that transport to Cyp26s is crucial for robustness in the RA system. Budhu, A., Gillilan, R. and Noy, N. (2001). Localization of the RAR interaction domain of cellular retinoic acid binding protein-II. J. Mol. Biol. 305, 939-949. Budhu, A. S. and Noy, N. (2002). Direct channeling of retinoic acid between cellular retinoic acid-binding protein II and retinoic acid receptor sensitizes mammary carcinoma cells to retinoic acid-induced growth arrest. Mol. Cell. Biol. 22, 2632-2641. DEVELOPMENT Chen, A. C., Yu, K., Lane, M. A. and Gudas, L. J. (2003). Acknowledgements h k h Acknowledgements We thank Lei Zhang, Tailin Zhang and Ines Gehring for technical assistance. Acknowledgements We thank Lei Zhang, Tailin Zhang and Ines Gehring for technical assistance. Competing interests statement The authors declare no competing financial interests. Supplementary material Supplementary material available online at http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.077065/-/DC1 Funding g This study was supported by The National Institutes of Health [P50 GM-76516 to K.R., A.D.L., Q.N., T.F.S.; R01 GM-67247 to Q.N.; R01 NS-41353 to T.F.S.]; and the National Science Foundation [DMS-0917492 to Q.N.]. Deposited in PMC for release after 12 months. g This study was supported by The National Institutes of Health [P50 GM-76516 to K.R., A.D.L., Q.N., T.F.S.; R01 GM-67247 to Q.N.; R01 NS-41353 to T.F.S.]; and the National Science Foundation [DMS-0917492 to Q.N.]. Deposited in PMC for release after 12 months. Competing interests statement The authors declare no competing financial interests. Models suggest that Crabps facilitate RA degradation Crabp2 in mice has a putative nuclear localization signal and can form a complex with RA and RARs, while Crabp1 enhances the formation of RA degradation products in vitro (Dong et al., 1999). Although our model does not distinguish between different Crabps, it can address questions regarding how positive or negative roles influence gradient robustness. We hypothesize three such roles: (1) Crabps transport RA to receptors, jA in our models (Fig. 3A); (2) Based on these models, we tested the concentration range over which Crabp2a promotes optimal robustness in the hindbrain experimentally, by co-injecting Crabp2a-MO with a range of doses 2154 RESEARCH REPORT Development 139 (12) Development 139 (12) Fig. 4. Robustness gains require Crabp-mediated RA degradation. (A,B)E value distributions for models with either a 5-fold increase (A) or decrease (B) of RA synthesis rate with respect to 10- (green), 5- (magenta), or 2-fold (red) increases in Vbp. (C)Percentages (x-axis) of wild-type (blue bars), mild (red bars), moderate (green bars) and severe (black bars) phenotypes resulting from injection of different amounts of Crabp2a mRNA per embryo (y-axis) treated with 5 nM RA. (D,E)Simulations of 5-fold increases in RA synthesis rate in which binding proteins: (1) have all three mechanisms; (2) do not transport RA to receptors (jB0); (3) do not sequester RA (jA0); (4) do not transport RA to Cyp26 degradation (rabpdeg0); (5) must bind RA to allow degradation (radeg0); and (6) are absent. These results are consistent with Crabps both facilitating interactions between RA and RARs and promoting RA degradation (Fig. 3A) (Dong et al., 1999). They help explain how hindbrain patterning can be robust to a 20-fold range of RA concentrations (Hernandez et al., 2007). They also reveal an essential role for these proteins that can help explain why Crabps are so highly conserved – zebrafish Crabp2a is 74% similar in protein sequence to human CRABP2 (Sharma et al., 2003). The apparent lack of a requirement for Crabps in mice might reflect differential redundancies among fatty acid-binding proteins or differential regulation in placental mammals at the level of maternal and extra- embryonic tissues (Sapin et al., 1997; Zheng and Ong, 1998). References Homozygous deletion of the CRABPI gene in AB1 embryonic stem cells results in increased CRABPII gene expression and decreased intracellular retinoic acid concentration. Arch. Biochem. Biophys. 411, 159-173. Delva, L., Bastie, J. N., Rochette-Egly, C., Kraïba, R., Balitrand, N., Despouy, G., Chambon, P. and Chomienne, C. (1999). Physical and functional interactions between cellular retinoic acid binding protein II and the retinoic acid-dependent nuclear complex. Mol. Cell. Biol. 19, 7158-7167. p p Dong, D., Ruuska, S. E., Levinthal, D. J. and Noy, N. (1999). Distinct roles for cellular retinoic acid-binding proteins I and II in regulating signaling by retinoic acid. J. Biol. Chem. 274, 23695-23698. Crabps and retinoid signaling 2155 RESEARCH REPORT Dupé, V. and Lumsden, A. (2001). Hindbrain patterning involves graded responses to retinoic acid signalling. Development 128, 2199-2208. Rupp, R. A., Snider, L. and Weintraub, H. (1994). Xenopus embryos regulate the nuclear localization of XMyoD. Genes Dev. 8, 1311-1323. p g g p Eldar, A., Rosin, D., Shilo, B. Z. and Barkai, N. (2003). Self-enhanced ligand degradation underlies robustness of morphogen gradients. Dev. Cell 5, 635-646. Sample, C. and Shvartsman, S. Y. (2010). Multiscale modeling of diffusion in the early Drosophila embryo. Proc. Natl. Acad. Sci. USA 107, 10092-10096. Sapin, V., Ward, S. J., Bronner, S., Chambon, P. and Dollé, P. (1997). Differential expression of transcripts encoding retinoid binding proteins and retinoic acid receptors during placentation of the mouse. Dev. Dyn. 208, 199- 210. Fiorella, P. D. and Napoli, J. L. (1994). Microsomal retinoic acid metabolism. Effects of cellular retinoic acid-binding protein (type I) and C18-hydroxylation as an initial step. J. Biol. Chem. 269, 10538-10544. Häcker, U., Nybakken, K. and Perrimon, N. (2005). Heparan sulphate proteoglycans: the sweet side of development. Nat. Rev. Mol. Cell Biol. 6, 530- 541. Sharma, M. K., Denovan-Wright, E. M., Boudreau, M. E. and Wright, J. M. (2003). A cellular retinoic acid-binding protein from zebrafish (Danio rerio): cDNA sequence, phylogenetic analysis, mRNA expression, and gene linkage mapping. Gene 311, 119-128. Hernandez, R. E., Putzke, A. P., Myers, J. P., Margaretha, L. and Moens, C. B. (2007). Cyp26 enzymes generate the retinoic acid response pattern necessary for hindbrain development. Development 134, 177-187. Sharma, M. K., Saxena, V., Liu, R. Z., Thisse, C., Thisse, B., Denovan-Wright, E. M. and Wright, J. M. (2005). Differential expression of the duplicated cellular retinoic acid-binding protein 2 genes (crabp2a and crabp2b) during zebrafish embryonic development. Gene Expr. Patterns 5, 371-379. References Lampron, C., Rochette-Egly, C., Gorry, P., Dollé, P., Mark, M., Lufkin, T., LeMeur, M. and Chambon, P. (1995). Mice deficient in cellular retinoic acid binding protein II (CRABPII) or in both CRABPI and CRABPII are essentially normal. Development 121, 539-548. Thisse, B., Heyer, V., Lux, A., Alunni, V., Degrave, A., Seiliez, I., Kirchner, J., Parkhill, J. P. and Thisse, C. (2004). Spatial and temporal expression of the zebrafish genome by large-scale in situ hybridization screening. Methods Cell Biol. 77, 505-519. Lander, A. D. (2007). Morpheus unbound: reimagining the morphogen gradient. Cell 128, 245-256. Lander, A. D., Nie, Q. and Wan, F. Y. (2007). Membrane-associated non- receptors and morphogen gradients. Bull. Math. Biol. 69, 33-54. Umulis, D. M., Shimmi, O., O’Connor, M. B. and Othmer, H. G. (2010). Organism-scale modeling of early Drosophila patterning via bone morphogenetic proteins. Dev. Cell 18, 260-274. Linville, A., Radtke, K., Waxman, J. S., Yelon, D. and Schilling, T. F. (2009). Combinatorial roles for zebrafish retinoic acid receptors in the hindbrain and pharyngeal arches. Dev. Biol. 325, 60-70. Wartlick, O., Kicheva, A. and González-Gaitán, M. (2009). Morphogen gradient formation. Cold Spring Harb. Perspect. Biol. 1, a001255. Maves, L. and Kimmel, C. B. (2005). Dynamic and sequential patterning of the zebrafish posterior hindbrain by retinoic acid. Dev. Biol. 285, 593-605. White, R. J. and Schilling, T. F. (2008). How degrading: Cyp26s in hindbrain development. Dev. Dyn. 237, 2775-2790. Meinhardt, H. (2009). Models for the generation and interpretation of gradients. Cold Spring Harb. Perspect. Biol. 1, a001362. y White, R. J., Nie, Q., Lander, A. D. and Schilling, T. F. (2007). Complex regulation of cyp26a1 creates a robust retinoic acid gradient in the zebrafish embryo. PLoS Biol. 5, e304. Napoli, J. L. (1996). Biochemical pathways of retinoid transport, metabolism, and signal transduction. Clin. Immunol. Immunopathol. 80, S52-S62. y Won, J. Y., Nam, E. C., Yoo, S. J., Kwon, H. J., Um, S. J., Han, H. S., Kim, S. H., Byun, Y. and Kim, S. Y. (2004). The effect of cellular retinoic acid binding protein-I expression on the CYP26-mediated catabolism of all-trans retinoic acid and cell proliferation in head and neck squamous cell carcinoma. Metabolism 53, 1007-1012. Perz-Edwards, A., Hardison, N. L. and Linney, E. (2001). Retinoic acid-mediated gene expression in transgenic reporter zebrafish. Dev. Biol. 229, 89-101. Romand, R., Sapin, V., Ghyselinck, N. B., Avan, P., Le Calvez, S., Dollé, P., Chambon, P. and Mark, M. (2000). References Spatio-temporal distribution of cellular retinoid binding protein gene transcripts in the developing and the adult cochlea. Morphological and functional consequences in CRABP- and CRBPI-null mutant mice. Eur. J. Neurosci. 12, 2793-2804. Zheng, W. L. and Ong, D. E. (1998). Spatial and temporal patterns of expression of cellular retinol-binding protein and cellular retinoic acid-binding proteins in rat uterus during early pregnancy. Biol. Reprod. 58, 963-970. DEVELOPMENT
https://openalex.org/W3013188179	https://www.frontiersin.org/articles/10.3389/fmicb.2020.00415/pdf	English	null	Comparison of Bacterial DNA Profiles in Mid-Trimester Amniotic Fluid Samples From Preterm and Term Deliveries	Frontiers in microbiology	2,020	cc-by	10,063	ORIGINAL RESEARCH published: 24 March 2020 doi: 10.3389/fmicb.2020.00415 Lisa Stinson1,2†, Maria Hallingström3,4†, Malin Barman5, Felicia Viklund6, Jeffrey Keelan1,2, Marian Kacerovsky7,8, Matthew Payne1,2‡ and Bo Jacobsson3,4,9‡ 1 Division of Obstetrics and Gynaecology, Faculty of Health and Medical Sciences, The University of Western Australia, Crawley, WA, Australia, 2 Women and Infants Research Foundation, Subiaco, WA, Australia, 3 Department of Obstetrics and Gynecology, Sahlgrenska University Hospital, Gothenburg, Sweden, 4 Department of Obstetrics and Gynecology, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden, 5 Food and Nutrition Science, Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden, 6 Stockholm South General Hospital, Stockholm, Sweden, 7 Department of Obstetrics and Gynecology, Faculty of Medicine in Hradec Kralove, University Hospital Hradec Kralove, Charles University, Hradec Kralove, Czechia, 8 Biomedical Research Centre, University Hospital Hradec Kralove, Hradec Kralove, Czechia, 9 Department of Genetics and Bioinformatics, Area of Health Data and Digitalization, Institute of Public Health, Oslo, Norway Keywords: amniotic ﬂuid, bacteria, 16S rRNA, preterm birth, microbial invasion of amniotic cavity Edited by: Correspondence: Lisa Stinson lisa.stinson@uwa.edu.au †These authors have contributed equally to this work and share ﬁrst authorship †These authors have contributed equally to this work and share ﬁrst authorship ‡These authors have contributed equally to this work and share senior authorship Specialty section: This article was submitted to Evolutionary and Genomic Microbiology, a section of the journal Frontiers in Microbiology Received: 22 December 2019 Accepted: 27 February 2020 Published: 24 March 2020 Specialty section: This article was submitted to Evolutionary and Genomic Microbiology, a section of the journal Frontiers in Microbiology Specialty section: This article was submitted to Evolutionary and Genomic Microbiology, a section of the journal Frontiers in Microbiology Received: 22 December 2019 Accepted: 27 February 2020 Published: 24 March 2020 Comparison of Bacterial DNA Proﬁles in Mid-Trimester Amniotic Fluid Samples From Preterm and Term Deliveries Lisa Stinson1,2†, Maria Hallingström3,4†, Malin Barman5, Felicia Viklund6, Jeffrey Keelan1,2, Marian Kacerovsky7,8, Matthew Payne1,2‡ and Bo Jacobsson3,4,9‡ Edited by: Edited by: Andrés Moya, University of Valencia, Spain University of Valencia, Spain Reviewed by: Fabien Magne, University of Chile, Chile Maria Dzunkova, Joint Genome Institute, United States Correspondence: Lisa Stinson lisa.stinson@uwa.edu.au †These authors have contributed equally to this work and share ﬁrst authorship ‡These authors have contributed equally to this work and share senior authorship Reviewed by: Fabien Magne, University of Chile, Chile Maria Dzunkova, Joint Genome Institute, United States *Correspondence: Lisa Stinson lisa.stinson@uwa.edu.au †These authors have contributed equally to this work and share ﬁrst authorship ‡These authors have contributed equally to this work and share senior authorship Reviewed by: Fabien Magne, University of Chile, Chile Maria Dzunkova, Joint Genome Institute, United States Infection and inﬂammation are well recognized causes of spontaneous preterm delivery (PTD) (<37 gestational weeks) and adverse infant outcomes. To date, there has been very little investigation into bacterial communities in amniotic ﬂuid using next generation sequencing technology. In particular, it is important to characterize amniotic ﬂuid bacterial proﬁles in complicated pregnancies as well as in asymptomatic women to identify predictive bacterial DNA signatures. Here, 1198 mid-trimester amniotic ﬂuid samples from a cohort of Swedish women undergoing mid-trimester genetic amniocentesis were screened for bacterial DNA using qPCR protocols speciﬁcally designed to reduce the impacts of reagent contamination and human DNA mispriming. The majority of samples were devoid of detectable bacterial DNA; however, approximately a ﬁfth of the cohort (19.9%) were 16S rRNA gene positive in duplicate screening. Among these, nine women had a spontaneous PTD. These nine women were matched with 18 healthy women with a delivery at term. We used PacBio SMRT technology, coupled with appropriate negative extraction and PCR controls, to sequence the full-length 16S rRNA gene in this subset of 27 women. The amniotic ﬂuid samples contained low-abundance and low-diversity bacterial DNA proﬁles. Species typically associated with spontaneous PTD were absent. We were not able to identify any differences in the amniotic ﬂuid bacterial DNA proﬁles of women with a subsequent spontaneous PTD compared to women who delivered at term. The ﬁndings suggest that, in a minor proportion of pregnancies, DNA from non-pathogenic bacteria may be present in the amniotic ﬂuid far earlier than previously reported. Early detection of bacterial DNA in the amniotic ﬂuid was, in this study, not associated with spontaneous PTD. BACKGROUND The existence of an amniotic ﬂuid microbiome is contentious; however, there is some evidence to suggest that bacterial DNA is commonly found in amniotic ﬂuid at delivery (Collado et al., 2016; Urushiyama et al., 2017; Wang et al., 2018; Stinson et al., 2019a). It is not known when in gestation bacteria may ﬁrst be able to access the amniotic space. Apart from three 16S rRNA gene sequencing studies (Urushiyama et al., 2017; Kayem et al., 2018; Zhu et al., 2018), studies of microbial proﬁles in mid-trimester amniotic ﬂuid to date have largely relied on bacterial culture and targeted real-time PCR, and have primarily focused on identifying pathogenic bacteria involved in the etiology of spontaneous PTD (Watts et al., 1992; Bearﬁeld et al., 2002; Payne et al., 2014; Rowlands et al., 2017; Morimoto et al., 2018). High quality data on the full microbial ecology of mid-trimester amniotic ﬂuid is lacking, making it diﬃcult to speculate on the extent to which bacterial DNA is found in amniotic ﬂuid at this time point. Importantly, by comparing bacterial proﬁles in amniotic ﬂuid samples from women with a spontaneous PTD and those with a term delivery, we may be able to identify a predictive microbiome signature of high- risk pregnancies. The existence of a fetal or in utero microbiome (an ecosystem of interacting microorganisms) in normal pregnancy remains controversial (Collado et al., 2016; Stinson et al., 2016; de Goﬀau et al., 2019). Numerous studies have investigated bacterial colonization of the amniotic ﬂuid in both complicated and healthy pregnancies using culture-dependant and culture- independent techniques. These studies have reported a diverse range of results, with some ﬁnding that amniotic ﬂuid was devoid of bacteria (Rowlands et al., 2017), some ﬁnding that all amniotic ﬂuid samples contain bacteria (Collado et al., 2016; Urushiyama et al., 2017; Wang et al., 2018; Zhu et al., 2018), and yet others reporting a combination of positive and negative samples (Watts et al., 1992; Mandar et al., 2001; Bearﬁeld et al., 2002; Mendz et al., 2013; Cobo et al., 2017; Kayem et al., 2018; Lim et al., 2018; Morimoto et al., 2018; Stinson et al., 2019a). BACKGROUND While pathogenic colonization of the amniotic ﬂuid has been well-documented and thoroughly studied in the context of spontaneous preterm delivery (PTD; <37 gestational weeks) and fetal infection (DiGiulio, 2012; Mendz et al., 2013; Stinson and Payne, 2019; Thorell et al., 2019), little is known about the nature, origins, and signiﬁcance of any endemic amniotic ﬂuid bacteria in normal pregnancies. In particular, the timing and stability of such colonization remains largely unexplored. Such information is important, as bacterial populations in the amniotic ﬂuid are likely to inﬂuence fetal skin and gut colonization (via swallowing of the amniotic ﬂuid which begins at the end of the ﬁrst trimester) which could have important implications for the development and maturation of the fetal immune system. There is currently a need for robust, well-controlled studies of the bacterial content of amniotic ﬂuid in normal and high- risk pregnancies, particularly one which employs full-length 16S rRNA gene sequencing and appropriate measures to reduce external contamination so that low biomass microbiota can be described with conﬁdence. In the present study, we characterized the bacterial DNA proﬁles of amniotic ﬂuid collected during mid- trimester genetic amniocentesis from women with no clinical evidence of infection that went on to deliver at term or by spontaneous PTD. To date, there has been limited characterization of bacterial populations in amniotic ﬂuid from uncomplicated pregnancies using next generation sequencing (Collado et al., 2016; Urushiyama et al., 2017; Kayem et al., 2018; Lim et al., 2018; Wang et al., 2018; Zhu et al., 2018; Stinson et al., 2019a). Unfortunately, most previous studies are complicated by the fact that appropriate and stringent extraction and PCR controls were not used. None of these studies attempted to eliminate reagent-derived contamination using laboratory- based techniques, such as those previously described by our group (Stinson et al., 2018). This makes it diﬃcult to make conclusions on the true level of bacterial DNA in their samples. Apart from being inadequately controlled, these studies have all relied on short 16S rRNA gene amplicons with diﬀerent variable regions for their sequencing. While such an approach is somewhat useful for broad community proﬁling without detailed taxonomic information, it is not able to give accurate species level assignment (Martinez-Porchas et al., 2016). Additionally, the selection of the region of the 16S rRNA gene to be sequenced can introduce bias into the results (Cruaud et al., 2014; Tremblay et al., 2015). Citation: Stinson L, Hallingström M, Barman M, Viklund F, Keelan J, Kacerovsky M, Payne M and Jacobsson B (2020) Comparison of Bacterial DNA Proﬁles in Mid-Trimester Amniotic Fluid Samples From Preterm and Term Deliveries. Front. Microbiol. 11:415. doi: 10.3389/fmicb.2020.00415 March 2020 \| Volume 11 \| Article 415 1 Frontiers in Microbiology \| www.frontiersin.org Bacterial Proﬁles in Mid-Trimester AF Stinson et al. BACKGROUND Furthermore, some of these studies used QIIME v1 for operational taxonomic unit (OTU) clustering and taxonomic assignment, a platform which has been shown to give inaccurate taxonomic assignments and to inﬂate the number of OTUs in a sample when the default settings are used (Edgar, 2017). Frontiers in Microbiology \| www.frontiersin.org Cohort Selection the immediately preceding and following women in the cohort (Figure 1), according to the following criteria: 1. Parity (primiparous or multiparous), 1. Parity (primiparous or multiparous), 2. BMI (matched according to WHO deﬁnitions of BMI categories: underweight (<18.5); normal weight (18.5– 24.9); pre-obesity (25.0–29.9); and obesity class I–III (≥30.0), 3. Smoking (yes/no), 4. IVF (yes/no), 5. Ethnicity [categorized according to Burchard et al. (2003): Black or African American (Africans); White (Caucasians); and Asian]. Sample Collection Three milliliter of amniotic ﬂuid was collected for research purposes (in addition to that collected for diagnostic use) during mid-trimester genetic amniocentesis performed at 14– 20 gestational weeks at Sahlgrenska University Hospital/Östra, Gothenburg, Sweden. Amniotic ﬂuid samples were immediately stored at 4–8◦C. The samples were centrifuged at 12,000 × g for 20 min at 4◦C to pellet the cells. Pellets were resuspended in 1 mL Cohort Selection Between September 2008 and July 2017, 3126 women underwent a mid-trimester genetic amniocentesis at Sahlgrenska University Hospital/Östra, Gothenburg, Sweden (Figure 1). Amniocentesis was performed per clinical indications such as advanced maternal age, anxiety, abnormal ﬁrst-trimester combined screening or family history of chromosomal abnormalities or genetic diseases. Women ≥18 years of age with a viable singleton pregnancy were eligible. Women with multiple pregnancy, infectious diseases (such as HIV or hepatitis B), who had known or suspected fetal malformations as well as women undergoing amniocentesis during times when study samples could not be collected were considered ineligible (n = 763). Women who declined participation, who had language diﬃculties and therefore could not provide informed consent in Swedish, or if an insuﬃcient amount of ﬂuid was retrieved during amniocentesis, were considered excluded (n = 1148). The remaining 1215 women were enrolled into the study. 16S rRNA gene data was retrieved from 1198 women, of which 238 women tested positive in the qPCR screen. Among these, nine healthy women had a spontaneous PTD and they were matched with women who delivered in gestational weeks 38+0 to 41+6 to a 1:2 ratio using March 2020 \| Volume 11 \| Article 415 Frontiers in Microbiology \| www.frontiersin.org 2 Stinson et al. Bacterial Proﬁles in Mid-Trimester AF FIGURE 1 \| Flow chart of the cohort and selection of cases and controls. E 1 \| Flow chart of the cohort and selection of cases and controls sterile 1× PBS and frozen at −80◦C until processing. Samples were initially shipped on dry ice to the Institute of Molecular Biomedicine, Comenius University, Bratislava, Slovakia, where 200 µL of cell pellet resuspension from each sample was aspirated under aseptic conditions for downstream analyses unrelated to the current study. These samples were again frozen at −80◦C and shipped on dry ice to the Division of Obstetrics and Gynaecology, The University of Western Australia, Crawley, WA, Australia, for microbial proﬁling analyses used in the current study. Other samples were sent directly (on dry ice) to the Division of Obstetrics and Gynaecology, The University of Western Australia, Crawley, WA, Australia from Gothenburg, Sweden. These had thus not been thawed. There was no diﬀerence in the downstream 16S rRNA gene qPCR results between the samples that were shipped initially to Slovakia and those that were shipped directly to Australia. qPCR Screening The 1198 samples were screened for bacterial DNA using a custom Taqman qPCR protocol, adapted from Yang et al. (2002) with a new forward primer to improve coverage of Ureaplasma spp. and Mycoplasma spp. The V6 region of the 16S rRNA gene was ampliﬁed in 20 µL reactions containing 6 µL of template or water (negative template control), 1× TaqMan Fast Advanced Master Mix (Applied Biosystems), 0.9 µM each of the forward (917F 5′-GAATTGACGGGGRCCCGC-3′) and reverse (1033R 5′-TGCGGGACTTAACCCAACA-3′) primers, 0.25 µM of probe (5′-FAM-CACGAGCTGACGACARCCATGCA-TAMRA-3′), p y g g To obtain barcoded 16S rRNA gene amplicons, ampliﬁcation was carried out in two steps. The ﬁrst PCR was carried out in 50 µL reactions containing 7.5 µL of template or water (negative template control), 1× AccuStart II ToughMix (Quantabio), 0.3 µM each of the forward and reverse primers, 1.25 µL each of the ArcticZymes dsDNase and DTT, and 13.5 µL of water. The PCR ampliﬁcation program consisted of an initial heating step at 94◦C for 3 min; 40 cycles of 94◦C for 30 s, 55◦C for 30 s, and 72◦C for 2 min; and a ﬁnal extension step of 72◦C for 7 min. PCR reactions were performed in a Veriti Thermal Cycler (Applied Biosystems). PCR products were visualized on a QIAXcel automated electrophoresis system using a DNA high resolution gel cartridge (run parameters OM500) to conﬁrm the presence and size of amplicons. and 0.95 µL of water. Master mix solutions were treated with a PCR Decontamination Kit (ArcticZymes R⃝), which consisted of a double-stranded DNase (dsDNase) and DTT (which helps to inactivate the dsDNase), as previously described (Stinson et al., 2018). Brieﬂy, master mix solutions (including primers and probes) were treated with 0.5 µl of dsDNase and 0.5 µl of DTT per 20 µl reaction, then incubated at 37◦C for 20 min (dsDNase activation), followed by incubation at 60◦C for 20 min (dsDNase inactivation). This treatment eliminated ampliﬁcation of background microbial DNA, preventing negative template controls from reading as positive, and allowing positive/negative calls to be made conﬁdently. Each qPCR was run with three negative template controls and a 1 ng human DNA control (which reﬂected the average total DNA assay input per sample – combined microbial and human), as human DNA is a known confounder in low biomass 16S rRNA gene studies due to 16S rRNA gene primers/probes binding to human gDNA with varying aﬃnity (Kommedal et al., 2012). Ampliﬁcation and Barcoding KingFisher Flex platform as per manufacturer’s instructions. One blank extraction control (EC) was included in the center of each 96 deep-well plate. Ampliﬁcation and Barcoding Amniotic ﬂuid samples from nine women with subsequent spontaneous PTD and 18 matched controls with a delivery at term (all of which were positive for bacterial DNA by qPCR screen) were selected for full-length 16S rRNA gene sequencing. The primers 27F (5′-gcagtcgaacatgtagctgactcaggtcacAGRGTTY GATYMTGGCTCAG-3′) and 1492R (5′-tggatcacttgtgcaagcat cacatcgtagRGYTACCTTGTTACGACTT-3′) were used, with universal UNITAG sequences (in lower case) and a 5′ amine (NH4-C6) block attached to each primer. A set of three barcoded UNITAG-F and 15 barcoded UNITAG-R primers were designed to generate PacBio sequencing-ready amplicons, using an asymmetric barcoding strategy. All primers were synthesized and HPLC-puriﬁed by Integrated DNA Technologies. qPCR Screening For a sample to be called positive, it had to have a cycle threshold (Ct) value at least 1 cycle less than the 1 ng human DNA control that was included on the respective run. Utilizing this conservative approach, we were able to account for mis-priming with human DNA on a run-by-run basis. Samples were screened in duplicate to conﬁrm the ﬁndings, and only samples that produced positive results for both replicates were considered positive. All samples were run on a ViiA7 real-time PCR system (Life Technologies). Primary PCR products were puriﬁed using NucleoMag NGS magnetic beads (Macherey-Nagel), normalized to 1 ng/µL, then used as template in a secondary, nested PCR, in order to generate asymmetrically barcoded amplicons. Secondary PCR was carried out in 25 µL reactions containing 2 µL of template or water (negative template control), 1× AccuStart II ToughMix, 0.3 µM each of the forward and reverse primers, and 3 µL of water. The PCR ampliﬁcation program consisted of an initial heating step at 94◦C for 3 min; 10 cycles of 94◦C for 30 s, 55◦C for 30 s, and 72◦C for 2 min; and a ﬁnal extension step of 72◦C for 7 min. PacBio Sequencing Barcoded 16S rRNA gene amplicons obtained from the secondary PCR were pooled in equimolar concentrations based on QIAXcel quantitation of the target band. The pool was then concentrated using NucleoMag NGS magnetic beads and eluted in 50 µL of 1× TE. The pool was next visualized in a 1.2% agarose gel using SYBR Safe DNA stain (Invitrogen) and bands of the appropriate size were excised using a sterile disposable scalpel. Excised bands were puriﬁed using a QIAquick Gel Extraction Kit as per manufacturer’s instructions. 500 ng of puriﬁed DNA was used for library preparation at the Ramaciotti Centre for Genomics, University of NSW, Sydney, NSW, Australia. Here, SMRTbell adapters were ligated onto barcoded PCR products, and the library was sequenced on a PacBio Sequel system on a single SMRT cell. Sequence ﬁles were deposited to NCBI Sequence Read Archive under accession number PRJNA602788. DNA Extraction Amniotic ﬂuid suspensions were centrifuged at 40,000 × g for 5 min at 4◦C to pellet cells. Cell pellets were resuspended in 353 µL Buﬀer MBL/RNase A solution (QIAGEN) and DNA was extracted using a QIAGEN MagAttract Microbial DNA Kit on the March 2020 \| Volume 11 \| Article 415 Frontiers in Microbiology \| www.frontiersin.org 3 Bacterial Proﬁles in Mid-Trimester AF Stinson et al. Maternal and Neonatal Characteristics Maternal and Neonatal Characteristics The medical and demographic details of the women being screened for bacterial DNA are presented in Table 1. Cases of missing data reﬂect women being lost to follow-up, women who had an abortion or miscarriage, and women who were excluded post-enrollment. We are not the ﬁrst to screen amniotic ﬂuid samples from mid- trimester using universal 16S rRNA gene primers. Two previous such studies have reported that 0 and 3% of samples were positive for bacterial DNA (Rowlands et al., 2017; Kayem et al., 2018). Neither of these studies, however, reported any details of controls used and in the case of Rowlands et al. (2017), who used a qPCR assay, it is unclear if samples were truly negative or if Ct cut- oﬀvalue for a negative result was set based upon ampliﬁcation present in negative PCR controls (a common practice used for distinguishing “positive” samples from background noise). Without the use of decontaminated reagents, negative controls frequently appear at Cts similar to those of amniotic ﬂuid in 16S rRNA gene qPCR screening tests, making it incredibly diﬃcult to determine a true positive result. Data Analysis All qPCR data were analyzed using QuantStudioTM Real-Time PCR Software, version 3.1 (Thermo Fisher Scientiﬁc) using default settings (including both threshold and baseline set to AUTO) to generate Ct values. Diﬀerences between characteristics of subjects with positive or negative 16S rRNA gene results and diﬀerences between cases and controls were analyzed with Mann–Whitney U test for continuous variables, due to non- parametric distribution of the variables, and with χ2 test or Fishers exact test (if the expected value in any cell were 5 or less) for dichotomous variables. Analyses were performed with IBM SPSS Statistics, version 25). Sequence Data Analysis However, the absence of detectable bacterial DNA in the majority of samples suggests that our sample collection protocol did not introduce ubiquitous contamination. load was not quantiﬁed in this study, previous reports suggest that amniotic ﬂuid contains 100–1000 16S rDNA copies/mL (Urushiyama et al., 2017; Lim et al., 2018). Considering that there is substantial 16S rRNA gene copy number variation within the genomes of diﬀerent bacterial species however, these estimates do not correlate with CFU/mL and actual bacterial cell numbers may be as low as 1/15th of these values, depending on the bacterial species present (Kembel et al., 2012). Regardless, these data emphasize the low biomass nature of this sample type. In the present study, the average Ct value for the 16S rRNA gene positive samples was 34.1 (range: 28.5–43.9). Using a previously described enzymatic decontamination protocol (Stinson et al., 2018), we were able to produce completely negative extraction and PCR controls in our qPCR screen, and this, combined with the use of a biologically relevant (in terms of quantity) human DNA control with each run to account for any potential mis-priming, allowed us to make positive/negative calls with a higher level of sensitivity and accuracy. It should be noted that we did not collect negative controls during sampling, and so we are not able to assess the possibility of contamination during sample collection and initial processing, a limitation that is common in most microbiome studies. However, the absence of detectable bacterial DNA in the majority of samples suggests that our sample collection protocol did not introduce ubiquitous contamination. Ureaplasma Screening Ureaplasma Screening Samples that tested positive for bacterial DNA by 16S rRNA gene qPCR (n = 238) were further screened for Ureaplasma parvum and Ureaplasma urealyticum DNA. Reactions consisted of 6 µl of template or water (negative template control), 1× TaqMan Fast Advanced Master Mix (Applied Biosystems), 0.9 µM of each of the forward (5′-AAGGTCAAGGTATGGAAGATCCAA-3′) and reverse (5′-TTCCTGTTGCCCCTCAGTCT-3′) primers, 0.25 µM of the U. parvum and U. urealyticum probes (UU-parvo: 5′-FAM-TCCACAAGCTCCAGCAGCAATTTG-BHQ1-3′ and UU-T960: 5′-VIC-ACCACAAGCACCTGCTACGATTTGTTC- BHQ1-3′), and water to a ﬁnal volume of 20 µL. Positive controls consisting of puriﬁed U. parvum and U. urealyticum DNA were included in each batch. March 2020 \| Volume 11 \| Article 415 Frontiers in Microbiology \| www.frontiersin.org 4 Bacterial Proﬁles in Mid-Trimester AF Stinson et al. TABLE 1 \| Pregnancy demographic characteristics of the cohort (n = 1198). Characteristic Data available for n Mean (range) or n (%) Maternal age (y) 1198 36.4 (20–47) Nulliparity 1194 319 (27%) Preterm delivery 1159 78 (7%) GA at delivery (weekday) 1159 39+4 (22+1–43+0) GA at sampling (weekday) 1193 15+6 (13+6–22+1) BMI at ﬁrst prenatal visit 1171 24.5 (15.8–47.4) Smoking at ﬁrst prenatal visit 1189 62 (5.2%) Diabetes type 2 1198 4 (0.33%) Gestational diabetes 1198 15 (1.3%) Male/female fetus ratio 1160 592/606 (49.4/50.6%) Values are reported as mean (range) or n (percent). TABLE 1 \| Pregnancy demographic characteristics of the cohort (n = 1198). Sequence Data Analysis PacBio raw reads were processed using the SMRT Link Analysis software version 6.0 to obtain demultiplexed circular consensus sequence (CCS) reads with a minimum of three full passes and 99.9% sequence accuracy. Sequence data were processed using the software package GHAP v2.1. GHAP is an in-house amplicon processing pipeline developed by Paul Greenﬁeld (CSIRO, Australia) (Greenﬁeld, 2017) built around tools from USearch (Edgar, 2010) and The Ribosomal Database Project (RDP) (Cole et al., 2014), combined with locally written tools for generating OTU tables. BIOM ﬁles (McDonald et al., 2012) generated by GHAP were analyzed using MicrobiomeAnalyst – a web-based tool for statistical and visual analysis of microbiome data (Dhariwal et al., 2017). Reads were initially denoised using GHAP with a minimum number of three reads in a minimum of two samples required to retain an OTU. Alpha diversity was assessed as number of observed OTUs and Chao1 (species level analysis). Beta diversity was assessed using PERMANOVA and unweighted unifrac distances. Diﬀerential abundance was calculated univariately. All p-values were calculated using student’s t-test unless stated otherwise. load was not quantiﬁed in this study, previous reports suggest that amniotic ﬂuid contains 100–1000 16S rDNA copies/mL (Urushiyama et al., 2017; Lim et al., 2018). Considering that there is substantial 16S rRNA gene copy number variation within the genomes of diﬀerent bacterial species however, these estimates do not correlate with CFU/mL and actual bacterial cell numbers may be as low as 1/15th of these values, depending on the bacterial species present (Kembel et al., 2012). Regardless, these data emphasize the low biomass nature of this sample type. In the present study, the average Ct value for the 16S rRNA gene positive samples was 34.1 (range: 28.5–43.9). Using a previously described enzymatic decontamination protocol (Stinson et al., 2018), we were able to produce completely negative extraction and PCR controls in our qPCR screen, and this, combined with the use of a biologically relevant (in terms of quantity) human DNA control with each run to account for any potential mis-priming, allowed us to make positive/negative calls with a higher level of sensitivity and accuracy. It should be noted that we did not collect negative controls during sampling, and so we are not able to assess the possibility of contamination during sample collection and initial processing, a limitation that is common in most microbiome studies. Ureaplasma spp. Were Not Detected in Mid-Trimester Amniotic Fluid Samples p Ureaplasma parvum and Ureaplasma urealyticum infection of the intra-amniotic cavity is typically associated with spontaneous PTD (Gray et al., 1992; Cassell et al., 1993; Horowitz et al., 1995). Therefore, all samples that tested positive for bacterial DNA by 16S rRNA gene qPCR (using primers optimized to improve coverage of Ureaplasma spp.) were further screened for U. parvum and U. urealyticum DNA using targeted qPCR (Yi et al., 2005). All of these were found to be negative. Further, no members of the Ureaplasma genus were identiﬁed by 16S rRNA gene sequencing. These results are similar to those reported by Payne et al. (2014) and Rowlands et al. (2017), both of whom failed to detect Ureaplasma spp. DNA in mid-trimester amniotic ﬂuid from Australian women. Payne et al. (2014), however, did detect Ureaplasma spp. DNA in two women (0.42%) from their Chinese cohort. In contrast, Kayem et al. (2018) screened 980 amniotic ﬂuid samples taken for genetic testing at 16–20 weeks gestation and reported that 12 samples (1.22%) were positive for Ureaplasma spp. DNA (10 U. urealyticum and 2 U. parvum) by qPCR screening. Collectively the evidence suggests that Ureaplasma spp. colonization of the amniotic cavity in mid-gestation is a rare event. Values are reported as mean (range) or n (percent). Characteristic Cases (n = 9) Controls (n = 18) p-Value Maternal age (y) 36 (21–43) 36 (23–44) 0.94 Nulliparitya 2 (22%) 4 (22%) 1.00 GA at delivery (week+day) 34+6 (30+5–36+6) 39+3 (37+1–42+1) < 0.001 GA at sampling (week+day) 15+4 (14+4–16+5) 15+5 (14+2–16+5) 0.78 Pre-pregnancy BMIa 24 (21–27) 24 (19–29) 0.78 Smoking during pregnancya 0 (0%) 0 (0%) – IVFa 1 (11%) 2 (22%) 1.00 Diabetes type 2 0 (0%) 0 (0%) – Gestational diabetes 0 (0%) 0 (0%) – Male/female fetus ratio 4/5 (44/56%) 7/11 (39/61%) 1.00 Values are reported as mean (range) or n (percent). aMatching criteria. Characteristic Cases (n = 9) Controls (n = 18) p-Value Frontiers in Microbiology \| www.frontiersin.org Bacterial DNA Is Present in Amniotic Fluid in Mid-Pregnancy Of the samples that tested negative for bacterial DNA 6 9% were from women with a spontaneous PTD There TABLE 2 \| Pregnancy demographic characteristics for 16S rRNA gene positive (n = 238) and negative (n = 960) samples. Characteristic n Positive Negative p-Value Maternal age (y) 1198 37 (21–46) 37 (20–47) 0.90 Nulliparity 1194 65 (27%) 254 (27%) 0.81 Preterm delivery 1159 64 (7%) 14 (6%) 0.68 GA at delivery (week+day) 1159 39+4 (30+5–43+0) 39+3 (22+1–43+0) 0.36 GA at sampling (week+day) 1193 15+6 (14+2–20+4) 15+6 (13+6–22+1) 0.47 BMI at ﬁrst prenatal visit 1171 23.1 (16.9–45.4) 23.6 (15.8–47.4) 0.18 Smoking at ﬁrst prenatal visit 1189 15 (6%) 47 (5%) 0.38 Diabetes type 2 1198 4 (0.4%) 0 (0)% 1.0 Gestational diabetes 1198 1 (0.%) 14 (1.5%) 0.33 Male/female fetus ratio 1160 111/118 (48.5/51.5%) 481/450 (51.7/48.3%) 0.39 Values are reported as mean (range) or n (percent). TABLE 2 \| Pregnancy demographic characteristics for 16S rRNA gene positive (n = 238) and negative (n = 960) samples. to movement of the amniotic ﬂuid, which can be thought of as an extension of the fetal extra-cellular ﬂuid. After fetal skin keratinization occurs, the amniotic ﬂuid changes in osmolality and can no longer equilibrate with the fetus. The eﬀect that fetal skin keratinization has on the amniotic ﬂuid microbiome is unknown. The samples examined in the present study were primarily taken prior to gestational week 20. The fetal skin would not have acted as an eﬀective barrier between the amniotic ﬂuid microbiome and the fetal extracellular ﬂuid at this stage in development. Description of Bacterial DNA Proﬁles in Mid-Trimester Amniotic Fluid Overall, the amniotic ﬂuid samples proﬁled here contained low-abundance and low-diversity bacterial DNA proﬁles, as was expected based on previous studies (Collado et al., 2016; Urushiyama et al., 2017; Lim et al., 2018; Wang et al., 2018; Figure 2). Of the 27 amniotic ﬂuid samples sequenced, ﬁve (one case and four controls) returned insuﬃcient reads (≤50). Each of these samples gave a clear positive signal on the qPCR screen, but failed to produce an amplicon with the full-length 16S rRNA gene primers used for sequencing, suggesting that the bacterial DNA present in these samples were not covered by these primers. Overall, the amniotic ﬂuid samples proﬁled here contained low-abundance and low-diversity bacterial DNA proﬁles, as was expected based on previous studies (Collado et al., 2016; Urushiyama et al., 2017; Lim et al., 2018; Wang et al., 2018; Figure 2). Of the 27 amniotic ﬂuid samples sequenced, ﬁve (one case and four controls) returned insuﬃcient reads (≤50). Each of these samples gave a clear positive signal on the qPCR screen, but failed to produce an amplicon with the full-length 16S rRNA gene primers used for sequencing, suggesting that the bacterial DNA present in these samples were not covered by these primers. This raises the possibility that the full breadth of the amniotic ﬂuid bacterial communities present were not captured in this study. Of the remaining samples, we recovered an average of 6503 reads per sample (range 1284–9563) and 12 OTUs per sample (range 2–33). Here, our negative PCR controls were completely negative, while our negative ECs were sporadically positive. Seven taxa were detected in six ECs: Pelomonas puraquae, Casaltella massiliensis, Staphylococcus pasteuri, Bosea eneae, Bosea vestrisii, Acinetobacter beijerinckii, and Rhodobacter blasticus (Table 4). the 238 positive samples (6.1%) were from women with a spontaneous PTD, four of which were iatrogenic (medically indicated) PTD. Of the samples that tested negative for bacterial DNA, 6.9% were from women with a spontaneous PTD. There was also no diﬀerence in the average gestational age at sampling for those that tested positive for bacterial DNA compared to those that tested negative (p = 0.47). Our data indicate that in some pregnancies, bacterial DNA is present in the amniotic ﬂuid at 14–20 weeks gestation. However, in the majority of cases, the amniotic ﬂuid appears sterile at this gestational age. Presence of bacterial DNA in the amniotic cavity at mid-trimester did not predict spontaneous PTD in our cohort. Bacterial DNA Is Present in Amniotic Fluid in Mid-Pregnancy Of the 1198 mid-trimester amniotic ﬂuid samples screened by qPCR, 238 (19.9%) contained detectable levels of bacterial DNA. Each of these samples was conﬁrmed as positive for bacterial DNA in duplicate screening. Pregnancy demographic characteristics for 16S rRNA gene positive (n = 238) and negative (n = 960) samples are presented in Table 2, with no signiﬁcant diﬀerences between the groups. As in Table 1, missing data is due to lost to follow-up, women with an abortion or miscarriage and women who were excluded post-enrollment. Although bacterial Interestingly, our detection of bacterial DNA in the amniotic ﬂuid was not associated with spontaneous PTD. Only 14 of March 2020 \| Volume 11 \| Article 415 Frontiers in Microbiology \| www.frontiersin.org 5 Bacterial Proﬁles in Mid-Trimester AF Stinson et al. TABLE 2 \| Pregnancy demographic characteristics for 16S rRNA gene positive (n = 238) and negative (n = 960) samples. Characteristic n Positive Negative p-Value Maternal age (y) 1198 37 (21–46) 37 (20–47) 0.90 Nulliparity 1194 65 (27%) 254 (27%) 0.81 Preterm delivery 1159 64 (7%) 14 (6%) 0.68 GA at delivery (week+day) 1159 39+4 (30+5–43+0) 39+3 (22+1–43+0) 0.36 GA at sampling (week+day) 1193 15+6 (14+2–20+4) 15+6 (13+6–22+1) 0.47 BMI at ﬁrst prenatal visit 1171 23.1 (16.9–45.4) 23.6 (15.8–47.4) 0.18 Smoking at ﬁrst prenatal visit 1189 15 (6%) 47 (5%) 0.38 Diabetes type 2 1198 4 (0.4%) 0 (0)% 1.0 Gestational diabetes 1198 1 (0.%) 14 (1.5%) 0.33 Male/female fetus ratio 1160 111/118 (48.5/51.5%) 481/450 (51.7/48.3%) 0.39 Values are reported as mean (range) or n (percent). TABLE 3 \| Pregnancy demographic characteristics for cases (spontaneous preterm delivery (<37 gestational weeks; n = 9) and controls (delivery at term; n = 18). Characteristic Cases (n = 9) Controls (n = 18) p-Value Maternal age (y) 36 (21–43) 36 (23–44) 0.94 Nulliparitya 2 (22%) 4 (22%) 1.00 GA at delivery (week+day) 34+6 (30+5–36+6) 39+3 (37+1–42+1) < 0.001 GA at sampling (week+day) 15+4 (14+4–16+5) 15+5 (14+2–16+5) 0.78 Pre-pregnancy BMIa 24 (21–27) 24 (19–29) 0.78 Smoking during pregnancya 0 (0%) 0 (0%) – IVFa 1 (11%) 2 (22%) 1.00 Diabetes type 2 0 (0%) 0 (0%) – Gestational diabetes 0 (0%) 0 (0%) – Male/female fetus ratio 4/5 (44/56%) 7/11 (39/61%) 1.00 Values are reported as mean (range) or n (percent). aMatching criteria. the 238 positive samples (6.1%) were from women with a spontaneous PTD, four of which were iatrogenic (medically indicated) PTD. Description of Bacterial DNA Proﬁles in Mid-Trimester Amniotic Fluid the 238 positive samples (6.1%) were from women with a spontaneous PTD, four of which were iatrogenic (medically indicated) PTD. Of the samples that tested negative for bacterial DNA, 6.9% were from women with a spontaneous PTD. There was also no diﬀerence in the average gestational age at sampling for those that tested positive for bacterial DNA compared to those that tested negative (p = 0.47). Our data indicate that in some pregnancies, bacterial DNA is present in the amniotic ﬂuid at 14–20 weeks gestation. However, in the majority of cases, the amniotic ﬂuid appears sterile at this gestational age. Presence of bacterial DNA in the amniotic cavity at mid-trimester did not predict spontaneous PTD in our cohort. Demographic details of the cases and controls which were selected for bacterial proﬁling are presented in Table 3. There were no signiﬁcant diﬀerences between the groups. The potential exposure of the fetus to bacteria at this early gestational age is of particular interest, as the fetal skin does not keratinize until approximately 20 weeks gestation (Wiberg- Itzel, 2012). Prior to this stage, the fetal skin oﬀers no resistance March 2020 \| Volume 11 \| Article 415 6 Bacterial Proﬁles in Mid-Trimester AF Stinson et al. However, in cases where one of these OTUs was detected in an EC, it was not detected in the samples that were extracted in that batch, suggesting that extraction-based contamination was not a contributor to our results. However, in cases where one of these OTUs was detected in an EC, it was not detected in the samples that were extracted in that batch, suggesting that extraction-based contamination was not a contributor to our results. Saccharibacteria (TM7 human oral clone), an extremely small coccus (200–300 nm) that until recently has been uncultivated, but is ubiquitous in numerous environments (He et al., 2015). It is important to note that Saccharibacteria was not a particularly good match for this particular OTU, with only 92.6–93% sequence homology. This might indicate that these OTUs belong not to Saccharibacteria, but to a related, as-yet undescribed bacteria (for example, an uncultivated clade of At the species level, amniotic ﬂuid samples were dominated by reads that mapped to Saccharibacteria (TM7 oral clone), Acidovorax temperans, Tepidimonas taiwanensis, and P. puraquae (Figure 3). Description of Bacterial DNA Proﬁles in Mid-Trimester Amniotic Fluid Although the sequence matched reasonably well to this particular bacterium, a 2.8% diﬀerence in a 1.5 kb amplicon represents a 42 bp diﬀerence in the 16S rRNA gene. It is, therefore, possible that this OTU belongs to an as yet uncharacterized bacterium. This sequence was not detected in any of the negative controls. Four samples contained reads that mapped to P. puraquae with a high level of sequence homology (99.5%). P. puraquae is a relatively uncharacterized bacteria, ﬁrst isolated in 2007 from hemodialysis water (Gomila et al., 2007), and previously reported in amniotic ﬂuid samples collected during cesarean section (Stinson et al., 2019a) and in endometrial samples (Fang et al., 2016; Verstraelen et al., 2016). It is a Gram-negative, motile rod with a single polar ﬂagellum. Although P. puraquae is often thought of as a contaminant in microbiome research (Salter et al., 2014), it was present in only one of the eight ECs taken here and in neither of the two PCR controls. The ECs processed in the same batch as the samples that contained reads mapping to P. puraquae did not contain reads mapping to P. puraquae themselves. Although this species has not previously been identiﬁed in mid-trimester amniotic ﬂuid samples, Urushiyama et al. (2017) reported ﬁnding reads that mapped to Pelomonas Other less abundant OTUs identiﬁed here are in line with those previously documented in mid-trimester amniotic ﬂuid samples by others. Four of our samples contained reads that mapped to Propionibacterium acnes, which has previously been identiﬁed in mid-trimester amniotic ﬂuid (Urushiyama et al., 2017; Zhu et al., 2018) as well as in amniotic ﬂuid at term (Collado et al., 2016; Stinson et al., 2019a). We also identiﬁed reads that mapped to Bacillus sp. (which could not be diﬀerentiated between Bacillus anthracis, Bacillus cereus, Bacillus mycoides, Bacillus pseudomycoides, Bacillus thuringiensis, and Bacillus weihenstephanensis) and Bacillus niabensis. Reads that map to species of the Bacillus genus have been consistently reported in previous studies of the mid-trimester amniotic ﬂuid microbiome (Urushiyama et al., 2017; Kayem et al., 2018; Zhu et al., 2018). Other genera detected here that have also been detected using short sequence technology in mid-trimester amniotic ﬂuid include Arthrobacter sp., Streptococcus sp., Staphylococcus sp., Massilia sp., and Paenibacillus sp. (Urushiyama et al., 2017; Kayem et al., 2018; Zhu et al., 2018). Description of Bacterial DNA Proﬁles in Mid-Trimester Amniotic Fluid Interestingly, bacterial species typically associated with ascending intrauterine infection and preterm labor were not detected in this study (Keelan et al., 2016). This supports the view that invasion by such species represents a pathological event that likely occurs after 20 weeks’ gestation, whereas potential colonization by the species identiﬁed in this study occurs much earlier and is non-pathogenic. Description of Bacterial DNA Proﬁles in Mid-Trimester Amniotic Fluid Eleven samples contained reads that mapped to FIGURE 2 \| Abundance (number of reads) of bacterial species detected in mid-trimester amniotic ﬂuid samples from women with spontaneous preterm delivery (cases, n = 9) and women with a delivery at term (matched controls, n = 18). FIGURE 2 \| Abundance (number of reads) of bacterial species detected in mid-trimester amniotic ﬂuid samples from women with spontaneous preterm delivery (cases, n = 9) and women with a delivery at term (matched controls, n = 18). FIGURE 2 \| Abundance (number of reads) of bacterial species detected in mid-trimester amniotic ﬂuid samples from women wit (cases, n = 9) and women with a delivery at term (matched controls, n = 18). TABLE 4 \| Bacterial taxa (number of reads) detected in negative extraction controls (EC) and negative PCR controls (NTC, no template controls). Species EC1 EC2 EC5 EC6 EC7 EC8 EC10 EC11 NTC1 NTC2 Casaltella massiliensis 10170 Pelomonas puraquae 6075 Bosea eneae 1719 Bosea vestrisii 1338 Acinetobacter beijerinckii 2236 Staphylococcus pasteuri 208 601 Rhodobacter blasticus 525 Frontiers in Microbiology \| www.frontiersin.org 7 March 2020 \| Volume 11 \| Article 415 March 2020 \| Volume 11 \| Article 415 Stinson et al. Bacterial Proﬁles in Mid-Trimester AF FIGURE 3 \| Percent abundance (highest to lowest) of bacterial species in all mid-trimester amniotic ﬂuid samples (n = 27). FIGURE 3 \| Percent abundance (highest to lowest) of bacterial species in all mid-trimester amniotic ﬂuid samples (n = 27). saccharophila in mid-trimester amniotic ﬂuid samples using short read sequencing. TM7). Six samples contained reads that mapped to A. temperans with a reasonably good level of sequence homology (98.3%). A. temperans is a Gram-negative rod with mega-pili on its surface, and has previously been isolated from various clinical and environmental samples, including urine, cervical swabs, and wastewater (Willems et al., 1990). This species has not previously been identiﬁed in amniotic ﬂuid. Four samples contained reads that mapped to T. taiwanensis with 97.2% sequence homology. T. taiwanensis is a relatively uncharacterized bacteria that was ﬁrst described in 2006 in hot springs (Chen et al., 2006). It is a Gram-negative, motile rod with a single polar ﬂagellum. Given that T. taiwanensis is a thermophile found at moderately high temperatures (50–60◦C), it is unlikely to be the correct identity for this particular OTU. Frontiers in Microbiology \| www.frontiersin.org Contribution to the “Sterile Womb” Debate The idea that the womb is sterile continues to be hotly debated in both scientiﬁc and lay circles. The data presented here at the very least support the notion that exposure to bacterial DNA may occur prior to birth in some healthy pregnancies. Further, we have provided evidence that this exposure occurs early in pregnancy. Although we do not know if this DNA is representative of live, biologically active cells, due to our carefully controlled processing protocols, it is unlikely to be cell-free bacterial DNA. However, questions remain around the interpretation of such data. Low biomass samples are notoriously diﬃcult to accurately characterize, and the interpretation of data from these environments must be performed cautiously (Stinson et al., 2019b). As in all other microbiome studies, there is also always a risk of contamination. Although we have used rigorous experimental controls and decontaminated PCR reagents, it should be kept in mind that we did not include any negative controls during sampling. Further, the observed bacterial signatures in these samples do not necessarily imply the presence of a true “microbiome” (that is, an ecological system of interacting organisms and their environment). Our results might reﬂect the presence of bacteria that are transferred from mother to fetus, but are not suited to survive within the womb. These bacteria may therefore be transiently present, but not true colonizers. Indeed, this may explain the low abundance of bacteria in our samples. If a true microbiome was established this early in pregnancy, we might expect it to become increasingly abundant after months of incubation in utero. However, data from other studies of the amniotic ﬂuid microbiome suggest that it remains low abundance at term (Collado et al., 2016; Urushiyama et al., 2017; Stinson et al., 2019a). Another explanation for the low titer of bacteria found within the intra-amniotic space is the presence of both fetal and maternal immunological components in amniotic ﬂuid which may suppress bacterial growth [for instance maternal and fetal neutrophils (Gomez-Lopez et al., 2017)]. Alternatively, non- bacterial microbial elements such as bacteriophages may curate bacterial communities in utero and prevent them from expanding in biomass; a previous study has already reported that nearly all reads associated with viruses in amniotic ﬂuid were associated with bacteriophages (Lim et al., 2018). Previously, Urushiyama et al. (2017) proﬁled amniotic ﬂuid samples obtained during cesarean section from pregnancies with various stages of chorioamnionitis. Comparison of Mid-Trimester Bacterial DNA Proﬁles From Term and Preterm Pregnancies We were unable to identify any signiﬁcant diﬀerences in bacterial DNA proﬁles of amniotic ﬂuid from women with a spontaneous PTD or a delivery at term. There was no diﬀerence in alpha March 2020 \| Volume 11 \| Article 415 Frontiers in Microbiology \| www.frontiersin.org Frontiers in Microbiology \| www.frontiersin.org 8 Bacterial Proﬁles in Mid-Trimester AF Stinson et al. diversity of cases and controls (number of observed OTUs p = 0.87; Chao1 p = 0.44) (Figure 4). Nor was there any diﬀerence in beta diversity of cases and controls (PERMANOVA p = 0.534). None of the species detected here varied in abundance between cases and controls upon univariate analysis. not ﬁnd any diﬀerences in the alpha diversity of amniotic ﬂuid samples from women with a spontaneous PTD compared to women with a term delivery. However, given the low number of spontaneous PTD in our cohort and the lack of data on placental pathology, we are unable to clarify this question here. We may have been unable to identify any predictive bacterial DNA signatures for spontaneous PTD due to the gestational age at which these samples were taken. Spontaneous PTD-associated bacteria have previously been detected in amniotic ﬂuid samples early in gestation [Ureaplasma spp. at 20 weeks (Han et al., 2009; Romero et al., 2014), Fusobacterium spp. at 21 weeks (Han et al., 2009), and U. parvum, Citrobacter koseri and viridans group streptococcus at 22 weeks (Han et al., 2009; Romero et al., 2014)], but not prior to 20 weeks. The average gestational age at which the present samples were taken was 15+6 weeks. It may have been more informative for our purposes to collect amniotic ﬂuid samples later in the second trimester; however, amniocentesis is not routinely performed at this stage of pregnancy, making such a study unfeasible. Further, ours was a cohort of asymptomatic women presenting for genetic amniocentesis, while previous detection of early pathogenic colonization of amniotic ﬂuid has occurred in women presenting with spontaneous preterm labor and preterm rupture of membranes. Contribution to the “Sterile Womb” Debate As well as identifying pathogenic bacteria in cases of high grade chorioamnionitis, these authors also found an inverse relationship between alpha diversity and chorioamnionitis severity. This might suggest that amniotic ﬂuid with a low diversity of bacteria is more vulnerable to infection than amniotic ﬂuid with a high diversity of bacteria. Conversely, these data might also suggest that intra-amniotic infection with pathogenic bacteria reduces amniotic ﬂuid diversity. Their data therefore poses a chicken or the egg question: Does low diversity precede infection, or does infection result in low diversity? We did FIGURE 4 \| Alpha diversity (number of observed operational taxonomic units) in cases (n = 9) and controls (n = 18). REFERENCES Dhariwal, A., Chong, J., Habib, S., King, I. L., Agellon, L. B., and Xia, J. (2017). MicrobiomeAnalyst: a web-based tool for comprehensive statistical, visual and meta-analysis of microbiome data. Nucleic Acids Res. 45, W180–W188. doi: 10.1093/nar/gkx295 Bearﬁeld, C., Davenport, E. S., Sivapathasundaram, V., and Allaker, R. P. (2002). Possible association between amniotic ﬂuid micro-organism infection and microﬂora in the mouth. BJOG 109, 527–533. doi: 10.1111/j.1471-0528.2002. 01349.x DiGiulio, D. B. (2012). Diversity of microbes in amniotic ﬂuid. Semin. Fetal Neonatal. Med. 17, 2–11. doi: 10.1016/j.siny.2011.10.001 Burchard, E. G., Ziv, E., Coyle, N., Gomez, S. L., Tang, H., Karter, A. J., et al. (2003). The importance of race and ethnic background in biomedical research and clinical practice. N. Engl. J. Med. 348, 1170–1175. doi: 10.1056/NEJMsb025007 Edgar, R. C. (2010). Search and clustering orders of magnitude faster than Bioinformatics 26, 2460–2461. doi: 10.1093/bioinformatics/btq461 Edgar, R. C. (2017). Accuracy of microbial community diversity estimated by closed- and open-reference OTUs. PeerJ 5, e3889. doi: 10.7717/peerj.3889 Cassell, G. H., Waites, K. B., Watson, H. 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Gynecol. 217, 693.e1–693.e16. doi: 10.1016/j.ajog.2017.09.013 Cobo, T., Vives, I., Rodriguez-Trujillo, A., Murillo, C., Angeles, M. A., Bosch, J., et al. (2017). Impact of microbial invasion of amniotic cavity and the type of microorganisms on short-term neonatal outcome in women with preterm labor and intact membranes. Acta Obstet. Gynecol. Scand. 96, 570–579. doi: 10.1111/aogs.13095 Gomila, M., Bowien, B., Falsen, E., Moore, E. R., and Lalucat, J. (2007). Description of Pelomonas aquatica sp. nov. and Pelomonas puraquae sp. nov., isolated from industrial and haemodialysis water. Int. J. Syst. Evol. Microbiol. 57(Pt 11), 2629–2635. doi: 10.1099/ijs.0.65149-0 Cole, J. ETHICS STATEMENT The study was approved by the Ethics Review Board at the University of Gothenburg, Sweden (Dnr Ö 639-03, T 318-08, T 694-11, and Dnr 2019-0602) and written informed consent was obtained from all participants. DATA AVAILABILITY STATEMENT This study was supported by grants from the agreement concerning research and education of doctors, Sweden (Grant Numbers: ALFGBG-11522, ALFGBG-136431, ALFGBG-426411, ALFGBG-507701, ALFGBG-671611, and ALFGBG-717501; grant receiver: BJ), the Health & Medical Care Committee of the Regional Executive Board, Region Västra Götaland, Sweden (Grant Numbers: VGFOUREG-231311, VGFOUREG-308151, and VGFOUREG-368351; grant receiver: BJ), Sahlgrenska University Hospital Foundations, Sweden (grant receiver: MH), the foundations “Linnéa och Josef Carlssons Stiftelse” (grant numbers: 2011:1 nr:21, 2012 nr:15, and 2014 nr:12; grant receiver: BJ), “Kungl och Hvitfeldtska Stiftelsen” (grant receiver: MH), “Alice Swenzons stiftelse för vetenskaplig forskning” (grant receiver: MH), and “Stiftelsen Wilhelm och Martina Lundgrens vetenskapsfond” (grant receiver: MH). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The datasets generated for this study can be found in the PRJNA602788. AUTHOR CONTRIBUTIONS BJ and MH designed the study and were responsible for sample collection. MH and FV collected the data. MB and LS performed the statistical analyzes and graphical presentation of data. LS CONCLUSION Here we have provided evidence that, while most mid-trimester amniotic ﬂuid samples are sterile, bacterial DNA is present in amniotic ﬂuid at this time point in approximately one in ﬁve pregnancies. Mid-trimester amniotic ﬂuid bacterial DNA proﬁles FIGURE 4 \| Alpha diversity (number of observed operational taxonomic units) in cases (n = 9) and controls (n = 18). FIGURE 4 \| Alpha diversity (number of observed operational taxonomic units) in cases (n = 9) and controls (n = 18). March 2020 \| Volume 11 \| Article 415 Frontiers in Microbiology \| www.frontiersin.org 9 Bacterial Proﬁles in Mid-Trimester AF Stinson et al. performed the DNA extractions, PCR, sequencing, microbiome data analysis, and drafted the manuscript. BJ, MH, and MP received funding for the project. MP, JK, MK, and BJ supervised the work. All authors participated in the planning stages of analyzes and interpretation of data, and contributed to the manuscript by critical review and editions. are of low biomass and low diversity. We were not able to identify any diﬀerences in the bacterial DNA proﬁles of samples from pregnancies that had a spontaneous PTD and matched term controls. The developmental and immunological signiﬁcance of the presence or absence of bacterial DNA in mid-trimester amniotic ﬂuid samples remains unclear. 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Horowitz, S., Mazor, M., Romero, R., Horowitz, J., and Glezerman, M. (1995). Infection of the amniotic cavity with Ureaplasma urealyticum in the midtrimester of pregnancy. J. Reprod. Med. 40, 375–379. Stinson, L. F., Keelan, J. A., and Payne, M. S. (2018). Identiﬁcation and removal of contaminating microbial DNA from PCR reagents: impact on low biomass microbiome analyses. Lett. Appl. Microbiol. 68, 2–8. doi: 10.1111/lam.13091 Kayem, G., Doloy, A., Schmitz, T., Chitrit, Y., Bouhanna, P., Carbonne, B., et al. (2018). Antibiotics for amniotic-ﬂuid colonization by Ureaplasma and/or Mycoplasma spp. to prevent preterm birth: a randomized trial. PLoS One 13:e0206290. doi: 10.1371/journal.pone.0206290 Stinson, L. REFERENCES doi: 10.1111/aji.12189 Yi, J., Yoon, B. H., and Kim, E. C. (2005). Detection and biovar discrimination of Ureaplasma urealyticum by real-time PCR. Mol. Cell Probes. 19, 255–260. doi: 10.1016/j.mcp.2005.04.002 Zhu, L., Luo, F., Hu, W., Han, Y., Wang, Y., Zheng, H., et al. (2018). Bacterial communities in the womb during healthy pregnancy. Front. Microbiol. 9:2163. doi: 10.3389/fmicb.2018.02163 Rowlands, S., Danielewski, J. A., Tabrizi, S. N., Walker, S. P., and Garland, S. M. (2017). Microbial invasion of the amniotic cavity in midtrimester pregnancies using molecular microbiology. Am. J. Obstet. Gynecol. 217, e71. doi: 10.1016/j. ajog.2017.02.051 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. Salter, S. J., Cox, M. J., Turek, E. M., Calus, S. T., Cookson, W. O., Moﬀatt, M. F., et al. (2014). Reagent and laboratory contamination can critically impact sequence-based microbiome analyses. BMC Biol. 12:87. doi: 10.1186/s12915- 014-0087-z Copyright © 2020 Stinson, Hallingström, Barman, Viklund, Keelan, Kacerovsky, Payne and Jacobsson. 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. Stinson, L. F., Boyce, M. C., Payne, M. S., and Keelan, J. A. (2019a). The not-so- sterile womb: evidence that the human fetus is exposed to bacteria prior to birth. Front. Microbiol. 10:1124. doi: 10.3389/fmicb.2019.01124 Stinson, L. F., Keelan, J. A., and Payne, M. S. (2019b). Proﬁling bacterial communities in low biomass samples: pitfalls and considerations. Microbio. Aust. 40, 181–185. March 2020 \| Volume 11 \| Article 415 Frontiers in Microbiology \| www.frontiersin.org 11
W2775893206.txt	https://iptek.its.ac.id/index.php/tekno/article/download/2413/1975	en		PENGGUNAAN METODE STATISTIK DALAM PERUMUSAN STRATEGI PEMASARAN PRODUK BERDASARKAN PREFERENSI DAN PERSEPSI KONSUMEN	Jurnal teknobisnis/Teknobisnis	2,017	cc-by-sa	0
https://openalex.org/W4386009741	https://czasopisma.uksw.edu.pl/index.php/s/article/download/12876/11115	Polish	null	Ks. Henryk Skorowski , Moralność społeczna. Wybrane zagadnienia z etyki społecznej, gospodarczej i politycznej, Wydawnictwo Salezjańskie, Warszawa 1996, ss. 250 + 2 nlb.	Seminare	1,997	cc-by	15,525	R E C E N Z J E R Clemens Br e ue r , Mensch von Anfang an. Fragen der Gentechnologie und der Bioethik, Köln 1995, ss. 16. Jedną z najbardziej kontrowersyjnych kwestii naukowych ostatnich lat jest problem etycz nych granic postępu w dziedzinie medycznej. Wiele sporów wzbudzają propozycje eksperymen tów, w których tzw. dobro nauki stawiane jest ponad dobrem osoby ludzkiej. Głośna była w roku 1996 sprawa zniszczenia w Wielkiej Brytanii ludzkich embrionów, uznanych za nieprzydatne do dalszych prac naukowych. Dyskusje na tym polu dotyczą także wszelkich manipulacji genetycz nych, diagnostyki prenatalnej i oczywiście prawnego zakazu aborcji. Tymi właśnie, istotnymi z etycznego punktu widzenia zagadnieniami zajął się w swym opracowaniu Clemens Breuer z katedry teologii moralnej Uniwersytetu w Augsburgu. Tekst ukazał się w 225 zeszycie z serii „Kirche und Gesellschaft” , firmowanej przez Katholische Sozialwissenschaftliche Zentralstelle Mönchengladbach. Ważnym impulsem do powstania tej publikacji było pojawienie się w roku 1994 projektu konwencji bioetycznej Rady Europy. Został on wprawdzie odrzucony, ale niektóre zaproponowane w nim rozwiązania i argumenty częstokroć powracają w dyskusjach. Tak jest np. ze sprawą podejmowania działań medycznych wobec osób niezdolnych do samodzielnego wyrażenia swej zgody lub swego sprzeciwu. Zwolenników ma także koncepcja, aby wykrycie nieprawidłowości rozwojowych płodu było usprawiedliwieniem jego spędzenia. Autor omawianego artykułu pod kreśla przeto absolutną konieczność uwzględniania podstawowych norm moralnych przy uchwala niu przepisów, regulujących obszar bioetyki i technologii genetycznej. U podstaw tych rozstrzyg nięć tkwi obowiązek poszanowania godności każdego człowieka. Odnosząc to do problemu początku życia ludzkiego C. Breuer przypomina wypowiedź Federalnego Sądu Konstytucyjnego z roku 1974, który stwierdził, iż „tkwiące w ludzkim bycie od samego początku potencjalne zdolności (możliwości) wystarczają, aby uzasadnić mówienie o godności człowieka” . Godność człowieka jest bowiem jakością właściwą wszelkiemu ludzkiemu życiu i dlatego ochronie godności podporządkować trzeba także cały proces rozwoju człowieka jeszcze w jego fazie embrionalnej. Przekonującym argumentem za ochroną życia poczętego pozostaje dla autora stwierdzenie, że „wkrótce po połączeniu komórki jajowej i plemnika uruchomiony zostaje spontaniczny proces, w którym następuje jakościowy przeskok od jedynie biologicznego do osobowego życia człowieka” . Konieczność ochrony życia od momentu poczęcia nie dla wszystkich jest jednak oczywista, mimo wagi przytaczanych argumentów. W omawianym tekście znajdujemy więc polemikę z poglądami australijskiego filozofa moralności Petera Singera. Twierdzi on między innymi, że 0 wartości danego życia decyduje stan zdrowia lub pewne właściwości organizmu. W obec tego rodzaju poglądów potrzebne jest więc ciągłe wskazywanie na szczególną wartość życia ludzkiego, na jego godność i nienaruszalność w każdej fazie. R E C E N Z J E I oczywiście na jego poza-biologiczny wymiar. Kolejnym problemem, jakim zajmuje się w swym artykule C. Breuer, są diagnostyczne 1 terapeutyczne metody technologii genetycznej. Jako kryteria etycznej dopuszczalności diagnos tyki prenatalnej wymienia się: chęć uspokojenia rodziców w kwestii stanu zdrowia ich nienaro dzonego jeszcze dziecka, odsunięcie lęków matki związanych z możliwością wystąpienia powik łań, przygotowanie rodziców na trud przyjęcia dziecka chorego lub niepełnosprawnego i umoż liwienie rozpoczęcia terapii w przypadku stwierdzenia nieprawidłowości. Chociaż diagnostyczne 334 RECENZJE metody wywołują na ogół oddźwięk pozytywny, to jednak w praktyce okazuje się, że przy niekorzystnym wyniku badań podejmowana jest decyzja o aborcji. Efekt końcowy rozmija się zatem ze szczytnymi założeniami tych działań. Zdaniem autora opracowania trzeba również pamiętać o tym, że wciąż jeszcze możliwości diagnozy znacznie przekraczają możliwości leczenia. Tak więc diagnostyka prenatalna stoi nierzadko u podstaw późniejszej aborcji z przyczyn genetycznych. Trudnym i kontrowersyjnym problemem są również analizy genetyczne po urodze niu. Istnieje bowiem niebezpieczeństwo, że wyniki tychże analiz wykorzystywane będą przez pracodawców w celu określenia przydatności danego pracownika lub przez towarzystwa ubez pieczeniowe w celu określenia ryzyka, związanego z ubezpieczeniem danego klienta. Wśród metod terapii w genetyce wyróżnić można terapię somatyczną i terapię embrionu. Założeniem jest, że dokona się wymiany określonego nieprawidłowego genu na zdrowy. Niebez pieczeństwo terapii somatycznej polega ńa tym, że z prób naprawienia jakiegoś defektu genetycz nego przerodzić się ona może w działania mające na celu ulepszanie natury. Groźba jest tym bardziej realna, że istnieje wiele osób, które gotowe są poświęcić duże pieniądze dla realizacji swych kaprysów. W ykonywanie tego rodzaju zabiegów na embrionach jest jeszcze niemożliwe technicznie, ale - zdaniem C. Breuera - nawet gdy stanie się możliwe, to z etycznego punktu widzenia należy je i tak bezwzględnie odrzucić. Nie jest ono bowiem leczeniem istniejącej już osoby ludzkiej, ale manipulowaniem jej tożsamością. Ingerencja w rozwój embrionu stanowi otwarcie drzwi dla eugeniki. W dziedzinie technologii genetycznej dzieje się wiele rzeczy, świadczących o rozejściu się etyki i nauki. Jak stwierdza autor omawianego tekstu, wielu badaczy usprawiedliwia swe działania wysokiej rangi celami naukowymi i - jeśli trzeba - mogą oni o tym przekonać gremia opiniodawcze. Dziwne jest też, że dąży się do postępu w genetyce i w ogóle w biotechnologii, podczas gdy wielu ludzi nie ma zaspokojonych nawet elementarnych potrzeb w przypadku choroby. Wolność badawcza tam znajduje swe granice, gdzie dotyka ona dobra najwyższego. Tak więc na gruncie chrześcijańskiej wizji człowieka używanie technologii genetycznej dopuszczalne jest tylko w bardzo ograniczonym zakresie. R E C E N Z J E Nie wolno przy tym naruszać integralności, tożsamo ści, i kontyngencji osoby ludzkiej. Pluralizm nie jest możliwy w odniesieniu do wartości podstawowych, jeśli więc pozwolimy na niczym nieograniczony rozwój technologii genetycznej, to konsekwencją może być, wg Breuera, zniszczenie godności człowieka pod sztandarami człowie czeństwa. Opracowanie autorstwa augsburgskiego teologa, mimo iż niezbyt obszerne, stanowi wyraźną i jednoznaczną odpowiedź na dylematy technologii genetycznej z chrześcijańskiego punktu widzenia. Jest to zarazem punkt widzenia jak najbardziej ogólnoludzki, gdyż u podstaw rozstrzyg nięć stawia godność każdego człowieka i jego niezbywalne prawo do życia. Tadeusz Kamiński Jerzy G oc ko, Ekonomia a moralność. Poszukiwania teologicznomoralne, Lub lin 1996, ss. 397. Misją, czyli podstawową drogą Kościoła, jest człowiek jako osoba. Nie jest on istotą wyizolowaną, ale tkwiącą, funkcjonującą i działającą w określonej rzeczywistości polityczno- - społeczno-gospodarczo-kulturowej. Ta rzeczywistość stanowi nieodzowny kontekst życia i dzia łania człowieka. Jeśli zatem misją Kościoła jest służba zbawieniu człowieka, to Kościół wierny temu posłannictwu nie może być obojętny na kontekst życia i działania człowieka. Tu też znajduje swoje uzasadnienie potrzeba wypowiadania się Kościoła na szeroko rozumiane sprawy społeczno-gospodarcze, przy czym nie chodzi o prowadzenie gospodarki, kierowanie sprawami życia społecznego, zajmowanie się działalnością polityczną, ale o dobro człowieka, któremu oWa 335 RECENZJE rzeczywistość może utrudniać albo ułatwiać zbawienie. Jednym z fundamentalnych obszarów owego kontekstu życia człowieka jest obszar życia gospodarczego. Musi on także być uwzględ niany we współczesnym nauczaniu Kościoła, tym bardziej, iż podobnie jak wszystkie inne płaszczyzny życia ludzkiego, także i ta płaszczyzna rodzi wiele moralnych i społecznych dylematów. W tym kontekście należy widzieć publikację książkową Jerzego Gocko zatytułowaną: Ekonomia a moralność. Poszukiwania teologicznomoralne. Jest to rozprawa doktorska tego autora. W swej obecnej treści niewiele odbiega ona od pierwowzoru noszącego tytuł: Życie gospodarczo - -społeczne w wybranych posoborowych publikacjach teologicznomoralnych. Głównym przedmiotem zainteresowania autora jest próba ukazania życia gospodarczo - -społecznego z perspektywy chrześcijańskiej, jako jednego z podstawowych obszarów zaan gażowania człowieka, uczyniona w oparciu o wybrane posoborowe publikacje teologicznomoralne (s. 18). Można powiedzieć, że chodzi o odpowiedź na pytanie: na ile i w jakim zakresie życie gospodarczo-społeczne, jako jeden z podstawowwych obszarów funkcjonowania człowieka, znala zło swoje odbicie w posoborowych publikacjach teologicznomoralnych? Chodzi zatem o próbę syntezy propozycji moralności gospodarczej, jakie zostały wypracowane w okresie posoborowym. To jest główny cel publikacji. Dotyczy on moralności gospodarczej w obrębie katolickiej teologii moralnej. W tym miejscu podkreślić należy, że autor podejmuje zagadnienie jakościowo ważne na gruncie analiz naukowych. R E C E N Z J E W tym miejscu podkreślić należy, że zgromadzona przez autora literatura obejmuje praktycznie wszystkie najważniejsze ośrodki teologiczno moralne w świecie i dlatego może być uważana za reprezentatywną dla całości posoborowej teologii moralnej. Autor zatem musiał wykonać olbrzymią pracę dotyczącą nie tylko zestawu literatury, ale także jej gruntownej analizy. O dobrej analizie literatury świadczy bowiem faktyczne jej wykorzystanie w rozprawie. y j y y y j j y y p W niniejszej recenzji trudno silić się na ukazanie pełnej treści omawianej publikacji, a tym bardziej ustosunkować się do poszczególnych tez. Musi wystarczyć ogólne zapewnienie o istotnej wartości poznawczej i naukowej przedstawionych w niej analiz, wniosków i tez. W niniejszej recenzji trudno silić się na ukazanie pełnej treści omawianej publikacji, a tym bardziej ustosunkować się do poszczególnych tez. Musi wystarczyć ogólne zapewnienie o istotnej wartości poznawczej i naukowej przedstawionych w niej analiz, wniosków i tez. W podsumowaniu podkreślić należy, iż mamy przed sobą interesującą publikację dotyczącą związku ekonomii z moralnością. Ta problematyka jest dziś szczególnie ważna i domaga się także naukowych refleksji. Stąd też z radością odnotować należy, iż publikacja o takiej tematyce ukazała się na polskim rynku wydawniczym. Jest ona znaczącym wkładem w toczącą się dyskusję na temat życia społeczno-gospodarczego w jego etycznym wymiarze. Należy zatem wyrazić przekonanie, iż stanowić ona będzie cenne źródło naukowych przemyśleń dla znawców tej problematyki, jak również odpowiedzialnych za całokształt życia gospodarczego. W ykorzystana także być winna w wykładach tej problematyki zarówno w uczelniach ekonomicznych, jak i humanistycznych. ks. Henryk Skorowski SDB ks. Henryk Skorowski SDB Andrzej K o p i c z k o , Kościół Warmińskii a polityka wyznaniowa po II wojnie światowej, Olsztyn 1996, Ośrodek Badań Naukowych im. Wojciecha Kętrzyńs kiego w Olsztynie, s. 244. Jubileusze i rocznice są często okazją do przypomnienia dziejów instytucji, diecezji bądź monografii miast. Dzięki zaangażowaniu i pomocy Ośrodka Badań Naukowych im. W ojciecha Kętrzyńskiego w Olsztynie, ukazują się ostatnio prace dotyczące historii Kościoła także poza wydawnictwem diecezjalnym. Przykładem tego są chociażby pozycje książkowe ks. A. Kopiczki, wykładowcy historii Kościoła w W yższym Seminarium Duchownym, Instytucie Kultury Chrześ cijańskiej im. Jana Pawła II w Olsztynie, W yższym Studium Katechetycznym w Gietrzwałdzie. Pierwsza praca: Ustrój i organizacja diecezji warmińskiej w latach 1525 -1772, Olsztyn 1993 ukazała się w 750 rocznicę powołania do istnienia tej jednostki kościelnej. Jest ona zmodyfikowa ną wersją pracy doktorskiej przygotowanej pod kierunkiem ks. prof. Bolesława Kumora. Ksiądz A. R E C E N Z J E Próba syntezy moralności gospodarczej w publikacjach teologicz nomoralnych, jest zagadnieniem o ogromnym ciężarze naukowym. Jest to także zagadnienie naukowo trudne. Dotyczy bowiem z jednej strony całego obszaru literatury posoborowej, z dru giej zaś strony istotnego okresu czasu (30 lat). Podkreślić także należy, że autor podejmuje problem nowatorski w ścisłym tego słowa znaczeniu. Ani bowiem w literaturze polskiej, ani też zagranicznej nie ma całościowej syntezy moralności gospodarczej w posoborowych publikacjach teologicznomoralnych. Na podkreślenie zasługuje także doskonałe uzasadnienie podjętego przez autora zagadnienia. Z jednej strony uzasadnienie to stanowi system pytań kreślących szeroką płaszczyznę dylematów i wezwań, przed którymi staje także chrześcijanin, który musi uczest niczyć w skomplikowanej i m oralnie nieobojętnej rzeczywistości, jaką jest współczesne życie gospodarcze, z drugiej zaś strony uzasadnienie to stanowi wykazanie braku w literaturze wspomnianej już syntezy moralności gospodarczej. Stwierdzić zatem należy, że ks. J. Gocko w swojej publikacji podejmuje zagadnienie jakościow o ważne, znaczącej rangi naukowej, w pełni nowatorskie. Na podkreślenie zasługuje jasna i logiczna konstrukcja publikacji. Jest ona podporząd kowana głównemu założeniu badawczemu, jakim jest synteza propozycji moralności gospodarczej wypracowanej w okresie posoborowym w publikacjach teologicznomoralnych. Dlatego autor z przejrzystą i jasną konsekwencją w rozdziale I (s. 29-65) ukazuje najważniejsze perspektywy rzutujące na teologiczno-eklezjalny charakter nauczania społecznego Kościoła. Chodzi o od powiedź na pytanie: czym jest nauczanie społeczne Kościoła? Drugi rozdział (s. 67-123) ukazuje tendencje rozwojowe w ramach katolickiej teologii moralnej, zapoczątkowane jeszcze pod koniec ubiegłego stulecia, a dopełnione posoborową odnową. Refleksja kościelna nad problematyką życia gospodarczo-społecznego, dokonywana w obszarze teologii moralnej nie może pominąć analizy teologiczno-antropologicznych podstaw tej dziedziny ludzkiego zaangażowania. Stąd też w rozdziale III (s. 125-193) autor podejmuje próbę określenia tego, na jakie fundamentalne perspektywy teologiczne zwracają uwagę w swoich opracowaniach teologowie moraliści okresu posoborowego. Konsekwentnie dalej w dwóch pozostałych rozdziałach autor podejmuje zagadnienia bar dziej szczegółowe, które tworzą zasadniczy obszar analiz i badań dla opracowania moralności gospodarczej. I tak rozdział IV (s. 195-269) jest poświęcony moralno-społecznym aspektom życia gospodarczego. Z kolei rozdział V (s. 271-339) dotyczy zasadniczo moralnej oceny 336 RECENZJE głównych systemów społeczno-ekonomicznych. Publikacja zatem w swej strukturze jest jasna i logiczna. Poszczególne rozdziały wyczerpują problem postawiony w jej tytule. W badaniach dotyczących zasadniczego problemu publikacji jej autor musiał przeprowadzić szeroka kwerendę bibliograficzną. Zgromadził i wykorzystał ogromny zestaw literatury w postaci: podręczników teologii moralnej, podręczników zbiorowych, słowników, samodzielnych monografii, licznych artykułów. R E C E N Z J E Kopiczko aktualnie zajmuje się najnowszymi dziejami archidiecezji warmińskiej, co zaowoco wało już publikacją książkową, wydaną w 50 lat po powrocie Warmii i Mazur do Polski. W cale to nie znaczy, że okres ten został zaniedbany przez historyków z kręgu „Hosianum” . Historio grafia dysponuje wieloma publikacjami obrazującymi życie i działalność Kościoła na tym terenie, co stawia Olsztyn w czołówce w porównaniu z innymi diecezjami. Książka ks. Kopiczki: Kościół Warmiński - opracowana na niezwykle bogatym materiale źródłowym zgromadzonym w archiwach państwowych i kościelnych w Olsztynie, jest wprost pionierska pod tym względem w Polsce. Jednak ma ona pewne niedociągnięcia. Przede wszystkim odnosi się wrażenie że powstała w wielkim pośpiechu. Dowodem tego jest choćby brak indeksu. źródłowym zgromadzonym w archiwach państwowych i kościelnych w Olsztynie, jest wprost pionierska pod tym względem w Polsce. Jednak ma ona pewne niedociągnięcia. Przede wszystkim odnosi się wrażenie że powstała w wielkim pośpiechu. Dowodem tego jest choćby brak indeksu. Autor podzielił całość na siedem rozdziałów, zaopatrzył dodatkowo wstępem, zakończeniem, dziesięcioma aneksami, bibliografią, wykazem ważniejszych skrótów, streszczeniem w języku p p p g j y Autor podzielił całość na siedem rozdziałów, zaopatrzył dodatkowo wstępem, zakończeniem, dziesięcioma aneksami, bibliografią, wykazem ważniejszych skrótów, streszczeniem w języku 337 RECENZJE niemieckim i spisem treści. Pozycja wydrukowana na dobrym papierze, w pięknej szacie graficznej i prawie bez błędów literowych. Dodatkowo walor tekstu i wyniki badań podkreśla 19 zamieszczonych tabel. Pewnym mankamentem jest brak map diecezji z podziałem na dekanaty, województw uwzględniających powiaty z rozmieszczeniem ludności pod względem wyznaniowym i etnicznym. Niestety, sam temat pracy nie został sformułowany precyzyjnie, tak pod względem terytorium jak i chronologii. leżeli chodzi o terytorium, to czytelnik nie powinien mieć żadnych wątpliwości, że chodzi o całą diecezję. Tymczasem ks. Kopiczko ograniczył się w większości tylko do województwa olsztyńskiego, niejednokrotnie wykraczając poza diecezję. Widać tu brak konsekwencji, dlaczego kilka razy podawał dane powiatu działdowskiego (s. 103, 110, 136) a pomijał nowolubawski, chociaż oba powiaty wchodziły w skład tej samej jednostki ad ministracyjnej. Terminus ad quem powinien być określony przynajmniej w przybliżeniu. Co to znaczy „po II wojnie światowej” ; pierwsze lata, czy cały okres aż do upadku komunizmu? Bardziej adekwatnym tematem, odpowiadającym treści, byłby ten zapowiedziany we wstępie (s. 6) i odpowiednio zmodyfikowany, np. Stanowisko (polityka) władz państwowych wobec Kościoła warmińskiego w latach 1945-1980 (na przykładzie województwa olsztyńskiego). Rozdział wstępny (historyczny) przedstawia w skondensowanym skrócie tło wydarzeń. R E C E N Z J E Mimo iż zagadnienia te zostały już wcześniej opracowane, to jednak umieszczenie ich na początku książki było wprost konieczne. Niezwykle cenną informację powtórzył Autor za o. Cz. Baranem, że to pierwsze władze państwowe (Ministerstwo Administracji Publicznej) dostrzegły potrzebę nowych duszpasterzy dla osiedleńców. W tym miejscu nasuwa się pytanie, czy tylko franciszkanie i ks. Franciszek Borowiec od początku stawili się do pracy w tak trudnym okresie? Brakuje wyraźnie informacji podającej liczbę księży diecezjalnych i zakonnych przybywających razem z napływową ludnością, czy też oddelegowanych przez swoich przełożonych. Jakie zakony, pozostawiając swoje puste klasztory na W armii i Mazurach, przekazały swoją własność polskim współbraciom? Skoncentrowanie się Autora głównie na rządcach diecezji tworzy niepełny obraz i nie wyczerpuje tematu. Kolejny, trzeci paragraf ukazuje genezę i mechanizm działania polityki wyznaniowej. Sprawami Kościoła katolickiego i inwigilacją duchowieństwa w Polsce Ludowej zajmowano się od początku „w pionie politycznym” . Z prezentowanej pracy dowiadujemy się, że najpierw działały Referaty do Spraw W yznań (Wydziały do Spraw W yznań) powołane do istnienia na szczeblu wojewódzkim, następnie Referaty przy powiatach (17 IV 1950) i wreszcie organ centralny, Urząd do Spraw W yznań (19 IV 1950), chociaż tej ostatniej daty Autor nie precyzuje, a szkoda, bo przecież to wszystko działo się tuż po podpisaniu Porozumienia (14 IV 1950). Już z samego zestawienia dat widać zaplanowaną, zakłamaną i dwulicową postawę państwa względem Kościoła. Trudno zrozumieć intencję Piszącego oraz odczytać trafnie, która informacja dotycząca powołania Olsztyńskiej W ojewódzkiej Rady Narodowej jest prawdziwa; 24 maja 1950 czy 14 sieipnia 1947 (s. 19). Wśród tej mnogości dat skomasowanych na jednej stronicy, powinno być też odnotowane ustanowienie przez Radę Ministrów województwa olsztyń skiego (29 V 1946). Korzystanie z materiałów archiwalnych jednej proweniencji przy opracowywaniu poszczegól nych zagadnień przypomina okres tworzenia się władzy ludowej na Warmii i Mazurach, gdy jedno starostwo (powiat) powierzano pieczy PPR a drugie PPS. Taki jednostronny sposób posługiwania się źródłami - raz kościelne, raz państwowe - nie może dać pełnego obrazu rzeczywistości. Informacje zaczerpnięte ze zbiorów państwowych powinny być natychmiast uzupełnione i skorygowane z materiałami zgromadzonymi w Archiwum Akt Nowych Archidiece zji Warmińskiej. Przykładowo, Autor podaje za Wyzn. 11. (s. 21) nazwiska czterech księży usuniętych z diecezji, „a kilku innych osadzono w więzieniu” . Od razu powstaje pytanie, którzy z nich byli bardziej represjonowani i więcej cierpieli; imienni czy bezimienni? Czyżby „Oazy” znane były w diecezji warmińskiej dopiero od 1979 r. (s. 32)? Drugi rozdział dotyczy represjonowania duchownych (s. 34-78). R E C E N Z J E Tak duża liczba duchownych (20-25% ) zaangażowa nych w różnym stopniu po stronie „reżim u” świadczy o tym, że na Ziemie Północne nie przybywali najbardziej wartościowi księża. Kolejny punkt poświęcono zakonom i zgromadzeniom zakonnym. Wyraźnie brak w nim kilku zdań wstępnych, informujących krótko o ilości zakonów męskich i żeńskich oraz charak terze ich pracy: duszpasterstwo czy realizacja swojego posłannictwa. Bez znajomości powojen nych dziejów tej diecezji lub lektury artykułu ks. Jana Obłąka opublikowanego w „Naszej Przeszłości” , 22, 1965, ten krótki tekst niewiele wnosi. Ponadto oparty na źródłach państwowych, dotyczy tylko województwa olsztyńskiego, a nie całej diecezji. Do tego dochodzi nieproporcjonal ne rozłożenie materiału, zdecydowanie więcej miejsca poświęcono siostrom zakonnym. Autor nic nie wspomniał, że mimo nacisku władz rządcy diecezji nie pozbawiali zakonników pracy duszpasterskiej lecz szukali możliwości zatrzymania ich na terenie diecezji. Jednym z przykładów takich „wybiegów” było m.in. pisanie fikcyjnych podań do wyższych przełożonych o zwolnienie ze ślubów. Przy wymienianiu takich domów zakonnych, jak Święta Lipka czy Gietrzwałd (s. 70), powinny znaleźć się też nazwy zakonników; jezuici, kanonicy regularni. Oczywiście są to już szczegóły, jednak konieczne, jeśli książka jest przeznaczona dla szerszego odbiorcy, a nie do mieszkańców jednego regionu. Ostatnie dwa paragrafy tego rozdziału dotyczą trudności stawianych przez państwo w przy gotowaniu nowych kadr duchowieństwa diecezjalnego (Seminarium Duchowne, Jednostka wojs kowa w Bartoszycach). Recenzent ma w tym miejscu pytanie; czy wizytacje WSD przez WdSW i Kuratorium miały miejsce, jak często i jakich dziedzin dotyczyły, a może to są tylko „osiągnięcia” W dSW w sprawozdaniach do UdSW? W przypisie 211 (s. 77) podano liczby alumnów odbywąjących służbę wojskową w poszczególnych latach (1958-1979). Czytelnik chciałby dowiedzieć się także o konsekwencjach skuteczności działania tej instytucji, np. ilu kleryków już nie wróciło do seminarium? Rozdział trzeci poświęcono polityce władz wobec grup narodowościowych i stowarzyszeń katolickich. Z tych pierwszych zauważono tylko autochtonów i grekokatolików. Pełniejsze informacje o losach duchowieństwa miejscowego pochodzenia powinny znaleźć się wcześniej w paragrafie Prześladowanie Księży, tym samym Autor uniknąłby zbędnych powtórzeń. Inte resujących danych na temat ilości grekokatolików w poszczególnych parafiach dostarczają proto koły z wizytacji kanonicznych. Z ich treści widać wyraźnie, jak z upływem lat coraz więcej ludzi przyznawało się do swojego wyznania. Stowarzyszenia katolickie i „Caritas” były żywo obecne w diecezji od początku (1945), ale miały krótki żywot, zniszczone podobnie jak w całym kraju. W kolejnym rozdziale omówiono laicyzację życia społecznego. R E C E N Z J E Na początku Autor nawią zuje do skomplikowanych stosunków państwo - Kościół, ukazując przykłady podburzania wier 338 RECENZJE nych przeciwko hierarchii oraz wprowadzania restrykcji podatkowych, wydawniczych itd. Dlacze go więcej miejsca poświęcono ks. Stefanowi Biskupskiemu, który nie był prześladowany przez władze państwowe, niż pierwszym rządcom administracji apostolskiej, pozbawionym urzędu i usuniętym z diecezji (ks. Teodor Bensch, ks. Wojciech Zink)? W paragrafie Prześladowanie księży czytelnik ma okazję dowiedzieć się, jak okrutny los spotkał duchowieństwo miejscowe i napływowe. Ponadto znajdujemy dużo ciekawych informacji dotyczących sposobu ingerencji państwa w wewnętrzne sprawy Kościoła (szykanowanie i usuwanie z parafii niewygodnych księży, zatwierdzanie duszpasterzy i próby ich zastraszania), ale są też i przykłady świadczące 0 niezbyt chlubnym zachowywaniu się niektórych duchownych (s. 55). Niezwykle interesujący 1 dość odważny jest punkt poświęcony „Księżom Patriotom” , a więc tym, którzy z różnych względów podporządkowali się polityce państwa socjalistycznego, a tym samym wykazali niechęć do Stolicy Apostolskiej i Episkopatu Polski. W yszczególniono organizacje („Caritas” , ZBoWiD, PAX, Komisja Duchownych i Świeckich Działaczy przy W ojewódzkim Komitecie Frontu Naro dowego) grupujące d u c h o w n y c h „postępowych” , operując nawet konkretnymi liczbami a także akcjami, do których byli oni angażowani. Tak duża liczba duchownych (20-25% ) zaangażowa nych w różnym stopniu po stronie „reżim u” świadczy o tym, że na Ziemie Północne nie przybywali najbardziej wartościowi księża. nych przeciwko hierarchii oraz wprowadzania restrykcji podatkowych, wydawniczych itd. Dlacze go więcej miejsca poświęcono ks. Stefanowi Biskupskiemu, który nie był prześladowany przez władze państwowe, niż pierwszym rządcom administracji apostolskiej, pozbawionym urzędu i usuniętym z diecezji (ks. Teodor Bensch, ks. Wojciech Zink)? W paragrafie Prześladowanie księży czytelnik ma okazję dowiedzieć się, jak okrutny los spotkał duchowieństwo miejscowe i napływowe. Ponadto znajdujemy dużo ciekawych informacji dotyczących sposobu ingerencji państwa w wewnętrzne sprawy Kościoła (szykanowanie i usuwanie z parafii niewygodnych księży, zatwierdzanie duszpasterzy i próby ich zastraszania), ale są też i przykłady świadczące 0 niezbyt chlubnym zachowywaniu się niektórych duchownych (s. 55). Niezwykle interesujący 1 dość odważny jest punkt poświęcony „Księżom Patriotom” , a więc tym, którzy z różnych względów podporządkowali się polityce państwa socjalistycznego, a tym samym wykazali niechęć do Stolicy Apostolskiej i Episkopatu Polski. W yszczególniono organizacje („Caritas” , ZBoWiD, PAX, Komisja Duchownych i Świeckich Działaczy przy W ojewódzkim Komitecie Frontu Naro dowego) grupujące d u c h o w n y c h „postępowych” , operując nawet konkretnymi liczbami a także akcjami, do których byli oni angażowani. R E C E N Z J E Punkt pierwszy o usuwaniu religii ze szkół - powinien być inaczej zatytułowany, ponieważ Autor zajmuje się także katechizacją dzieci i młodzieży prowadzoną przy parafiach. Co więcej, połowa opracowania 339 RECENZJE dotyczy właśnie wysiłków władz skierowanych przeciwko punktom katechetycznym. Dalsze trzy paragrafy poświęcono sprawom likwidacji kaplic w szpitalach, publikacji kościelnych (cenzura), krzyży, kaplic przydrożnych, cmentarzy katolickich (upaństwowienie). Niestety, tutaj Autor przeoczył likwidację Niższych Seminariów Duchownych, o których istnieniu wspominał wcześniej (s. 17). Piąty rozdział ukazuje napięcia w polityce wyznaniowej. Pierwsze dwa krótkie paragrafy - List papieża Piusa XII, Orędzie do biskupów niemieckich, Dwudziestolecie Kościoła na Ziemiach Zachodnich i „Sacrum Poloniae M illennium” - wyraźnie wykraczają poza granice diecezji i jest to w zasadzie zrozumiałe. Razi tutaj brak precyzji w podawaniu dat i tytułów np, nieszpory w Gnieźnie 16 a nie 15 kwietnia 1966, przewodniczył metropolita Karol W ojtyła, (kardynałem został rok później), zaś Marian Spychalski był już od 1963 r. marszałkiem (s. 128). Natomiast dość szczegółowo i interesująco przedstawił Autor wysiłki i inicjatywy władz państwo wych zmierzające do torpedowania uroczystości diecezjalnych (koronacje obrazów NM P w Giet rzwałdzie i Świętej Lipce). Nasuwa się pytanie, ile te atrakcyjne imprezy sportowe, rozrywkowe, kulturalne kosztowały i z jakich funduszów były pokrywane? W punkcie ostatnim - inne uroczystości kościelne - zaprezentowano inwigilację i trudności stawiane księżom w wykonywa niu ich pracy duszpasterskiej, np. odpusty, rekolekcje, procesje, pielgrzymki autokarowe itd. W tym paragrafie powinny być odnotowane także akcje o donioślejszym znaczeniu, jak: pereg rynacja obrazu po parafiach, piesze pielgrzymki (w tym na Jasną Górę). Trudno zrozumieć intencje Autora, dlaczego pominął także obchody kościelne Roku Kopernikowskiego, które były we Fromborku (17 VI 1973) wydarzeniem w skali międzynarodowej, i to w tamtym okresie. Problematyce materialnej Kościoła poświęcono najobszerniejszy, szósty rozdział. Z powodu ogromnych utrudnień budowy i remontów na Ziemiach Odzyskanych diecezja warmińska nie mogła mieć większych sukcesów na tym polu pracy. Z treści wynika, jakby kościół filialny w Dzietrzychowie był ważniejszy od kościoła św. Katarzyny (katedry) w Braniewie. Co więcej, brak nawet informacji, czy w ogóle rozpoczęto odbudowę pierwszej katedry. Paragraf „świątynie poewangełickie” powinien zostać zmieniony na: „Proces przejmowania obiektów poewangelic- kich” , bo przecież Kościół katolicki zagospodarowywał nie tylko same świątynie. Bardziej należało przy tym wyszczególnić sposoby uzyskiwania tych budynków: przyznane przez władze państwowe, spontanicznie zajęte przez ludzi, uzyskane drogą zamiany lub wykupione przez kurię. Szczególnie ten ostatni sposób został słabo zauważony (przypis 137, s. R E C E N Z J E 16, kupiono jeden kościół), a przecież była to udana akcja związana z dużym wysiłkiem finansowym całej diecezji. Kolejne dwa paragrafy - Zabór mienia kościelnego oraz podatki i czynsze oraz ich egzekucje administracyjne - opracowano w oparciu o źródła kościelne i państwowe. Być może zagadnienia te byłyby bardziej zrozumiałe, gdyby umieszczono dodatkowo osobny punkt omawiający pod stawy prawne własności Kościoła katolickiego na Warmii i Mazurach. y p g Ostatni rozdział przeznaczono innym Kościołom: ewangelicko-augsburskiemu, metodystycz- nemu, prawosławnemu i polskokatolickiemu. Piętą Achillesową obniżającą wartość książki są zbędne powtórzenia i przedruki treści zamieszczonych przez Autora w innych publikacjach. Czy konieczne było kopiowanie artykułu Ks. prof. S. Biskupski - objęcie urzędu wikariusza kapitulnego drukowanego w „Studiach W armińskich” , 32, 1995, albo też całego paragrafu Represje ze strony państwa wchodzącego w skład książki pt. Wyższe Seminarium Duchowne ,,H osianum ", Olsztyn 1995, s. 143-148? Czytelnik jest wprost zaskakiwany ilością powtórzeń; „..jednak doszło do podpisania porozumie nia między państwem i Kościołem. Nieco później został utworzony Urząd do Spraw W yznań” (s. 35); „Ten ostatni (UdSW) powstał wkrótce po podpisaniu porozumienia między Kościołem a państwem” (s. 19). Co więcej, podobne przykłady znajdujemy na tej samej stronicy (133), na której kończył się jeden paragraf a zaczynał następny; pielgrzymki, pojazdy, tablice rejestracyjne. j y j p g y p y p g y p j y j yj Omawiana publikacja nie jest wolna od pewnych nieścisłości i omyłek. Błędnie podano nazwisko historyka diecezji gdańskiej, powinno być „Baciński” , a nie „Buciński” (s. 12, 233). 340 RECENZJE Koło Lublina znajduje się miejscowość „Zemborzyce” a nie „Zembrzyce” (s. 52). W wykazie skrótów brakuje PWRN, podczas gdy umieszczono tak znane w publicystyce, jak PGR czy PZU. s ótów b a uje W N, podc as gdy u es c o o ta a e w pub cystyce, ja G c y U. Wydaje się, że Autor podjął się zbyt trudnego zadania do wykonania. Może korzystniej byłoby zawęzić problematykę do zagadnień jeszcze nie opracowanych i nie publikowanych i spróbować je wszechstronnie zanalizować. Najmocniejszą stroną opracowania są niewątpliwie źródła, które wykorzystał ks. Kopiczko. Do większości z nich na pewno po raz pierwszy dotarł badacz z kręgów kościelnych. Niewątpliwie solidną bibliografię autorów polskich i niemieckich, dotyczącą diecezji warmińskiej i województwa olsztyńskiego oraz Kościoła w PRL, można z pewnością uzupełnić pozycją - Państwowe prawo wyznaniowe PRL. Wybór tekstów źródłowych. Zebrał ks. M. Fąka, W arszawa 1978. R E C E N Z J E Supernaturalizm teologiczny, który nadprzyrodzoność ukazywał 341 RECENZJE jako nadbudowany dodatek do natury, w pewien sposób sam się przyczynił d o ' tego, że zwolennicy oświecenia wykazywali nieprzydatność tego, co nadprzyrodzone lub wręcz widzieli w nadprzyrodzoności przeszkodę na drodze rozwoju. W deistyczny światopogląd wpisany jest jednak pewien paradoks, który zdaje się polegać na tym, że podkreślając doświadczenie jako jedyne źródło wiedzy, jednocześnie neguje doświadczenie Boga, jakie stoi u podstaw wiary biblijnej. Objawienie przekazane w Biblii jawi się przecież jako wydarzenie i doświadczenie - jako słowo, które nadaje i zmienia bieg historii, ludu i poszczególnych osób. Szkoda, że owa zależność między historią, przez którą doświadcza się Boga objawiającego, a artykułowaną wiarą, nie została szerzej przedstawiona i zilustrowana. Rozdział trzeci koncentruje się na Jezusie Chrystusie, który stanowi szczyt objawienia się Boga w historii. Chrześcijaństwo głosząc Chrystusa spotykało się ze sprzeciwem. O ile naturalny wydaje się sprzeciw wychodzący ze świata pogańskiego, o tyle początki postawy krytyczno- liberalnej domagają się szczególnego omówienia. Ks. Kalisz wskazuje na protestantyzm, który odrzucając tradycję pozbawił biblię oparcia i uzasadnienia jej autorytetu. Autor prezentuje poglądy całej plejady oświeconych umysłów na temat historyczności Jezusa (H. S. Reimarus, E. G. Paulus, D .F. Straus, F. C. Baur, B. Bauer, oraz marksistów, J. Keller, Z. Poniatowski, J. Wipper) - do kompletu brakuje tylko E. Renana, który swoim Życiem Jezusa wywarł przecież ogromny wpływ na postawy religijne przełomu XIX i XX wieku. Krytyka liberalna odrzucając tradycję chrześcijańską, a wraz z nią historyczny obraz Chrystusa, na podstawie różnych apriory cznie przyjętych założeń historycznych, filozoficznych i socjologicznych zaczęła tworzyć różnego rodzaju teorie często bardziej cudowne niż prezentuje to samo chrześcijaństwo. Na podstawie teorii historii proponowanej przez H. Lacordaire uzasadnia, że „wydarzenie Jezus” należy do historii, gdyż dokumenty stwierdzające ten fakt mają charakter publiczny, jak również samo wydarzenie ma charakter publiczny i stanowi element historii powszechnej. Przyjęcie wiary w Jezusa jako Chrystusa nie może się dokonać na drodze oczywistości naukowej. Poznanie tej prawdy dokonuje się na drodze wiary, rozumianej jako zaufanie i powie rzenie się Bogu, a więc na tej samej drodze, na jakiej Apostołowie doświadczyli Jezusa jako Boga. Pytanie zasadnicze więc nie dotyczy dowodu naukowego mającego stwierdzić bóstwo Jezusa, ale racjonalności wyboru wiary w Jezusa jako Boga. W tym celu należałoby przyjrzeć się doświadczeniu Apostołów, którzy zachowali w tym względzie, jak to określa ks. Kulisz, „prawo autorskie” . R E C E N Z J E Na zakończenie trzeba stwierdzić, że recenzowana praca, wnosząc wiele interesującego materiału nie wyczerpuje w pełni zagadnienia. Może już w niedługim czasie poznamy bardziej dokładne opracowanie tej wyjątkowej diecezji w Polsce, której nazwa wiąże się ściśle z Ziemią i ludźmi na niej żyjącymi, a nie z siedzibą biskupa. ks. Jan Pietrzykowski SDB Ks. Józef Kulisz SJ, W kręgu zagadnień i problemów teologii fundamentalnej, Bobolanum 1994, ss. 223. Dziekan Papieskiego Wydziału Teologicznego w W arszawie i wykładowca teologii fun damentalnej na sekcji Bobolanum, oraz historii dogmatów na W ydziale Teologicznym ATK w Warszawie, oddał w ręce czytelników książkę, w której podejmuje istotne problemy dla życia chrześcijańskiego, są nimi fakt religii, objawienia oraz historyczność Jezusa. Książka składa się z autorskiego wprowadzenia, które jest zwięzłą ilustracją zawartości, pięciu rozdziałów, posłowia, streszczenia w języku niemieckim oraz indeksu nazwisk, Poszczególne rozdziały powstawały w latach osiemdziesiątych w dyskusji i polemice z religioznawstwem marksistowskim w formie małych książeczek: Jezus w świetle historii, oraz Bóg w Jezusie w 1989 r., Spór o religię sporem o człowieka w 1992 r. i Tęsknota za nieskończonym w 1993 r. Jak zaznacza Autor, rozdziały zostały przepracowane i uzupełnione. Możemy więc się spodziewać, że książka stanowi jednolite pod względem logicznym i myślowym dzieło. Marksistowscy religioznawcy nie są jedynymi adwersarzami, z jakim i podejmuje ks. Kulisz polemikę, raczej zamykają oni tylko całą plejadę krytyków, którzy od Laktancjusza i Celsusa w starożytności poprzez racjonalistów i modernistów w czasach nowożytnych stanowią doskonałe i zawsze aktualne świadectwo sprzeciwu, z jakim spotyka się chrześcijaństwo. Rozdział pierwszy zarysowuje podstawowy problem ludzkiej egzystencji, który wyraża się w dychotomii skończoności doświadczanej ostatecznie w śmierci i nieprzejednanego pragnienia nieśmiertelności. Człowiek szuka nieustannie przezwyciężenia owego rozdarcia i samotności przez twórczość techniczną i artystyczną, a przede wszystkim przez odniesienia międzyludzkie. Od krycie niewystarczalności tych relacji dla usprawiedliwienia swojego istnienia otwiera człowieka na transcendencję. „Doświadczenie religijne jest zatem przeżyciem totalnego kryzysu swej egzystencji przez człowieka i zarazem jego wzorcowym rozwiązaniem” (s. 27). Autor zwraca uwagę na historyczność i powszechność tego doświadczenia. Opierając się na danych religio znawczych (M. Eliade), psychologicznych (E. Fromm, V. Franki) i filozoficznych (Teilhard de Chardin) podejmuje polemikę z religioznawstwem redukcjonistycznym, wskazując, że religia ma swoje źródło w samej naturze człowieka. W problematykę rozdziału drugiego Autor wprowadza nas przytoczeniem poglądów deistów negujących możliwość faktu objawienia, wskazując również na pewien kontekst teologiczny negacji tego, co nadprzyrodzone. R E C E N Z J E Wiary Apostołów nie da się wytłumaczyć ani zniewalającą oczywistością Synostwa Bożego Jezusa, ani tym, że Jezus został przez pierwotną wspólnotę ubrany w szaty bóstwa. W iarygodności tego najbardziej negowanego dogmatu wiary chrześcijańskiej należy szukać w śro dowisku życiowym, tak wewnętrznym jak i zewnętrznym wspólnoty przedpaschalnej. Apostołowie byli uwarunkowani od wewnątrz powołaniem z jakim zwrócił się do nich Jezus, wspólnotą, w której wzrastał ich szacunek, podziw i zrozumienie. „Dzięki temu możemy dotrzeć nie tylko do nauk Jezusa, ale też do niektórych Jego słów - logiów, które zapadły głęboko w serca i pamięć Apostołów i były później ze czcią powtarzane” (s. 132). Uwarunkowaniem zewnętrznym była misja, jaką Jezus powierzył Apostołom, której do chowali wierności i dali temu świadectwo W tym celu ks. Kulisz zwraca naszą uwagę na słownictwo i postawy pierwotnego Kościoła. Takie pojęcia, jak: „otrzymać” , „przekazać” , „świadek” , „apostoł” dobitnie wyrażają świadomość pierwotnego Kościoła, której streszczeniem są słowa św. Piotra: „Nie za wymyślonymi bowiem mitami postępowaliśmy wtedy, gdy daliśmy wam poznać moc i przyjaźń Pana Jezusa Chrystusa, ale nauczaliśmy jako naoczni świadkowie Jego wielkości” (2 P 1, 16). Świadectwo Apostołów daje nam dostęp do samoświadomości Jezusa, którą wyrażają tytuły chrystologiczne („M esjasz” , „Syn Ojca” i „Syn Człowieczy” ). Szczególnymi znakami boskiej świadomości Jezusa są również cuda. g y j Rozdział piąty stanowi niejako odpowiedź na problem postawiony w rozdziale pierwszym. W historię człowieka zmagającego się ze swoją skończonością wkracza Chrystus zmartwych wstały. Oto odpowiedź na tajemnicę, którą człowiek jest sam dla siebie. W szystkie religie 342 RECENZJE i kultury ukazują, że człowiek od samych początków był przekonany, że „nie cały umrze” . Tęsknota za nieśmiertelnością nie była negowana nawet przez filozofów marksistowskich, znaj dowała jednak odpowiedź w pesymistycznej wizji np.: w „tragicznej koncepcji życia” B. Suchodolskiego. g W świecie biblijnym, wiara w Boga nie pozwalała na pogodzenie się z tragiczną wizją losów człowieka. W iara złożona w Jezusie Chrystusie przynosi ostateczne rozwiązanie odwiecz nego dylematu. W iara ta nie była prostym następstwem doświadczenia pustego grobu. Dopiero objawienie Jezusa - nie bez trudu przyjmowane - wyjaśniło znaczenie wielkanocnego doświad czenia i zrodziło pełną wiary postawę świadectwa. Kościół żyje od tej pory nie tyle przypomina niem tamtego wydarzenia, „przeciwnie Kościół jest czasem powolnego realizowania przyszłości, która zaistniała już w zmartwychwstałym Jezusie.., ” (s. 202). Po prezentacji struktury i treści pozostaje nam dokonać pewnej krytycznej oceny książki ks. J. Kalisza. R E C E N Z J E We wstępie znajdujemy zastrzeżenie, że książka, której tytuł mógłby sugerować, że mamy tu do czynienia z podręcznikiem teologii fundamentalnej, podręcznikiem nie jest. Już pobieżne przeglądnięcie spisu treści uwidacznia, że Autor stawia sobie inny cel, aniżeli publikację podręcznika. Dopiero jednak lektura książki daje pojęcie o jej specyfice. Jest to dzieło o charak terze apologijnym i jeśli można tak określić światopoglądowym. Charakter jej argumentacji i poruszana problematyka jest uwarunkowana polemicznie i skupia się na kwestiach fundamental nych. Dotyczy otwarcia człowieka na transcendencję i spełnienia religijnych pragnień w Jezusie Chrystusie. Więcej o charakterze książki i jej adresatach możemy się dowiedzieć dopiero z posłowia. Ks. Kulisz pisze, że: „człowiek, każdy człowiek, spotykając Boga w Jezusie Chrystusie, we wspólnocie Kościoła szuka uzasadnienia dla swojej wiary. Pragnie znaleźć racje, które uczyniły by rozumnym jego całkowite zawierzenie Jezusowi. Książka ta chciałaby pomóc w poszukiwaniu rozumowych i przekonujących racji, że tajemnica Chrystusa zmartwychwstałego, obecna w Koś ciele, nie tylko wyjaśnia sens ludzkiej egzystencji, ale ją realizuje i dopełnia” (s. 211). Niedopatrzeniem wydaje się, że Autor omawiając problemy religioznawcze, a zwłaszcza uzasadniając powszechność faktu religii, pomija dorobek W. Smitha. Chociaż dzisiaj hipoteza o praobjawieniu napotyka wiele trudności, to jednak wywarła ona ogromny wpływ. W śród wielu prezentowanych opinii ta wymagałaby przypomnienia Zastrzeżenie budzi interpretacja kantowskiego rozumienia religii (s. 16-17). Jego teoria poznania doprowadziła w konsekwencji do krytyki dowodów na istnienie Boga. Faktycznie religię z dziedziny poznania przeniósł w dziedzinę moralności. Nie było to jednak spowodowane odrzuceniem istnienia Boga, ale krytyką czystego rozumu, przez którą wykazywał, że prawdy religijne, jak: Bóg, dusza, nie są bezpośrednio poznawalne, lecz stanowią horyzont naszego poznania. Skoro więc rozum teoretyczny nie może poznać Boga, to staje się On postulatem rozumu praktycznego. Rzeczywiście Kant w ten sposób redukuje całkowicie religię do moralno ści1. Moralność zatem postuluje religijność, a nie odwrotnie, jak to twierdzi ks. Kulisz, jakoby rozwój moralności według Kanta mial prowadzić do zanikania religii, Tylko w pewnym sensie można powiedzieć, że moralność w ujęciu Kanta może istnieć bez religii, a mianowicie wówczas, gdy mówi, że powinność moralna jest bezwarunkowa i nawet Bóg nie jest potrzebny dla jej wzmocnienia. W skazuje w ten sposób, że to nie moralność ma źródło w religii, ale religia ma swoje źródło w doświadczeniu imperatywu moralnego, ponieważ nie da się w żaden sposób uzasadnić powinności moralnej na drodze empirycznej. Kant stwierdza, że bez religii człowiek nie może się obejść. 1 Por. Z . Z d y b i c k a , Człowiek i religia, Lublin 1984, s. 53. 2 Por. I. K a n t , Religia w obrębie samego rozumu, Kraków 1993, s. 11, 12. 2 Por. I. K a n t , Religia w obrębie samego rozumu, Kraków 1993, s. 11, 12. 1 Por. Z . Z d y b i c k a , Człowiek i religia, Lublin 1984, s. 53. R E C E N Z J E Podkreśla to, szukając przezwyciężenia antynomii pomiędzy moralnością a naturalnym pragnieniem szczęścia i widzi taką możliwość w istnieniu różniącej się od natury przyczyny, co jest jednocześnie postulatem za istnieniem Boga2. 343 RECENZJE Autor, mimo poprawek, które musiał nanieść, aby zestawić w jedną całość dotychczas oddzielnie wydane książeczki, nie uchronił się od zbędnych powtórzeń. Są to: powtórzenia w cytowaniu (na s. 73 i 122 J, Keller, Chrześcijaństwo pierwotne, s. 578-579), dwa razy pojawia się zagadnienie świadectwa apostolskiego (s. 90-91 i 135-137, podpunkt 3a rozdziału V jest streszczeniem podpunktu lb rozdziału III). Książka ks. J. Kalisza W kręgu zagadnień i problemów teologii fundamentalnej jest godna polecenia szerokiej rzeszy czytelników, dla których jest adresowana. Może stać się cenną lekturą dla każdego człowieka szukającego uzasadnienia swojej wiary. Przejrzysty styl języka i schemat uwzględniający historyczny i naturalny tok stawiania problemu dotyczącego ludzkiej egzystencji i Boga, jest najlepszym zaproszeniem do podjęcia tej lektury. Autor bez zbytecznego wnikania w zagadnienia czysto akademickie przekazuje czytel nikowi pomoc w odszukaniu odpowiedzi na podstawowe pytania, wskazując na zgodność Ob jawienia z naturalnymi dążeniami człowieka. ks. M ichał Rychert SDB Alessandro M an et i, Vivere insieme, Edizioni Dehoniane, Bologna 1991, ss. 173. Minęło przeszło 90 lat od czasu, kiedy Ch. Cooley w swojej książce Social Organization zwrócił uwagę na istnienie i funkcjonowanie w ramach szerszych struktur społecznych grup podstawowych. Od tego czasu zagadnienie to stało się częścią socjologicznej teorii większości socjologów. Nawet ci, którzy z racji odmiennych zainteresowań kładli nacisk na inne wymiary życia społecznego, nigdy nie kwestionowali Cooleyowskiego odkrycia. Życie bowiem i najprost sza obserwacja pozytywne weryfikowały teorię grup podstawowych. Natomiast zadanie, jakie stawiało sobie wielu socjologów, szło raczej w kierunku coraz wszechstronniejszego dokumen towania tej teorii i wskazywania roli grup podstawowych, tak dla jej członków, jak i nawet dla większych tworów społecznych. Społeczny charakter człowieka uwidacznia się w jego zależności od innych w fakcie zrodzenia, opieki, potrzeby współpracy, ale także osobowego wzrostu i woluntarystycznych zaangażowań. Środowisko grup podstawowych, bardziej czy mniej bezpośrednio zahacza o nie wszystkie. Ich obecność jednak najbardziej uwidacznia się tam, gdzie ludzi jednoczą wspólne wartości, cele, a nawet metody działań. Grupy podstawowe są takim forum par excellence. To, co indywidualne, nabiera tu wymiaru grupowego, a grupowe jest internalizowane przez jednostkę. Osobiste cele członków nabierają charakteru grupowego, a cele grupy stają się celami poszczegól nych jednostek. Jednostka w grupie podstawowej stanowi znacznie coś więcej niż jedno z jej ogniw; bez niej grupa nie byłaby tym, czym jest. R E C E N Z J E I na odwrót, jednostka ulega wpływowi grupy, z nią się identyfikuje i jest przez nią socjalizowana. Jako bezpośrednie środowisko społecznego życia człowieka, grupa podstawowa jest płaszczyzną, gdzie jednostkowe spotyka się ze społecz nym, a „psychologiczne” z „socjologicznym ” . Vivere insieme A. M anentiego podejmuje te aspekty w sposób przystępny, ale jednocześnie precyzyjny; teoretyczny, ale i poparty przykładami konkretnymi z życia. Jego wizja grup podstawowych ma charakter wybitnie dynamiczny. Świadome, dobrowolne i całościowe całą swoją osobowością zaangażowanie się jednostek daje początek nowej rzeczywistości. Z kolei w jej ramach i na jej forum rozgrywa się życie członków grupy. Tu „ja” i „m y” przenikają się dogłębnie, nie okradając jedno drugiego z właściwej sobie podmiotowości. Książka składa się z 11 rozdziałów. Trzy pierwsze mają charakter wprowadzający. Rozdział pierwszy przybliża pojęcie grupy podstawowej; rozdział drugi podkreśla odniesienie do wartości absolutnych wspólnot religijnych, a rozdział trzeci wyjaśnia podstawowe założenia teoretyczne. Osiem następnych rozdziałów dotyczy dynamiki grupy, konfliktów wspólnotowych - rozdziały: 344 RECENZJE czwarty i piąty, wewnątrzgrupowe porozumiewanie się - rozdział szósty, dojrzałe relacje - roz dział siódmy, tożsamość osobowa i interakcyjna - rozdział ósmy, formowanie się grupy - roz dział dziewiąty, fazy umacniania się grupy - rozdział dziesiąty oraz mediacyjna rola grupy - rozdział jedenasty. W reszcie apendyks przedstawia różne sposoby funkcjonowania grup. A. Manenti jest księdzem, a więc teologiem, ale ma też wykształcenie psychologi wyniesione z gregoriańskiej szkoły L. Rulli. Obydwie te dziedziny wiedzy spotkały się w analizie religijnych wspólnot, będących dla autora szczególnym przedmiotem zainteresowania. Religijna wspólnota jest wspólnotą bazującą na wartościach; sednem zaś tego systemu wartości jest aktualizacja Królestwa Bożego. To wyartykułowanie i wyakcentowanie wartości nie tylko nie koliduje z tym, co społeczne, ale go zakłada i uzupełnia. Zycie we wspólnocie religijnej bowiem nie jest celem samym w sobie; jest drogą, sposobem realizowania wartości transcendentalnych. Autor jest świadomy tego, że liczne aspekty życia jednostek i grupy mogą powodować, iż wartości przyjęte na płaszczyźnie teoretycznej nie znajdują do końca swego praktycznego odzwierciedlenia w konkretnym życiu grupy; ideał odbiega znacząco od rzeczywistości. Dzieje się tak z dwóch powodów: pierwszym jest to, iż niektórzy zapominają dlaczego żyją we wspólnocie; drugim - nieznajomość zasad życia wspólnotowego. Pierwszy problem, zdaniem autora Vivere insieme powinien być rozwiązywany na drodze nigdy nie kończącej się refleksji członków grupy: dlaczego tworzymy tę konkretną wspólnotę; drugi - na drodze swoistej dynamiki grupy, której „krwiobiegiem” jest system komunikowania się wewnątrzgrupowego. R E C E N Z J E Braki na tym poziomie prowadzą niechybnie do wewnętrznego zamknięcia się jednostek i obniżenia jakości grupy albo nawet jej całkowitego rozpadu. W arunkiem, by jakaś grupa mogła być znakiem, jest jej czytelność przez innych. To zaś wymaga ciągłego aggiornamento w dawaniu świadectwa tak, by być czytelnym znakiem oraz wyzbywania się postawy samozadowolenia: osiągnęliśmy to, co zamierzaliśmy; rozwiązaliśmy wszystkie problemy! Teraz zaczyna się rola innych: patrzeć i naśladować. Dużą rolę w tym względzie mają do spełnienia ci, którzy bardziej niż inni dostrzegają potrzebę dostosowania się do nowych oczekiwań otoczenia. Ich rola polega na prowokowaniu, stawianiu pytań, zmuszaniu do zastanowienia się, odrzuceniu postawy samozadowolenia i poczucia bycia nieomylnym. W tej roli „budzenia” są niepopularni, ale dla grupy zbawienni. Wspólnota religijna stawiająca sobie ciągle pytania dotyczące wartości, celów i środków, ma szanse bycia dla Królestwa Bożego, ma szanse osiągnięcia zbawiennej dynamiki, która umożliwia duchowy wzrost jej członków. Żeby wzrastać nie wystarczy być razem. Trzeba zdawać sobie sprawę z tego, dlaczego jest się razem i wiedzieć, jak to robić. To zaś możliwe jest do osiągnięcia na drodze usilnej wspólnotowej refleksji, mającej na celu dynamizację poszczególnych jej członków, jak i samej grupy Oto kwintesencja książki Vivere insieme. Jej lektura wydaje się być szczególnie pożyteczną dla liderów wszelkiego rodzaju grup. ks. Władysław Majkowski ks. Władysław Majkowski Monika M a z u r c z a k , Święty Mikołaj. Kim jest, skąd przychodzi i dlaczego jest taki dobiy, Warszawa, Jacek Santorski & Co 1995, ss. 141. Pod koniec ubiegłego roku w wydawnictwie Jacka Santorskiego ukazała się w formie książki praca magisterska Moniki Mazurczak. Warto zaznaczy, że powyższa praca została napisana z zakresu pedagogiki. Chociaż tematyka dotycząca św. Mikołaja, jak również sama jego postać, nie jest dla nas czymś obcym, jednak sam tytuł książki zachęca nas do zapoznania się z jej treścią. Treść książki autorka przedstawiła w dziewięciu rozdziałach, które następnie podzieliła na cztery części. Pierwsza z nich zajmuje się życiem i dziejami św. Mikołaja opisanym przez historię, legendy i mity. 345 RECENZJE W naszych czasach prawie wcale nie modlimy się do tego świętego. Nie prosimy o miłość w małżeństwie, szczęśliwy poród czy o opiekę nad dziećmi. Obecnie święty ten, jako dostojny staruszek, bardzo często stoi w witrynach sklepowych i reklamuje nowe samochody, coca-colę, czy inne najnowsze osiągnięcia techniki. Raz w roku, w okresie Bożego Narodzenia rozdaje prezenty. R E C E N Z J E Na przestrzeni zaś wieków był patronem od zwykłych i niezwykłych spraw i był znany z tego, że jest zawsze blisko potrzebującego człowieka. Jego postać potrafiła także godzić sprzeczności. Był opiekunem biednych i patronem złodziejaszków. Autorka idąc za holenderskim badaczem De Groot’em, przedstawia cztery dziedziny, którym patronował omawiany święty. Pierwsza grupa problemów to płodność i rodzicielstwo. Święty ten opiekował się osobami zamężnymi jak i niezamężnymi, dziećmi od poczęcia aż do osiągnięcia przez nie dojrzałości. Druga grupa, która czuła się szczególnie uprzywilejowana opieką św. Mikołaja, to byli ludzie biedni. Był patronem także bogatych. Czuwał on nad dobrami, przynosił powodzenie materialne, przywracał utracone dobra. Zaliczani do tej grupy społecznej złodzieje i kłusownicy obrali sobie za patrona właśnie św. Mikołaja. Opiekował się ponadto przewoźnikami i budowniczymi mostów. Za swego patrona uważali go piekarze, krawcy, tkacze, szewcy, stolarze, malarze drzeworytnicy i kupcy handlujący ziołami, olejem i lekarstwami. Swoim patronem obrali go także literaci, księża i mnisi. Autorka próbuje opracować, i robi to w dość interesujący sposób, curriculum vitae św. Mikołaja. Faktów jest mało. Z drugiej zaś strony, dzieje tej osoby obrosły w tak liczne legendy i opowiadania, że trudno dziś odróżnić wydarzenia rzeczywiste, od tych stworzonych przez czas. Sama nawet historyczność jest poddawana pod wątpliwość. Wielu historyków twierdzi, że to wszystko, co napisano o św. M ikołaju, jest wytworem legend i przekazów historycznych dotyczących innych postaci, jak na przykład Mikołaja, opata klasztoru w Syjonie. Musimy przyznać, że najwięcej informacji o tej postaci, dostarczają jednak legendy. I choć ich wiarygodność jest znikoma, pokazują one, kim była postać czczona przez wielu ludzi. Legendy mówią, że urodził się w Patarze, w rodzinie bogatej i pobożnej. Już w dzieciństwie odznaczał się powagą, na przykład w dni postne pozwalał się karmić tylko raz dziennie, wiele czasu spędzał na czytaniu Pisma Świętego. Biskupem Miry został przez przypadek. Pewnego dnia udał się do kościoła, nie wiedząc nic o uchwale biskupów, którzy uradzili dać mitrę pierwszemu spotkanemu człowiekowi w kościele. Za panowania cesarzów Dioklecjana i M aksymiana został uwięziony. Odzyskał wolność dzięki edyktowi mediolańskiemu. W krótce potem ocalił życie trzem rzymskim żołnierzom, oskarżonym o rabunek. Podczas Soboru Nicejskiego grupa żeglarzy będąca w niebezpieczeństwie, wzywała na pomoc sławnego biskupa. Św. Mikołaj natychmiast pojawił się przy nich i wybawił ich z opresji. Są to najstarsze legendy o św. Mikołaju, pochodzą bowiem z V -V III wieku. Jedną z najstarszych legend i bardziej znanych jest udzielenie pomocy, jakiej udzielił św. Mikołaj ojcu trzech córek. R E C E N Z J E Marynarze holenderscy przyczynili się do poznania osoby św. M ikołaja przez Amerykę. W yob rażenie o nim zmieniało się bardzo szybko w „nowym świecie” . Przyczynił się do tego najbardziej W ashington Irving na początku XIX wieku. Uczynił on ze św. Mikołaja postać pełną energii, życia i radości. Ponadto wyposażył go w nowy atrybut, fajkę. Amerykanie przenieśli też jego dzień wizyt, tj. 6 grudnia, na Święta Bożego Narodzenia. W latach dwudziestych naszego stulecia w Ameryce powstał nowy zawód. Santa Claus wyszedł na ulice, był widoczny i każdy mógł z nim porozmawiać. Jednak wygląd zewnętrzny świętych Mikołajów życzył sobie wiele do życzenia. Problemem tym zajął się amerykański żołnierz Jim Yelling, który opracował szereg zasad, które miały być przestrzegane przez absol wentów „szkół dla M ikołajów” . We Francji pod koniec zeszłego wieku, konkurencję św. M ikołajowi zrobił „ojciec Noel” , odrzucony jednak definitywnie w latach powojennych. Tymczasem siedzibę stałego zamieszkania św. Mikołaja zorganizowała Finlandia, Szwecja i Norwegia. Od czasu do czasu odbywają się Światowe Kongresy Świętych Mikołajów, podczas których omawiane są palące problemy doty czące ich działalności. Kolejny rozdział poświęcony jest kultowi św. M ikołaja w Polsce. Ślady kultu tegoż świętego znajdujemy już w XIII wieku w tzw. Kazaniach Świętokrzyskich. Powstaje wiele kościołów pod wezwaniem świętego Mikołaja. Staje się on początkowo patronem pasterzy, chłopów, a następnie opiekunem uczniów i panien. Po II wojnie światowej św. Mikołaj stał się „Dziadkiem M rozem ” , który został importowany ze Związku Radzieckiego. Od lat 90-tych powstaje nowa profesja. M ikołaja można zamówić, Są nimi najczęściej studenci dorabiający na studia. Na terenie Polski powstało szereg akcji, którym patronuje św. Mikołaj. Rozdział czwarty przedstawia wyniki rozmów autorki z dziećmi na temat wyobrażeń św. Mikołaja. Rozmowy, w liczbie kilkudziesięciu, zostały przeprowadzone z dziećmi klas zerowych dwóch przedszkoli i klas pierwszych dwóch szkół warszawskich. Dzieci bardzo różnie pojmują i wyobrażają tę postać. Dla jednych jest człowiekiem, dla innych krasnoludkiem, duchem, ma wspaniałą pamięć i nie zapomina o żadnym dziecku. Dla wielu dzieci Mikołaj potrafi prawie to, co Bóg. W edług niektórych św. Mikołaj jest człowiekiem, żyje między ludźmi i przebiera się, by rozdawać prezenty. M ikołajem może zostać każdy, kto posiada odpowiedni strój. Wiele dzieci uważa, że Mikołaj to człowiek, ale trochę inny, bo długo żyje, okazuje miłość, jest zawsze, jest niewidzialny, jest trochę człowiekiem, trochę duchem, żyje wiecznie. Na pytanie, gdzie mieszka św. Mikołaj, padały różne odpowiedzi. Najczęściej odpowiadano, że miejscem za mieszkania św. R E C E N Z J E Ojciec z braku środków materialnych, nie mógł wydać za mąż swoich córek. Św. Mikołaj dowiedziawszy się o tym, przez trzy kolejne noce wrzucał przez okno domu woreczki ze złotem, które odziedziczył po swoich rodzicach. Za trzecim razem ojciec biednych panien przyłapał dobrodzieja na gorącym uczynku. Ten ostatni prosił o zachowanie tajemnicy. Niektórzy twierdzą, że to właśnie ta legenda tłumaczy najbardziej rolę św. Mikołaja, jaką jest rozdawanie prezentów po kryjomu. Rozdział drugi mówi o szerzeniu się kultu św. Mikołaja. Możemy wyróżnić dwie ścieżki: wschodnią i zachodnią. W schodnia idzie wyraźnie w kierunku sacrum. Postać św. Mikołaja zajmuje w tradycji Wschodu bardzo ważne miejsce. Już w VI wieku zauważa się kult św. Mikołaja. Poświęca się mu nowo wybudowane kościoły, pisze hymny i pieśni. W Mirze powstaje sank tuarium, do którego, udają się liczni pątnicy. W IX wieku jego kult jest zaraz po kulcie Maryi. Poświęca się mu nowo wybudowane kościoły, pisze hymny i pieśni. W Mirze powstaje sank tuarium, do którego, udają się liczni pątnicy. W IX wieku jego kult jest zaraz po kulcie Maryi. Osobną tradycję posiada św. Mikołaj na Rusi. Był tam patronem chroniącym od najazdów tatarskich. W tygodniowym cyklu liturgicznym w Kościele prawosławnym czwartek jest dniem, Osobną tradycję posiada św. Mikołaj na Rusi. Był tam patronem chroniącym od najazdów tatarskich. W tygodniowym cyklu liturgicznym w Kościele prawosławnym czwartek jest dniem, 346 RECENZJE w którym oddaje się cześć apostołom, św. M ikołajowi i innym świętym. W iek XVIII i XIX to rozkwit kultu św. Mikołaja. Zaczyna się wierzyć w niego jak w Boga, a jego imię utożsamiane jest z „ikoną” . W kulturze zachodniej, podobnie jak we wschodniej, kult św. M ikołaja wyszedł z Miry. Jednak tutaj mówi się dużo mniej o nim jako o świętym. Kultura Zachodu idzie w kierunku pro f amon. W iadomość o świętym Mikołaju dotarła do Rzymu dzięki wymianie kulturowej między światem greckim a rzymskim, W XI wieku, po zajęciu Miry przez muzułmanów, przeniesiono jego ciało do Bari. Jego osoba staje się też dość szybko znana w Europie. Od końca XII wieku postać św. Mikołaja zaczyna pojawiać się w teatrze. Staje się on bohaterem krótkich utworów dramatycznych. Powstaje szereg nowych wątków legendarnych. Głównie uważa się św. M ikołaja za dawcę nagród i kar. Okres Reformacji desakralizuje św. Mikołaja. Zostaje on „biskupem szkolnym” (Anglia) lub „futrzanym M ikołajem” (Niemcy). Kult św. M ikołaja uratowała Holandia, która uważała go za patrona swoich statków. RECENZJE RECENZJE Rozdział piąty zawiera wypowiedzi dzieci na temat wyglądu zewnętrznego św. Mikołaja. Ogólnie dzieci mają następujące wyobrażenie: jest tłuściutki, ma czerwoną czapkę, czerwony płaszcz, wysokie kozaki, worek z zabawkami i laskę. Ponadto musi posiadać czapkę z pompo nem, długą i białą brodę. Rozdział szósty porusza problem listów dzieci w Polsce wysyłanych do św. Mikołaja. Autorka przedstawia treść życzeń i ich wygląd zewnętrzny. W oparciu o przestudiowaną korespondencję można powiedzieć, że dla dzieci św. Mikołaj jest bardziej przyjacielem, niż patronem. Jest gościem oczekiwanym, wyrozumiałym i kochanym. W dwóch następnych rozdziałach autorka próbuje dać odpowiedź na pytanie, po co ludziom św. Mikołaj? Jedną z odpowiedzi jest poszukiwanie historycznych związków między obyczajami dawnymi a współczesnymi. W edług niektórych kult św. Mikołaja jest związany z wierzeniami pogańskimi i ich późniejszymi transformacjami. W edług innych postać św. Mikołaja starca, sprzyja umacnianiu autorytetu przodków. W ielu rodziców wykorzystuje tradycję i postać św. Mikołaja - starego Mędrca, jako skuteczną metodę wychowawczą: nagroda i kara. Inną od powiedzią jest kwestia potrzeby u człowieka sacrum (New Age), kontaktu z Absolutem, które człowiek znajduje w obcowaniu z symbolami. Ostatni rozdział zawiera zestawienie „za i przeciw” odnośnie do św. Mikołaja. Jest to próba podsumowania i dania odpowiedzi, jaki wpływ na dzieci, dorosłych i kształtowanie się społeczeń stwa ma postać św. Mikołaja. Książka kończy się jego adresem. Niewątpliwie omawiana tutaj książka dotyczy dość ważnego problemu. Spotykamy się tak często z postacią św. Mikołaja, że często nie zdajemy sobie sprawy z bogatej treści, jaka kryje się za tą postacią. Autorka w sposób dość dokładny studiuje aspekt historyczny dotyczący tej postaci. Umiejętnie stara się rozdzielić historię od legend i przekazów ludowych. Technika pytań zastosowana rozmowie z dziećmi na pewno odzwierciedla nam w sposób klarowny ich widzenia i wyobrażenia o św. Mikołaju. W części końcowej, podsumowującej, autorka próbuje osadzić to wszystko na klasycznej filozofii i na prawdach wiary chrześcijańskiej. Po skończonej lekturze czuje się pewien mały niedosyt. Niektóre rozdziały mogłyby być bardziej rozbudowane. Szczególnie te mówiące o reakcjach dzieci na św. Mikołaja, o wpływach pozytywnych i skutkach wychowawczych, jakie ta postać wywiera na najmłodszych. Książka zasługuje w pełni na uważną lekturę z kilku powodów. Jednym z nich jest fakt, że przecież każdy z nas był małym dzieckiem. Każdy z nas wierzył na swój sposób w św. Mikołaja i niecierpliwie oczekiwał dnia szóstego grudnia lub choinki, pod którą św. Mikołaj zostawiał nam podarki. A czy teraz, już jako dorośli, nie wierzymy choć trochę w św. Mikołaja? R E C E N Z J E Mikołaja jest Biegun Północny, Laponia, Finlandia. Natomiast według niektórych dzieci św. Mikołaj mieszka w pałacu, w chmurach lub w domu z garażem dla reniferów. Gdy cho dzi o podarki, dzieci uważają, że dobrym św. Mikołaj przynosi dobre rzeczy, złym przynosi rózgi. 347 RECENZJE Lektura książki na pewno ubogaci nasze dziecięce wyobrażenia o tej postaci. Z drugiej strony, choć na trochę, przeniesie nas w szczęśliwy świat dzieciństwa, w którym wszystko było piękne i możliwe. Tego życzę. ks. Jan Niewęgłowski SDB Bronisław M i e r z w i ń s k i , Mężczyzna, mąż, ojciec, Wydawnictwo „Posłaniec” , Otwock 1996, ss. 266. Jest prawie truizmem stwierdzenie, że ludzka rzeczywistość ma zawsze wymiar płciowości. Człowiek nie może żyć poza płcią, w oderwaniu od płci. Stąd każda ludzka jednostka jest albo mężczyzną albo kobietą, a nie istotą apłciową. Oznacza to, że płciowość ma dwie swoje formy: kobiecość i męskość. W ypadki „pośrednie” , o ile się zdarzają, należy traktować w kategoriach biologicznych dewiacji. Ludzka płciowość (seksualność) będąc integralną częścią osobowości człowieka - przenika ją całą: uczucia, myśli, działanie... Jest sposobem rozumienia nas samych jako mężczyzny 348 RECENZJE i kobiety, jest sposobem komunikowania się jednostki z innymi. Mając charakter wielowymiaro wy: fizyczny, osobowy, poznawczy, wartościujący, duchowy i społeczny płciowość przenika całą ludzką egzystencję. W następstwie podziału ludzi według płci, mężczyzna jako mężczyzna i kobieta jako kobieta m ają swoje funkcje w poczęciu, noszeniu, wychowaniu dziecka, nawet w życiu ekonomicznym i społecznym. Na szczególną uwagę zasługuje komplementarny charakter ludzkiej płciowości. Płciowość jest nastawiona na dopełnienie w ramach ludzkiej egzystencji. M ężczyzna potrzebuje kobiety, a kobieta potrzebuje mężczyzny. Najważniejszym forum tego dopełnienia się mężczyzny i kobiety jest małżeństwo i rodzina. Na tym forum mężczyzna staje się mężem i ojcem, a kobieta żoną i matką. Książka B. M ierzwińskiego Mężczyzna, mąż, ojciec podejmuje problematykę mężczyzny w tej perspektywie, poszerzając jej wizję o wymiar katolickiej doktryny opartej na Bożym Objawieniu dotyczącej powołania mężczyzny. Cała analiza zmierza ku temu, by na jej podstawie, wziąwszy pod uwagę zmieniające się warunki życia oraz zmiany społeczno-kulturowe (znaki czasu), wyartykułować pastoralne postulaty, tak by mężczyzna stał się tym, kim z powołania (z woli Bożej) jest. Pierwsza część książki koncentruje się na problematyce: mężczyzna - mąż - ojciec w świetle nauk empirycznych, a w szczególności psychologii i socjologii. Dorobek nauk em pirycznych autor traktuje jako niezbędny dla zrozumienia mężczyzny i jego roli, tak w społeczeń stwie, jak i w rodzinie. Na poziomie osoby autor akcentuje trzy jej wymiary: płciowość, psychikę i religijność. W wymiarze społecznym akcent położony jest na jego roli jako męża i ojca, czyli na małżeństwo i rodzinę. RECENZJE Mierzwiński w książce M ężczyzna - mąż - ojciec zaprezentował się szczególnie analitk. Lubuje się w daleko idących podziałach i rozróżnieniach. Dla czytelnika o innej nieco intelektualnej orientacji może to stanowić pewną trudność. M ożna też zastanawiać się, czy zaprezentowane nauczanie Jana Pawła II nie należałoby raczej usytuować w szerszej perspek tywie, to jest nauczania posoborowego, do którego autor i tak z konieczności się odwołuje. ks. W ładysław Majkowski ks. W ładysław Majkowski ks. W ładysław Majkowski Ks. Bogusław Mi g u t , Znaki Misterium Chrystusa. Historiozbawcze ujęcie sakramentów według Salvatore Marsilego O SB (1910-1983), Lublin 1996, ss. 330. Od śmierci prof. Salvatore M arsilego mija prawie 15 lat. W pamięci tych, którzy mieli szczęście go w życiu spotkać, czy słuchać jego wykładów, pozostaje ciągle jego żywa, bardzo skromna postać, właściwa wielkim ludziom. Z radością odnotowujemy, że zarówno we Włoszech jak i poza nimi, nie maleje zainteresowanie uprawianą przez Marsilego teologią liturgii. Recenzowana książka ks. Bogusława Miguta: Znaki Misterium Chrystusa. Historiozbawcze ujęcie sakramentów według Salvatore Marsilego... jest pierwszym, tak obszernym, drukowanym studium teologiczno-liturgicznym, godnym szczególnej uwagi, ze względu na całościowe opraco wanie koncepcji sakramentów według prof. Marsilego. Książka jest rzeczywistym wkładem Autora w wypełnienie dużej luki na polu badań nad przemianami w liturgice posoborowej. Jak zaznacza sam Autor we W stępie do swojej książki (s. 31), stawia on sobie za cel prezentację historiozbawczej koncepcji sakramentów Salvatore Marsilego, jako czołowego włos kiego teologa, doby dojrzałego ruchu liturgicznego i posoborowej odnowy liturgicznej. Jako że książka powstała z rozprawy doktorskiej, towarzyszy jej także próba oceny teologii liturgii wg Marsilego z punktu widzenia innych, rozwiniętych po Soborze W atykańskim II koncepcji sakramentów. Ks. B. Migut swoje dociekania naukowe ujął w pięciu rozdziałach. W rozdziale pierwszym ukazuje ogólną koncepcję sakramentu wypracowaną przez Dom Marsilego, w której sakramenty jaw ią się w zbawczych znakach jako kontynuacja historii zbawienia. Interesująca jest tu szczegól nie historiozbawcza perspektywa sakramentalnych znaków, oraz ich relacja do „Chrystusa-Znaku- Sakramentu” , który jest nie tylko źródłem samych sakramentów Kościoła, ale i sakramentalności liturgii (s. 35-91). W rozdziale drugim, zatytułowanym: Chrzest wszczepieniem w Misterium Chrystusa i Koś cioła, Autor omawia wypracowaną przez prof. Marsilego historiozbawczą koncepcję chrztu św. Już w tytule tego rozdziału, mówiącym o wszczepieniu w Misterium, podkreśla teologiczną głębię i nadzwyczajny wymiar znaczeniowy tego paschalnego sakramentu. Ks. M ig u t, zauważa, że Marsili odcina się od jurydycznego pojmowania sakramentów, a tym samym od praw no-kon stytutywnego ujmowania ich ustanowienia. Ks. RECENZJE Autor podejmuje też problematykę kulturowo uwarunkowanych „męs kich ról” społecznych (gender) i związanej z tym uprzywilejowanej w stosunku do kobiety pozycji społecznej mężczyzny, co obecnie jest przedmiotem szczególnej krytyki ruchów feminist- cznych, jako relikt patriarchalnego systemu. Druga część publikacji prezentuje zarys teologii mężczyzny. Autor ukazuje specyfikę powołania mężczyzny, do jakiego został wezwany w akcie stworzenia go przez Stwórcę. Treść tego powołania zapisana jest na kartach Pisma Świętego, której oficjalną interpretację odnaj dujemy w M agisterium Kościoła, a w szczególności w bogatym w tym zakresie nauczaniu obecnego Papieża Jana Pawła II. Niezmienne w swej zasadniczej strukturze i treści powołanie mężczyzny realizuje się jednak w konkretnych uwarunkowaniach społecznych i kulturowych. Ich podstawową cechą jest dynami ka i zmienność; obecnie bardziej niż w poprzednich epokach. Jest to jednocześnie element nadziei, ale i zagrożenia. Nadzieja z wiary w prawie nieograniczone możliwości, jakie jaw ią się przed człowiekiem, tak w wymiarze osiągnięć technologicznych, jak i doskonalszych form organizacji życia. Jednak racjonalizacja życia we wszystkich jego sferach: odniesienia człowieka do przyrody, organizacji społecznej, kulturowej oraz polityki stanowi dla człowieka, a szczególnie dla mężczyzny, niebezpieczną pułapkę. Może on nie być w stanie odczytać w tych okolicznościach swego powołania albo nie być w stanie go realizować. Tym bardziej zachodzi takie niebezpieczeń stwo, że - jak zauważa autor - „wokół m ężczyzny i jego problemów zapanowała zmowa milczenia, obojętność: brak badań naukowych brak całościowych publikacji, brak potrzebnych decyzji ze strony kompetentnych instytucji” (s. 10). Naprzeciw tym potrzebom musi wyjść teologia praktyczna (pastoralna). W tej perspektywie autor książki formułuje szereg postulatów (10), mających na celu podjęcie duszpasterskich działań, adekwatnych do zaistniałych na tym polu potrzeb. Książka B. M ierzwińskiego prezentuje się jako bardzo ważna pozycja w poruszanej przez niego tematyce, tym bardziej, że teologia mężczyzny ciągle potrzebuje pełniejszych opracowań. Poza tym autor potrafił w sposób adekwatny spożytkować wiedzę dostarczaną przez nauki empiryczne jako punkt wyjścia do swoich dociekań teologiczno-pastoralnych, przestrzegając przy tym autonomii obydwu dziedzin wiedzy. Stanowi to niewątpliwie dodatkowy atut książki. Wreszcie wysuwane przez niego postulaty pastoralne m ogą stanowić podstawę do opracowania 349 RECENZJE stosownego do dzisiejszych potrzeb programu duszpasterstwa m ężczyzny-m ęża-ojca, Jestem przekonany, że książka ta będzie ciekawą i pożyteczną lekturę, tak dla wytrawnych pastoralistów, jak i mniej doświadczonych studentów. stosownego do dzisiejszych potrzeb programu duszpasterstwa m ężczyzny-m ęża-ojca, Jestem przekonany, że książka ta będzie ciekawą i pożyteczną lekturę, tak dla wytrawnych pastoralistów, jak i mniej doświadczonych studentów. B. RECENZJE M igut potrafi znaleźć delikatną granicę prof. Marsilego w akcentowaniu zbawczych wydarzeń, dla rozumienia np. profetycznego charakteru chrztu Janowego, będącego przygotowaniem chrztu Jezusa, pojmowanego przecież jako nowa rzeczywistość eschatologiczno-mesjańska, będącą „chrztem Duchem” i umożliwiająca wejście do Królestwa Bożego (s. 93-110). Autor dobrze odczytuje intencje Marsilego, który chce, aby chrześcijanin dzisiaj lepiej rozumiał na czym polega nowość w rozumieniu chrztu, dlatego podkreśla, że chrzest umieszcza człowieka w nowym wymiarze odniesienia do Boga i to Boga Osobowego, który przez swoją dynamis włącza człowieka w wewnętrzne bogactwo swojego 350 RECENZJE życia. Taka wizja chrztu pomaga w rozumieniu eklezjalnego wymiaru chrztu (s. 119-133). Tu ks. Migut zamieszcza także wyjątkowo aktualny materiał refleksyjny na temat ujmowania przez Marsilego chrztu niemowląt. Rozdział trzeci książki Autor poświęca proponowanej przez Marsilego tematyce bierz mowania. Widzi on ten sakrament jako żywe uczestnictwo w misterium Ducha Świętego. Ks. B. Migut przedstawia tu najpierw misterium Ducha Świętego w szerokiej panoramie całej historii zbawienia. Udowadnia, w jaki sposób Zesłanie Ducha Świętego staje się rzeczywistym bierz mowaniem Kościoła. Autor akcentuje jedną z wielkich trosk M arsilego, by w przygotowaniu do tego sakramentu uświadomić wszystkim, że w bierzmowaniu chodzi nie tylko o zetknięcie się chrześcijanina z działaniem Ducha Świętego, lecz także o prawdziwą wiarę bierzmowanego, że naprawdę otrzymuje Ducha Świętego w darze. W tym temacie zostaje odnotowana jedna z batalii Marsilego, odnośnie do kolejności udzielania sakramentów. Marsylii był zdania, że racje teologi czne i tradycja patrystyczna przemawiają za kolejnością następującą: chrzest, bierzmowanie i Eucharystia, oraz że ta kolejność nie powinna być modyfikowana. Autor książki nie jest bezkrytyczny w tym względzie, porównując najnowszą literaturę z zakresu teologii liturgii stwierdza, że po tylu latach nadal nie ma ostatecznych rozstrzygnięć w tej kwestii (s. 156-167). Celebracja misterium paschalnego Chrystusa mocą Ducha Świętego, to tytuł czwartego rozdziału książki. W tym rozdziale teologiczno-liturgiczna myśl M arsilego jaw i się najobficiej. Autor, na podstawie literatury bardzo wiernie prezentuje Misterium Paschalne Chrystusa według S. Marsilego. W skazuje On na M isterium Paschalne Chrystusa jako wielowymiarowe wypełnienie się Paschy Starego Testamentu (wymiar historyczny, rytualny i celebratywny). Dłużej zatrzymuje się na samej koncepcji Eucharystii, która według Marsilego najlepiej wyraża się w anamnezie śmierci i zmartwychwstania Chrystusa. W Eucharystii chodzi więc przede wszystkim o Paschę Chrystusa, o „Nową pam iątkę” , będącą „zbawczą rzeczywistością Chrystusa, która dokonała się dla zbawienia ludzi” . W ten sposób Pascha Chrystusa staje się również Paschą Kościoła. RECENZJE Cały sposób interpretacji celebrowania Misterium Paschalnego u M arsilego zmierza systematyc człowiekowi, by pomóc mu w nieustannym odczytywaniu, ciągle aktualnego zaproszen tusa do uczestnictwa w wielkim misterium Paschy. Rozdział piąty książki ks. B. Migut poświęca nauce S. Marsilego, odnoszącej się do uobecniania Misterium Chrystusa w innych sakramentach: tj. w sakramencie pokuty, w sakramen cie namaszczenia chorych, sakramencie kapłaństwa i sakramencie małżeństwa. Znajdujemy tu materiał proporcjonalnie mniej obszerny, chociaż bardzo interesujący. Autor podsumowując swoje badania na temat nauczania Dom Salvadore w odniesieniu do misterium pojednania w sakramencie pokuty stwierdza, że Profesor uczy nas tu przede wszystkim dwóch rzeczy: Pierwsza odnosi się do faktu kontynuacji w sakramencie pokuty zbawczej misji Chrystusa przychodzącego: znaleźć, zbawić i uzdrowić człowieka. Druga zaś, wskazuje na niezwykłą łączność sakramentu pokuty z sakramentami inicjacji chrześcijańskiej. Słusznie ks. Migut podkreśla entuzjastyczne podejście Marsilego do odnowionych obrzędów sakramentu namaszczenia chorych. Najważniejszą sprawą jest, by chory w sakramencie namaszczenia spotkał się z Chrystusem. Spotkanie z Chrystusem bowiem sprawia, że „chory wraz z Nim przedstawia Ojcu kielich swojego cierpienia, prosząc, by spełniła się wola Ojca i przyjmując w pełni Jego wolę” (s. 268). Autor bardzo inteligentnie wybrnął z tematu: Sakrament święceń włączeniem w kapłaństwo i misję Chrystusa. W idząc, że w odniesieniu do kapłaństwa nauka Marsilego nie stanowi pewnej całości, omawiając ten temat w przypisach, obficiej niż poprzednio, cytuje szerszy kontekst wypowiedzi Profesora. Dobrze też, że Autor ukazując historiozbawczy wymiar sakramentu małżeństwa według M arsilego, nie próbuje „na siłę” doszukiwać się w materiale tego, czego nie ma. Oryginalnym elementem w kontekście ujmowania i przeżywania tego sakramentu z pewnoś cią jest temat „Kościoła domowego” i sprawowanej przez rodzinę „liturgii domowej” , chociaż wydaje mi się, że o tym mówi nie tylko S. Marsili. 351 RECENZJE W zakończeniu książki Autor przedstawia owoce swoich badań. Trzeba przyznać, że zrobił to dobrze. Na szczególną uwagę zasługuje zgromadzona i wykorzystana przez Autora bogata bibliografia, w której po raz pierwszy pojawiły się skompletowane, a zagubione wcześniej niektóre materiały Profesora. Książka ks. B. M iguta nie tylko sprawia dobre wrażenie, ale jest rzeczywiście pozycją wartościową i interesującą. Napisana jest językiem naukowym bardzo komunikatywnym. Dobrze przybliża nam ona prezentowaną przez prof. S. Marsilego, posoborową koncepcję liturgii oraz metodę współczesnego uprawiania teologii liturgicznej, Książka zainteresuje z pewnością nie tylko liturgistów, godna jest uwagi szerokiego grona teologów i nie tylko. ks. Adam Durak SDB Ks. Henryk S k o r o w s k i , Moralność społeczna. RECENZJE Migut zamieszcza także wyjątkowo aktualny materiał refleksyjny na temat ujmowania przez Marsilego chrztu niemowląt. Rozdział trzeci książki Autor poświęca proponowanej przez M arsilego tematyce bierz mowania. Widzi on ten sakrament jako żywe uczestnictwo w misterium Ducha Świętego. Ks. B. Migut przedstawia tu najpierw misterium Ducha Świętego w szerokiej panoramie całej historii zbawienia. Udowadnia, w jaki sposób Zesłanie Ducha Świętego staje się rzeczywistym bierz mowaniem Kościoła. Autor akcentuje jedną z wielkich trosk M arsilego, by w przygotowaniu do tego sakramentu uświadomić wszystkim, że w bierzmowaniu chodzi nie tylko o zetknięcie się chrześcijanina z działaniem Ducha Świętego, lecz także o prawdziwą wiarę bierzmowanego, że naprawdę otrzymuje Ducha Świętego w darze. W tym temacie zostaje odnotowana jedna z batalii Marsilego, odnośnie do kolejności udzielania sakramentów. M arsylii był zdania, że racje teologi czne i tradycja patrystyczna przemawiają za kolejnością następującą: chrzest, bierzmowanie i Eucharystia, oraz że ta kolejność nie powinna być modyfikowana. Autor książki nie jest bezkrytyczny w tym względzie, porównując najnowszą literaturę z zakresu teologii liturgii stwierdza, że po tylu latach nadal nie ma ostatecznych rozstrzygnięć w tej kwestii (s. 156-167). Celebracja misterium paschalnego Chrystusa mocą Ducha Świętego, to tytuł czwartego rozdziału książki. W tym rozdziale teologiczno-liturgiczna myśl M arsilego jawi się najobficiej. Autor, na podstawie literatury bardzo wiernie prezentuje M isterium Paschalne Chrystusa według S. Marsilego. W skazuje On na M isterium Paschalne Chrystusa jako wielowymiarowe wypełnienie się Paschy Starego Testamentu (wymiar historyczny, rytualny i celebratywny). Dłużej zatrzymuje się na samej koncepcji Eucharystii, która według Marsilego najlepiej wyraża się w anamnezie śmierci i zmartwychwstania Chrystusa. W Eucharystii chodzi więc przede wszystkim o Paschę Chrystusa, o „Nową pam iątkę” , będącą „zbawczą rzeczywistością Chrystusa, która dokonała się dla zbawienia ludzi” . W ten sposób Pascha Chrystusa staje się również Paschą Kościoła. Cały sposób interpretacji celebrowania Misterium Paschalnego u Marsilego zmierza systematycznie ku człowiekowi, by pomóc mu w nieustannym odczytywaniu, ciągle aktualnego zaproszenia Chrys tusa do uczestnictwa w wielkim misterium Paschy. Rozdział piąty książki ks. B. M igut poświęca nauce S. Marsilego, odnoszącej się do uobecniania M isterium Chrystusa w innych sakramentach: tj. w sakramencie pokuty, w sakramen cie namaszczenia chorych, sakramencie kapłaństwa i sakramencie małżeństwa. Znajdujemy tu materiał proporcjonalnie mniej obszerny, chociaż bardzo interesujący. RECENZJE Wybrane zagadnienia z etyki społecznej, gospodarczej i politycznej, Wydawnictwo Salezjańskie, Warszawa 1996, ss. 250 + 2 nlb. Życie społeczne ze swej natury domaga się uporządkowania według pewnych reguł. Reguły te określane są przede wszystkim przez prawo stanowione. Podstawą jednak tego prawa muszą być zasady etyczne. W przeciwnym razie prawo nie będzie spełniać swej funkcji. Będzie słabe, albo będzie działać wręcz destruktywnie na daną społeczność. Między innymi z tego względu rodzi się ciągła potrzeba określania wymiaru etycznego we wszystkich dziedzinach życia społecz nego. Potrzeba ta dotyczy nie tylko bezpośrednio relacji międzyludzkich, ale również wszystkich tych odniesień, które na nie wpływają. Obejmuje więc ona zarówno niezwykle ważny sektor gospodarczy, a także wszystkie działania wchodzące w zakres najszerzej pojętej polityki, w tym również polityki międzynarodowej. Naprzeciw potrzebie przybliżeniu etycznych zasad w życiu społecznym wychodzi wydana przez W ydawnictwo Salezjańskie, książka ks. dr. hab. Henryka Skorowskiego SDB, M oralność społeczna, W arszawa 1996. Autor zaznacza w podtytule, że interesują go „wybrane zagadnienia z etyki społecznej, gospodarczej i politycznej” . I jest to zrozumiałe, gdyż nie sposób w jednej publikacji omówić wszystkich problemów, jakie pojawiają się w tym przedmiocie. Dodać należy, że ks. Skorowski, dziekan Wydziału Historyczno-Społecznego w ATK w Warszawie, od lat zajmuje się prob lematyką społeczną. Opublikował na ten temat szereg artykułów, a w ostatnich latach także książek, m.in. Być chrześcijaninem i obywatelem dziś. Refleksje o postawach moralno-społecz nych, W arszawa 1994 i Problematyka praw człowieka, W arszawa 1996. W kolejnej swej publikacji ks. Skorowski rozważa moralność społeczną „w świetle myśli społecznej Kościoła” , a dokładniej - jak pisze we W stępie - odniesienie człowieka, który jest osobą do procesów historyczno-kulturowo-społeczno-gospodarczo-politycznych (s. 5). Już samo zestawienie ze sobą tych relacji pokazuje skomplikowany splot rozmaitych zagadnień, które są poruszane w sześciu rozdziałach książki. W pierwszym z nich Autor przybliża chrześcijańską koncepcję osoby ludzkiej (s. 16). Odwołując się do niej ks. Skorowski stwierdza, że fundamentalnym elementem chrześcijańskiej wizji człowieka jest „godność osoby” . Nie zrozumie się jednak tego pojęcia „bez zrozumienia zasadniczych prawd chrześcijańskiej antropologii” (s. 17). Niepowtarzalną wartość osoby ludzkiej można odkryć wówczas, gdy zauważy się jej zarówno przyrodzoną, czyli cielesno-duchową sferę, jak również nadprzyrodzoną jej godność, wynikającą z prawdy o podobieństwie człowieka do Boga i usynowieniu go przez fakt odkupienia dokonany przez Chrystusa (s. 19). 350 RECENZJE życia. Taka wizja chrztu pomaga w rozumieniu eklezjalnego wymiaru chrztu (s, 119-133), Tu ks. RECENZJE Autor podsumowując swoje badania na temat nauczania Dom Salvadore w odniesieniu do misterium pojednania w sakramencie pokuty stwierdza, że Profesor uczy nas tu przede wszystkim dwóch rzeczy: Pierwsza odnosi się do faktu kontynuacji w sakramencie pokuty zbawczej misji Chrystusa przychodzącego: znaleźć, zbawić i uzdrowić człowieka. Druga zaś, wskazuje na niezwykłą łączność sakramentu pokuty z sakramentami inicjacji chrześcijańskiej. Słusznie ks. Migut podkreśla entuzjastyczne podejście Marsilego do odnowionych obrzędów sakramentu namaszczenia chorych. Najważniejszą sprawą jest, by chory w sakramencie namaszczenia spotkał się z Chrystusem. Spotkanie z Chrystusem bowiem sprawia, że „chory wraz z Nim przedstawia Ojcu kielich swojego cierpienia, prosząc, by spełniła się wola Ojca i przyjmując w pełni Jego wolę” (s. 268). Autor bardzo inteligentnie wybrnął z tematu: Sakrament święceń włączeniem w kapłaństwo i misję Chrystusa. Widząc, że w odniesieniu do kapłaństwa nauka Marsilego nie stanowi pewnej całości, omawiając ten temat w przypisach, obficiej niż poprzednio, cytuje szerszy kontekst wypowiedzi Profesora. Dobrze też, że Autor ukazując historiozbawczy wymiar sakramentu małżeństwa według Marsilego, nie próbuje „na siłę” doszukiwać się w materiale tego, czego nie ma. Oryginalnym elementem w kontekście ujmowania i przeżywania tego sakramentu z pewnoś cią jest temat „Kościoła domowego” i sprawowanej przez rodzinę „liturgii domowej” , chociaż wydaje mi się, że o tym mówi nie tylko S. Marsili. 351 RECENZJE W zakończeniu książki A utor przedstawia owoce swoich badań. Trzeba przyznać, że zrobił to dobrze. Na szczególną uwagę zasługuje zgromadzona i wykorzystana przez Autora bogata bibliografia, w której po raz pierwszy pojawiły się skompletowane, a zagubione wcześniej niektóre materiały Profesora. Książka ks. B. Miguta nie tylko sprawia dobre wrażenie, ale jest rzeczywiście pozycją wartościową i interesującą. Napisana jest językiem naukowym bardzo komunikatywnym. Dobrze przybliża nam ona prezentowaną przez prof. S. Marsilego, posoborową koncepcję liturgii oraz metodę współczesnego uprawiania teologii liturgicznej. Książka zainteresuje z pewnością nie tylko liturgistów, godna jest uwagi szerokiego grona teologów i nie tylko. ks. Adam Durak SDB Ks. Henryk S k o r o w s k i , Moralność społeczna. Wybrane zagadnienia z etyki społecznej, gospodarczej i politycznej, Wydawnictwo Salezjańskie, Warszawa 1996, ss. 250 + 2 nlb. Życie społeczne ze swej natury domaga się uporządkowania według pewnych reguł. Reguły te określane są przede wszystkim przez prawo stanowione. Podstawą jednak tego prawa muszą być zasady etyczne. W przeciwnym razie prawo nie będzie spełniać swej funkcji. Będzie słabe, albo będzie działać wręcz destruktywnie na daną społeczność. RECENZJE Między innymi z tego względu rodzi się ciągła potrzeba określania wymiaru etycznego we wszystkich dziedzinach życia społecz nego, Potrzeba ta dotyczy nie tylko bezpośrednio relacji międzyludzkich, ale również wszystkich tych odniesień, które na nie wpływają. Obejmuje więc ona zarówno niezwykle ważny sektor gospodarczy, a także wszystkie działania wchodzące w zakres najszerzej pojętej polityki, w tym również polityki międzynarodowej. Naprzeciw potrzebie przybliżeniu etycznych zasad w życiu społecznym wychodzi wydana przez W ydawnictwo Salezjańskie, książka ks. dr. hab. Henryka Skorowskiego SDB, M oralność społeczna, W arszawa 1996. Autor zaznacza w podtytule, że interesują go „wybrane zagadnienia z etyki społecznej, gospodarczej i politycznej” . I jest to zrozumiałe, gdyż nie sposób w jednej publikacji omówić wszystkich problemów, jakie pojaw iają się w tym przedmiocie. Dodać należy, że ks. Skorowski, dziekan Wydziału Historyczno-Społecznego w ATK w W arszawie, od lat zajmuje się prob lematyką społeczną. Opublikował na ten temat szereg artykułów, a w ostatnich latach także książek, m.in. Być chrześcijaninem i obywatelem dziś. Refleksje o postawach moralno - społecz nych, Warszawa 1994 i Problematyka praw człowieka, W arszawa 1996. W kolejnej swej publikacji ks. Skorowski rozważa moralność społeczną „w świetle myśli społecznej Kościoła” , a dokładniej - jak pisze we W stępie - odniesienie człowieka, który jest osobą do procesów historyczno-kulturowo-społeczno-gospodarczo-politycznych (s. 5). Już samo zestawienie ze sobą tych relacji pokazuje skomplikowany splot rozmaitych zagadnień, które są poruszane w sześciu rozdziałach książki. W pierwszym z nich Autor przybliża chrześcijańską koncepcję osoby ludzkiej (s. 16). Odwołując się do niej ks. Skorowski stwierdza, że fundamentalnym elementem chrześcijańskiej wizji człowieka jest „godność osoby” . Nie zrozumie się jednak tego pojęcia „bez zrozumienia zasadniczych prawd chrześcijańskiej antropologii” (s. 17). Niepowtarzalną wartość osoby ludzkiej można odkryć wówczas, gdy zauważy się jej zarówno przyrodzoną, czyli cielesno-duchową sferę, jak również nadprzyrodzoną jej godność, wynikającą z prawdy o podobieństwie człowieka do Boga i usynowieniu go przez fakt odkupienia dokonany przez Chrystusa (s. 19). 352 RECENZJE W odniesieniu do innych człowiek jest bytem społecznym. Jak zauważa ks. Skorowski „pojęcie społeczności kryje w sobie dwa istotne elementy” . Po pierwsze, życie społeczne potrzebne jest człowiekowi do jego funkcjonowania i rozwoju. Drugi element dotyczy potrzeby człowieka do udzielania siebie innym i do uczestniczenia w wartościach osobowych innych (s. 20). Te dwa elementy znajdują swój wyraz w wielu płaszczyznach, w których od urodzenia kształtuje się człowiek (s. 21-24). Podstawą tego kształtowania się, albo lepiej samostanowienia jest podmiotowość ludzka. Odwołując się do analiz K. RECENZJE W ojtyły, Autor omawianej pozycji twierdzi, że „w pojęciu osoby zawiera się całe pojęcie podmiotowości rozumianej jako oryginal ność człowieka i jego nieredukowalność” (s. 25). To zaś z kolei domaga się urzeczywistnienia przez aktywność i działanie człowieka. Cytując szereg autorów ks. Skorowski wyjaśnia, że aktywność i zaangażowanie człowieka „to po prostu wartościowość osoby dla otaczającej go rzeczywistości” (s. 27). Samo jednak pojęcie „wartościowości” osoby jest dość wieloznaczne. W edług katolickiej nauki społecznej bowiem nie ma jednostek „bezwartościowych” . Używając pojęć „wartościowość” , „jednostka wartościowa” trzeba dopowiedzieć, że chodzi o rzeczywiste uczestnictwo osoby w otaczającej ją rzeczywistości oraz o świadomą jej odpowiedzialność za współtworzenie własnego środowiska (s. 28). Tak rozumiana „wartościowość’ ’ jednostki jest wyrazem jej zaangażowania na rzecz dobra wspólnego, j j y j j g p g W rozdziale drugim swojej książki ks. Skorowski omawia zasady życia społecznego, według których powinny być tworzone struktury tego życia, aby było ono na miarę godności człowieka (s. 34). Do fundamentalnych zasad należą: zasada wolności (s. 35-40); zasada pomocniczości (s. 40-43); zasada solidarności (s. 43-46); zasada sprawiedliwości (s. 47-49). Kolejne rozdziały pracy Autor tytułuje podobnie Z problematyki... życia rodzinnego (s. 50-78); życia zawodowego (s. 79-117); narodu i państwa (s. 118-205), międzynarodowej 206-242). Już z tego układu widać wyraźnie, że treść rozważań ułożona jest według coraz szerszych kręgów społecznych. W arto zwrócić uwagę na fakt, że ks. Skorowski omawiając poszczególne kręgi spoleczneż dotyka zagadnień aktualnych i interesujących. Dla przykładu, rozpatrując problematykę rodziną omawia niezwykle ważny, a mało chyba znany i komentowany dokument Stolicy Apostolskiej, Kartę Praw Rodziny (s. 72-78). W zakresie życia zawodowego Autor przybliża między innymi prawa ludzi pracy (s. 93). Powołując się na dokumenty zarówno kościelne, jak i świeckie, omawia następujące prawa: prawo do pracy (s. 95); prawo do odpowiednich warunków pracy (s. 96); prawo do płacy (s. 97); prawo do tworzenia związków zawodowych (s. 99). Cenne jest również zauważenie i wyodręb nienie problematyki specyficznych społeczności zawodowych (s. 100). Ks. Skorowski wspomina jąc o zawodach mających swoją specyfikę, takich jak zawód lekarza, nauczyciela czy dzien nikarza, szerzej omawia społeczność wojskową (s. 101-117). W ydaje się, że tę część książki powinni przeczytać nie tylko fachowcy, ale także kapelani wojskowi, kadra wojskowa i sami żołnierze. Może łatwiej wówczas byłoby połączyć szczytne ideały z szarą i nie zawsze ciekawą rzeczywistością dnia codziennego. Na uwagę zasługują także rozważania dotyczące ojczyzny i patriotyzmu, a zwłaszcza punkty dotyczące „małych ojczyzn” , patriotyzmu lokalnego (s. RECENZJE 148-155) oraz funkcjonowania samo rządu terytorialnego w strukturach społeczności państwowej (s. 169-205). Zważywszy na fakt, że Autor prezentowanych refleksji swoją pracę habilitacyjną napisał na temat Antropologicmo- -etyczne aspekty regionalizmu (W arszawa 1990), marny do czynienia ze specjalistyczną i fachową analizą i oceną jakże aktualnych na gruncie polskim zjawisk i związanych z nimi problemów. W problematyce międzynarodowej istotne i ważne są sprawy dotyczące pokoju i wojny (s. 215-230); zadłużenia zagranicznego (s. 230-236); zagrożenie środowiska naturalnego (s. 236-242). Całość książki uzupełnia bibliografia, w której ks. Skorowski podaje dokumenty społeczne Kościoła, dokumenty świeckie oraz wybraną literaturę (s. 245-250). W ydana w ładnej szacie graficznej książka jest lekturą, po którą powinni sięgnąć wszyscy ci, którzy działają na różnych odcinkach życia społecznego. W ymiar etyczny tego życia naświet 353 RECENZJE lony z pozycji katolickiej nauki społecznej, ukazuje się nie tylko jako ideał, ale pobudza do głębszej refleksji, jak kształtować w oparciu o nie życie społeczne, gospodarcze i społeczne. ks. Andrzej Garbarz Reinhold W e i n s c h e n k , Podstawy pedagogiki Księdza Bosko, Warszawa, W ydawnictwo Salez jańskie 1996, ss. 208. Reinhold W e i n s c h e n k , Podstawy pedagogiki Księdza Bosko, Warszawa, W ydawnictwo Salez jańskie 1996, ss. 208. W iosną tego roku ukazała się w „W ydawnictwie Salezjańskim” , w tłumaczeniu na język polski, książka poświęcona ks. Bosko, dokładniej jego idei wychowawczej, jakim był system prewencyjny. Autorem książki jest niemiecki salezjanin, wykładowca W yższej Szkoły Pedagogi cznej w Monachium (oddział w Benediktbeuern). Książka jest dość obszerna, zawiera ponad dwieście stron. Treść książki autor przedstawia w pięciu rozdziałach. Przedtem jednak umieścił dość długi wstęp. Ukazał w nim w sposób syntetyczny postać ks. Bosko, jego historię życia i jego pracę na rzecz młodzieży, na tle innych ówczesnych systemów wychowawczych i pedagogów XIX wieku. Rozdział pierwszy poświęcony metodom wychowawczym, przedstawia ks. Bosko jako młodego księdza, który zauważył na ulicach i więzieniach Turynu niesamowitą nędzę młodych chłopców. Chciał dla nich coś zrobić. Powoli uświadamiał sobie, że może pomóc im ktoś, kto ich polubi, spróbuje zrozumieć ich sytuacje i znajdzie odpowiednią metodę, aby do nich trafić. Opracowując własną metodę działania, zmierzał do wypracowania jasnych celów wychowania, określonej podstawy antropologiczno - światopoglądowej i wychowawczej roli, jaką może spełnić instytucja ze swoim planem działania. Następnie autor opisuje dość szczegółowo koncepcję wychowawczą Aleksandra Sutherlanda N eill’a, autora projektu „Sum merhill” . RECENZJE Opracował on model wolnej szkoły, w której dzieci wychowuje się na szczęśliwych ludzi. Następną postacią, którą spotykamy na kartach książki, jest brazylijski pedagog Paulo Freire. Jest on przedstawicielem pedagogiki wolności, zmierzającej do wyzwolenia całych uciś nionych mas Ameryki Łacińskiej. Celem działań wychowawczych jest „uświadomienie” przez człowieka swojej aktywnej roli w życiu społeczeństwa. Fraire zajmował się także kształceniem ludzi dorosłych i zwalczaniem wśród nich analfabetyzmu. Po przedstawieniu powyższych dwóch sylwetek autor przechodzi do systemu wychowaw czego ks. Bosko, któremu dał nazwę „system prewencyjny” . Przez wiele lat ks. Bosko był raczej wychowawcą-praktykiem. Dopiero u schyłku swojego życia, i to wyraźnie na prośbę innych, utrwalił swoje idee na piśmie. „System prewencyjny” jako metoda wychowawcza Wraz z upływem czasu oratorium ks. Bosko stawało się coraz bardziej znane na terenie Turynu i W łoch. W rozmowie z ministrem Urbanem Ratazzi ks. Bosko wykazywał wyższość „systemu prewencyjnego” nad „systemem represyjnym” . Pierwszy z nich kładzie akcent na łagodność i pomaga młodemu człowiekowi przez przyjazną zachętę. Drugi wychowuje człowieka przy pomocy siły, nacisku i częstej kary, nawet za drobne przekroczenie. W 1884 roku ks. Bosko udziela wywiadu do „Journal de Rom e” . Na pytanie dotyczące istoty skuteczności jego systemu wychowawczego odpowiedział: „Jego istota jest bardzo prosta: chłopcom należy pozostawić zupełną wolność w czynieniu tego, co znajduje ich największe upodobanie. Wiele zależy też od odkrycia w nich zalążków dobra oraz troski o ich dalszy rozwój. Aby każdy z radością czynił to, co umie, postępuję .według tej zasady, a moi chłopcy współpracują ze mną nie tylko z zapałem, lecz także z miłością” (s. 42). Następny rozdział autor poświęcił perspektywom przyszłości w pedagogice, a konkretnie celom wychowawczym. W każdej aktywności człowieka cele odgrywają bardzo ważną rolę, które w praktyce dla jednostki stają się dążeniami i pragnieniami, W ychowanie jest takim procesem, który powinien pomóc młodemu człowiekowi w celowym wrośnięciu w otaczający go świat ludzi dorosłych. Ks. Bosko zajmując się w Turynie młodzieżą z marginesu społecznego, przeżył jej nędzę, zwątpienie, beznadziejność i brak przyszłości. W związku z powyższym celowo organizował im spotkania w niedziele i dni świąteczne, by w ten sposób wyrwać ich ze złych środowisk i zapełnić im czas pozytywnymi zajęciami, Pole pracy ks. Bosko dotyczyło więc wielu celów: wychowania, umiejętnego spędzania wolnego czasu, zdobycia kwalifikacji szkolnych, zawodo wych, nabywania ważnych życiowo przyzwyczajeń, aż do postępowania odznaczającego się stałością. Ks. Bosko stawiał jasny cel: wychować chłopca na „uczciwego obywatela i dobrego chrześcijanina” , Zmierzając do takiego celu ks. Bosko oparł wychowanie na religii. Często on podkreślał, jak już było wspomniane wcześniej, że religia jest jedną z kolumn wspierających jego system wychowawczy. W edług niego należy przyzwyczajać młodzież do praktyk religijnych, które mogą spełniać rolę wychowawczą. W ychowanie religijne powinno obejmować człowieka nie tylko w perspektywie jego celu ostatecznego, ale przede wszystkim ujmować go w całości jego istoty. Nie wymagał od wychowanków rzeczy nadzwyczajnych, lecz oczekiwał od nich zwykłego wypełniania codziennych obowiązków. „Przede wszystkim w naszym systemie - powie do ministra Ratazzi - próbujemy wszczepić w serca młodzieży świętą bojaźń Bożą, a także wlać w nie zamiłowanie do cnoty i odrazę do grzechu” (s. 69). Rozum i religia Filarami systemu prewencyjnego ks. Bosko uczynił religię i rozum, później dołączył do nich dobroć. Niewątpliwie, jak sam podkreślał, znaczny wpływ na jego powołanie pedagogiczne miał sen z dziewiątego roku życia. Sen opowiada o zachowaniu się Janka Bosko wśród wielkiej gromady chłopców, którzy bili się, przeklinali lub śmiali się. Janek Bosko próbował uciszyć ich i wprowadzić pokój przy pomocy pięści. Oczywiście to nie przyniosło żadnego skutku. Nagle zauważył ładnie ubranego pana, który powiedział mu: musisz pozyskać ich dobrocią i miłością, a nie pięściami. Ks. Bosko patrząc na ten dziwny sen z perspektywy wielu lat swojej pracy wśród biednych chłopców, dostrzegał między tymi dwoma faktami duże związki. W 1864 roku ks. Bosko przeprowadził rozmowę z Franciszkiem Bodrato, wykwalifikowa nym nauczycielem. Ten ostatni od dłuższego czasu obserwował ks. Bosko i jego pedagogiczne poczynania. W rozmowie ks. Bosko jasno zaznaczył, że „rozum i religia są podstawowymi filarami mojego systemu wychowawczego” (s. 41). 354 RECENZJE „System prewencyjny” jako metoda wychowawcza W śród sakramentów szczególną rolę wychowawczą przypisywał Eucharystii i spowiedzi. W swoim systemie wychowawczym ks. Bosko zmierzał do wszechstronnego wychowania młodego człowieka. Pamiętał nie tylko o dobrym chrześcijaninie, ale także mówił o zaan gażowanym obywatelu. Reprezentował stanowisko Kościoła, mając jednocześnie na oku dobro społeczeństwa. Można powiedzieć, że chodziło mu o praktyczną „caritas” . W rzeczywistości zajmował się ubogimi, by w ten sposób podnieść ich standart życiowy. W swoim systemie wychowawczym ks. Bosko zmierzał do wszechstronnego wychowania młodego człowieka. Pamiętał nie tylko o dobrym chrześcijaninie, ale także mówił o zaan gażowanym obywatelu. Reprezentował stanowisko Kościoła, mając jednocześnie na oku dobro społeczeństwa. Można powiedzieć, że chodziło mu o praktyczną „caritas” . W rzeczywistości zajmował się ubogimi, by w ten sposób podnieść ich standart życiowy. Rozdział trzeci autor poświęca stylowi wychowawczemu, jaki reprezentował ks. Bosko. W praktyce wiązało się to z realizacją celów. Na pierwszym miejscu zauważamy kwestię znajomości młodzieży, która stanowi punkt wyjścia do indywidualnego wychowania. Jako pod stawa stylu wychowawczego ks. Bosko umieszczał wyrozumiałe i pełne dobroci kontakty z młodym człowiekiem. To wszystko tworzyło właściwą atmosferę wychowawczą. Źródłem tego wszystkiego ks. Bosko czynił miłość: „M iłość cierpliwa jest, łaskawa jest...” (1 Kor 13, 4). Składnikiem stylu wychowawczego ks. Bosko było także zaufanie. Zaufanie było dla niego probierzem pozwalającym właściwie ocenić osiągane wyniki oraz ułatwiać wolny od zakłóceń przebieg dnia w domach, szkołach czy oratoriach. Stąd też dążył do wytworzenia wśród chłopców atmosfery rodzinnej, ta bowiem najlepiej rodzi zaufanie. RECENZJE 355 Na szczególną uwagę zasługuje w systemie prewencyjnym kwestia kar, które w tym systemie są zasadniczą sprzecznością. Do salezjanów pracujących w Ameryce napisze: „System prewencyjny niech będzie rzeczywiście naszą cechą. Nigdy żadnych przewidzianych prawem kar, nigdy żadnych poniżających słów, nigdy surowych wyrzutów w obecności innych” (s. 130). Narzędziem wychowawczym, które skutecznie broni przed stosowaniem kar jest asystencja, czyli nieustanna obecność i czujność wychowawcy. Rozdział czwarty poświęcony jest celom i strukturom wychowawczym. Zajmując się biednymi i opuszczonymi chłopcami tworzył dla nich oratoria, hospicja, szkoły i warsztaty. Znajdowali tam dach nad głową, chleb, wykształcenie, rozrywkę, wypoczynek, radość, zawód i nową rodzinę. Ostatni piąty rozdział mówi o wychowawcy i jego osobowości. W całokształcie działalności pedagogicznej zajmuje on znaczenie centralne. Ks. Bosko rozumiał rolę wychowawcy, stąd też nie tylko chętnie przebywał pośród chłopców, ale także uczestniczył w ich zabawach. Kładł akcent na obecność (asystencja) oraz czynne uczestnictwo we wszystkim, co się odnosiło do wychowanków. 1 P . S o r o k i n , Social and Cultural Dynamics, New York 1937, t. IV, s. 767. 2 E .W B u r g e s s , H . J . L o c k e , The Family: From Institution to Companionship, New York 1945. P . S o r o k i n , Social and Cultural Dynamics, New York 1937, t. IV, s. 767. „System prewencyjny” jako metoda wychowawcza Swoje rozważania o wychowawcy autor kończy cytatem z Romana Guardini: „Pierwszym elementem oddziaływania jest obecność wychowawcy; drugim, co czyni; trzecim, to co m ówi” . Książka, której poznaliśmy treść, przedstawia w sposób wyczerpujący „system prewencyj ny” św. Jana Bosko. Pokazuje go od strony teoretycznej i praktycznej. Potwierdzają to liczne cytaty z pism ks. Bosko. Autor w opracowywaniu omawianego zagadnienia sięgnął do dość licznych źródeł w języku włoskim. Nie poprzestał na tym. Dość często odwołuje się do współczesnych autorów i ich prac na temat metody wychowawczej ks. Bosko. Tymi autorami w większości są salezjanie, ale i nie tylko. Na kartach książki możemy znaleźć ich dość dużo. Omawiając dane zagadnienie dotyczące systemu prewencyjnego, autor wyjaśnia przedtem jego znaczenie ogólne, przyjęte na polu wychowania czy innej nauki, cytując wielu autorów. Zapewne jest to rzecz pozytywna, chociaż z drugiej strony ma się wrażenie, jakoby tracił na ostrości sam studiowany system prewencyjny. Inną rzeczą rzucającą się w oczy są drobne powtórzenia. Mimo tego książka stanowi bogate źródło informacji i niewątpliwie zasługuje na uważną lekturę. ks. Jan Niewęgłowski SDB Women, Men, and Marriage, Ch. Ciulów red., J Aronson Inc., Northvale-New Jersey-London 1996, ss. 168. Amerykański socjolog rosyjskiego pochodzenie, P. Sorokin, analizując sytuację rodziny amerykańskiej lat trzydziestych naszego stulecia, napisał: „Rodzina jako uświęcona jedność męża i żony, rodziców i dzieci będzie zdążała do upadku. Rozwody i separacje będą wzrastały, aż do zupełnego zatarcia się różnic między społecznie usankcjonowanym małżeństwem a nielegalnym związkiem seksualnym” 1. Niespełna dziesięć lat później E. W. Burgess i H .J Locke zatytułowali swoją książkę: Rodzina. Od -instytucji do partnerstwa2, zwracając uwagę na zmiany, jakie zaszły w rozumieniu rodziny i oczekiwaniach nupturientów względem niej. W tym samym czasie 356 RECENZJE T. Parsons, zastanawiając się nad ciągle wzrastającym stopniem rozwodów w Ameryce, stawia! pytanie: czy istnieje kryzys rodziny jako instytucji? Przewidywania Sorokina nie sprawdziły się; partnerski charakter rodziny akcentowany przez Burgessa i Locke’a nie wyeliminował jej wymiaru instytucjonalnego, a na stawiane przez siebie pytania dotyczące przyszłości rodziny Parsons sam znalazł odpowiedź: nie ma kryzysu rodziny jako instytucji; kryzys natomiast przeżywa wiele konkretnych rodzin. Podstawą tego rodzaju twierdzenia była obserwacja stwierdzająca, że większość rozwiedzionych zawiera nowy związek małżeński. Oznacza to, że zdecydowana większość ludzi wiąże swe życie z rodziną nawet wtedy, gdy wynieśli nie całkiem dobre doświadczenia z rodziny pochodzenia czy założonej przez siebie rodziny, obecnie zdezintegrowanej. „System prewencyjny” jako metoda wychowawcza Od czasu studium wspomnianych autorów wiele się zmieniło, tak w społeczeństwie amerykańskim, jak i innych społeczeństwach zindustrializowanych: zapom niano o kryzysie ekonomicznym lat trzydziestych, usunięto zgliszcza wojenne, zbudowano społeczeństwo „rów nych szans” ..., ale problem atyka kryzysu rodziny ciągle powraca. Niektóre jej aspekty sytuowa ne są w kontekście starych problemów; inne - w świetle nowych trendów życia rodzinnego, czy quasi-rodzinnego. Książka Women, Men, and Marriage pod redakcją Ch. Clulowa podejmuje problematykę idącą po tej linii. Aczkolwiek jest zbiorem artykułów wielu autorów (12), to jednak stanowi wyjątkowo harmonijną, jak na tego rodzaju kompilację, całość. Stało się to możliwe z dwóch powodów: pierwszym jest fakt podobnych zainteresowań wszystkich autorów książki: prob lematyka rodzinny pod kątem rodzinnego poradnictwa; drugim - wizja problematyki rodzinnej na tle zachodzących zmian w makrostrukturze, jak i zmian kulturowych. Analiza bazuje na danych z Wielkiej Brytanii, ale w mniejszym czy większym stopniu może być odniesiona do wszystkich społeczeństw zindustrializowanych i zurbanizowanych. Najbardziej znaczące trendy zmian we współczesnej rodzinie Wielkiej Brytanii to: - obniżenie się odsetek zamężnych i żonatych w stosunku do całej populacji; podczas gdy „złotym roku rodziny” (1961 r.) wynosił on 65, to 20 lat później został zredukowany do 47; - obniżenie się odsetek zamężnych i żonatych w stosunku do całej populacji; podczas gdy „złotym roku rodziny” (1961 r.) wynosił on 65, to 20 lat później został zredukowany do 47; - wzrost liczby samotnych, nigdy nie zamężnych matek: w 1971 - 7%; 1981 - 11% 1991 18% ół k „złotym roku rodziny” (1961 r.) wynosił on 65, to 20 lat później został zredukowany do 47; - wzrost liczby samotnych, nigdy nie zamężnych matek: w 1971 - 7%; 1981 - 11% a w 1991 - 18% ogółu matek; - wzrost liczby samotnych, nigdy nie zamężnych matek: w 1971 - 7%; 1981 - 11% a w 1991 - 18% ogółu matek; - wzrost liczby rozwodów: w 1981 r. na każdy 1000 małżeństw przypadało 11,9 rozwodów; w 1991 r. - 12,6; - wzrost liczby rozwodów: w 1981 r. na każdy 1000 małżeństw przypadało 11,9 rozwodów; w 1991 r. „System prewencyjny” jako metoda wychowawcza - 12,6; - bardzo gwałtowny wzrost liczby osób bez ślubu zamieszkujących wspólnie; dotyczy to tak tych, którzy w bardziej czy mniej określonej przyszłości zamierzają zawrzeć związek małżeński jak i tych, którzy nigdy tego nie zamierzają robić; jest tak niezależnie od faktu, że tylko 17% mieszkańców Wielkiej Brytanii uważa, iż małżeństwo jest przeżytą instytucją; tylko 17% mieszkańców Wielkiej Brytanii uważa, iż małżeństwo jest przeżytą - wzrost liczby dzieci urodzonych poza małżeństwem: w 1979 r. - 11% a w 1991 r. 30%; - obniżenie się wieku pierwszego współżycia seksualnego (średnio do 17 roku życia tak dla mężczyzn jak i kobiet); wzrost liczby dzieci urodzonych poza małżeństwem: w 1979 r. 11% a w 1991 r. 30%; - obniżenie się wieku pierwszego współżycia seksualnego (średnio do 17 roku życia tak dla mężczyzn jak i kobiet); - zaprzepaszczenie dziewictwa jako wartości; - zaprzepaszczenie dziewictwa jako wartości; - wzrost liczby ludzi mających homoseksualne doświadczenia; - wzrost częstotliwości pozamałżeńskich doświadczeń seksualnych, mimo że wyłączność małżeńska jest ciągle małżeńskim ideałem i w opinii większości (80%) najważniejszym wa runkiem małżeńskiego sukcesu; - wzrost częstotliwości pozamałżeńskich doświadczeń seksualnych, mimo że wyłączność małżeńska jest ciągle małżeńskim ideałem i w opinii większości (80%) najważniejszym wa runkiem małżeńskiego sukcesu; W spółczesne małżeństwo społeczeństw nowoczesnych balansuje między sprzecznościami: - partnerski charakter małżeństwa, a nawet jego prywatyzacja, wprowadza w nie element niestabilności; w tym samym kierunku działa emancypacja kobiety, zwłaszcza jej ekonomiczna niezależność; - ma miejsce zastępowanie wartości absolutnych relatywnymi; - ma miejsce zastępowanie wartości absolutnych relatywnymi; - ideałem małżeńskim pozostaje małżeńska wyłą kobiet przynajmniej raz tej wierności nie zachowało; - ideałem małżeńskim pozostaje małżeńska wyłączność, a około 60% mężczyzn i 40% kobiet przynajmniej raz tej wierności nie zachowało; 357 RECENZJE - istnieje napięcie pomiędzy rolami wynikającymi z podziału na płeć a tymi, które mają podłoże kulturowe; p - wysoko cenione życie rodzinne nie przeszkadza tolerancji, a nawet akceptacji dewiacyj nych form seksualnych doświadczeń, na przykład homoseksualnych; y y p y y - trwałość małżeństwa jest nieodzownym warunkiem małżeńskiego sukcesu, a jednocześnie rozwód jest powszechnie akceptowany. Mając na uwadze te wszystkie problemy rodziny współczesnej rodzi się na nowo pytanie sprzed 50 lat: czy rodzina społeczeństw zindustrializowanych i zurbanizowanych przeżywa kryzys? A może - jak to sugeruje książka Wemen, Men, and Marriage - należałoby zapytać: czy to nie jest dzisiejsze społeczeństwo, które ulega procesowi dezorganizacji, a w rodzinie tylko jak w soczewce najwyraźniej odzwierciedlają się jego problemy i najboleśniej dotykają poszczególne jednostki? „System prewencyjny” jako metoda wychowawcza Czytelnikowi książki Wemen, Men, and Marriage nie może ujść uwagi ogólna „atm osfera” książki - koniecznościowego charakteru zachodzących zmian w małżeństwie i rodzinie oraz nie wyartykułowanej, ale wyczuwanej bezsiły wobec nich, Dlatego nie ma w niej mowy o przygoto waniu do małżeństwa i życia w rodzinie, które nadawałoby pewien kierunek zmian, a jedynie o radzeniu sobie w sytuacjach kryzysowych. Na dodatek rolę powierzyciela osobistych prob lemów ma spełniać poradnia psychoanalityczna w wydaniu Freudowskim. ks. Władysław Majkowski
https://openalex.org/W4281264239	https://www.mdpi.com/1996-1944/15/10/3706/pdf?version=1653545803	English	null	Infill Density Influence on Mechanical and Thermal Properties of Short Carbon Fiber-Reinforced Polyamide Composites Manufactured by FFF Process	Materials	2,022	cc-by	16,001	Citation: Chicos, L.-A.; Pop, M.A.; Zaharia, S.-M.; Lancea, C.; Buican, G.R.; Pascariu, I.S.; Stamate, V.-M. Inﬁll Density Inﬂuence on Mechanical and Thermal Properties of Short Carbon Fiber-Reinforced Polyamide Composites Manufactured by FFF Process. Materials 2022, 15, 3706. https:// doi.org/10.3390/ma15103706 Academic Editor: Ricardo J. C. Carbas Received: 21 April 2022 Accepted: 19 May 2022 Published: 22 May 2022 Citation: Chicos, L.-A.; Pop, M.A.; Zaharia, S.-M.; Lancea, C.; Buican, G.R.; Pascariu, I.S.; Stamate, V.-M. Inﬁll Density Inﬂuence on Mechanical and Thermal Properties of Short Carbon Fiber-Reinforced Polyamide Composites Manufactured by FFF Process. Materials 2022, 15, 3706. https:// doi.org/10.3390/ma15103706 Academic Editor: Ricardo J. C. Carbas Received: 21 April 2022 Accepted: 19 May 2022 Published: 22 May 2022 Citation: Chicos, L.-A.; Pop, M.A.; Zaharia, S.-M.; Lancea, C.; Buican, G.R.; Pascariu, I.S.; Stamate, V.-M. Inﬁll Density Inﬂuence on Mechanical and Thermal Properties of Short Carbon Fiber-Reinforced Polyamide Composites Manufactured by FFF Process. Materials 2022, 15, 3706. https:// doi.org/10.3390/ma15103706 Keywords: fused ﬁlament fabrication; carbon ﬁber; polyamide; inﬁll density; mechanical properties; thermal properties Received: 21 April 2022 Accepted: 19 May 2022 Published: 22 May 2022 materials materials Article Inﬁll Density Inﬂuence on Mechanical and Thermal Properties of Short Carbon Fiber-Reinforced Polyamide Composites Manufactured by FFF Process Lucia-Antoneta Chicos 1,, Mihai Alin Pop 2 , Sebastian-Marian Zaharia 1 , Camil Lancea 1, George Razvan Buican 1, Ionut Stelian Pascariu 1 and Valentin-Marian Stamate 1 1 Department of Manufacturing Engineering, Transilvania University of Brasov, 500036 Brasov, Romania; zaharia_sebastian@unitbv.ro (S.-M.Z.); camil@unitbv.ro (C.L.); buican.george@unitbv.ro (G.R.B.); ionut.pascariu@student.unitbv.ro (I.S.P.); valentin_s@unitbv.ro (V.-M.S.) p ( ); _ ( ) 2 Department of Materials Science, Transilvania University of Brasov, 500036 Brasov, Romania; mihai.pop@unitbv.ro p ( ); _ ( ) 2 Department of Materials Science, Transilvania University of Brasov, 500036 Brasov, Romania; mihai.pop@unitbv.ro Correspondence: l.chicos@unitbv.ro Abstract: In three-dimensional (3D) printing, one of the main parameters inﬂuencing the properties of 3D-printed materials is the inﬁll density (ID). This paper presents the inﬂuence of ID on the microstructure, mechanical, and thermal properties of carbon ﬁber-reinforced composites, commer- cially available, manufactured by the Fused Filament Fabrication (FFF) process. The samples were manufactured using FFF by varying the inﬁll density (25%, 50%, 75%, and 100%) and were subjected to tensile tests, three-point bending, and thermal analyses by Differential Scanning Calorimetry (DSC) and Thermogravimetric Analysis (TGA). It was shown that the samples with 100% ID had the highest values of both tensile, 90.8 MPa, and ﬂexural strengths, 114 MPa, while those with 25% ID had the lowest values of 56.4 MPa and 62.2 MPa, respectively. For samples with inﬁll densities of 25% and 50%, the differences between the maximum tensile and ﬂexural strengths were small; therefore, if the operating conditions of the components allow, a 25% inﬁll density could be used instead of 50%. After DSC analysis, it was found that the variation in the ID percentage determined the change in the glass transition temperature from 49.6 ◦C, for the samples with 25% ID, to 32.9 ◦C, for those with 100% ID. TGA results showed that the samples with IDs of 75% and 100% recorded lower temperatures of onset degradation (approximately 344.75 ◦C) than those with inﬁll densities of 25% and 50% (348.5 ◦C, and 349.6 ◦C, respectively). 1. Introduction The mechanical and thermal performance of composite ﬁbers manufactured by FFF are inﬂuenced by a number of printing variables, matrix characteristics, ﬁber content, ﬁber orientation, ﬁber-matrix bonding, etc. In order to obtain components from high-strength ﬁber composites through FFF, it is of real interest to analyze, establish, and understand how these variables inﬂuence, especially in the case of ﬁber-reinforced polymeric composites about which there are no published analysis results. Investigations by many researchers have shown the effectiveness of the carbon ﬁber reinforcement (chopped) of thermoplastic polymers printed in FFF [1,2,6–8]. Liao et al. [6] reported the mechanical and thermal prop- erties of chopped carbon ﬁber (CF)-reinforced polyamide 12 (PA12) composites fabricated by FFF with different carbon ﬁber mass fractions. The addition of 10 wt% carbon ﬁbers into the PA12 matrix showed an increase in tensile strength (102.2%) and ﬂexural strength (251.1%) than pure PA12. It was also found that the crystallization peak temperature and the degradation temperature increased by 3.46 ◦C and 7.50 ◦C, respectively, after adding 10 wt% carbon ﬁbers [1]. In [9], the authors printed PLA composites reinforced with CF by varying the layers. The layers of carbon ﬁber PLA showed a maximum tensile strength of 1.16 times higher than that of pure PLA. Tian et al. [10] investigated the effect of FDM man- ufacturing parameters and carbon ﬁber content on the performance of samples produced from carbon-ﬁber reinforced composites. It has been found that composite PA has a better mechanical performance than ABS and PLA. Tekinalp et al. [11] compared the mechanical properties of parts made of carbon ﬁber-reinforced ABS fabricated by compression mold- ing and FDM. The results indicated that both the strength and the modulus of the parts increased signiﬁcantly with the increase in the carbon ﬁber content. g y The effect of various manufacturing parameters, such as layer thickness, inﬁll den- sity, inﬁll pattern, raster angle, and ﬁber orientation, on the mechanical properties was also investigated. Ning et al. [12] investigated the tensile characteristics of carbon ﬁber composites in terms of varying manufacturing parameters such as layer thickness, nozzle temperature, inﬁll speed, and raster angle. Samples printed at a raster angle of—45◦/45◦ showed a maximum modulus, whereas the maximum tensile strength was at an angle of 0◦/90◦. 1. Introduction Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Additive Manufacturing (AM), in recent years, has become one of the emerging and sustainable manufacturing processes. Its ability to manufacture complex, lightweight parts with a short manufacturing time and low material consumption has led to its use in various industries such as automotive, medical, and aerospace, in the manufacture of large portions or components of aircraft, rockets, and unmanned aerial vehicles (UAVs). The Fused Deposition Modeling (FDM) process, known as Fused Filament Fabrication (FFF), which uses the hot melt and adhesive properties of thermoplastics, is the most applied 3D printing technology due to its ﬂexibility, simplicity, and low cost in the production of complex shape parts used in various ﬁelds [1]. Copyright: © 2022 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/). Thermoplastic polymers are materials frequently used in various industries, also in the FFF process related to additive manufacturing technology, due to their high resistance to cooling and heating cycles, resistance to chemicals and mechanical stress, ease of produc- tion, and low costs [2]. In order to improve the properties and characteristics of polymers https://www.mdpi.com/journal/materials Materials 2022, 15, 3706. https://doi.org/10.3390/ma15103706 Materials 2022, 15, 3706 2 of 20 manufactured by AM, ﬁlled polymers have begun to be developed in recent years, and the most used ﬁllers are carbon ﬁbers, glass, carbon nanotubes, nanoparticles, and syn- thetic and natural ﬁbers [1–5]. Thermoplastic polymeric matrices commonly used in FFF are polylactides (PLA), acrylonitrile butadiene styrene (ABS), and polyamides (PA), such as PA6, PA12, and polyether-ether-ketone (PEEK). Although the additive manufacturing of polymer composites is considered to be cost-effective, it is still in its infancy because there are a number of problems that occur in the manufacturing process or factors that inﬂuence the process and therefore have an inﬂuence on the properties and characteristics of components manufactured by FFF. p y The 3D printing of ﬁber-reinforced polymer composites has become of real interest and current research claims the need to develop the process and composite materials used in this process. 1. Introduction The result showed that the 80% inﬁll density is the percentage that has the optimum strength and toughness. Terekhina et al. [23] showed that printed PA samples with a ﬁlling density above 60% have a signiﬁcant increase in strength. The correlation between FFF parameters and the results was studied by Belei et al. [24]. It was found that the temperature of the printing bed and layer height are the most inﬂuential parameters that affected the mechanical performance of the printed samples. Maszybrocka et al. [25] stated that inﬁll density is the main factor that inﬂuences the mechanical properties and found that strength properties increased with the increase in the ﬁlling density and they inﬂuenced both the core and outer layers ﬁlling density. y g y It is well known that the mechanical properties are inﬂuenced by the degree of crys- tallinity and, in the FFF, the temperature can directly affect the crystallinity of the printed part. Therefore, the thermal behavior of the composites reinforced with ﬁbers are also inves- tigated, although there are few studies. In the study of Liao et al. [6], it was demonstrated that the addition of carbon ﬁber in the PA12 composite reduced the thermal degradation of the matrix, and the authors concluded that carbon ﬁber acts as a thermal stabilizer, which protects the composite matrix. The thermal properties of the FDM polymeric Nylon 618, PLA, and PLA-reinforced carbon ﬁber in Kaur et al. [26] were studied. The 3D-printed carbon-based PLA exhibited better thermal behavior compared to the other polymeric materials. De Toro et al. [27] studied the thermal behavior and mechanical properties of the polyamide composite reinforced with 20% carbon ﬁber. The result showed the glass transition at 50 ◦C and the maximum degradation of the matrix at 450 ◦C. In [28], the addition of 20% carbon ﬁber to the ABS matrix led to a reduction in the glass transition tem- perature (from 110 ◦C to 105 ◦C) and also to a reduction in thermal stability by decreasing degradation on the set temperature (from 323 ◦C to 253 ◦C). Carbon ﬁber PA composite, compared to ABS and PLA, has a wide ﬁeld of use and superior mechanical performance, which justiﬁes the need to research the application of PA reinforced with carbon ﬁber in FFF. 1. Introduction In [13], the authors explored the effect of manufacturing parameters such as inﬁll density, layer thickness, and print speed on the mechanical properties of samples of Nylon material. It was found that inﬁll density has the highest inﬂuence on the mechanical characteristics because ﬂexural strength and UTS have maximum values at a 100% inﬁll density. Naranjo-Lozada et al. [14] investigated the effects of inﬁll density, inﬁll patterns, and ﬁber volume fraction on the tensile properties of 3D-printed composites reinforced with continuous and chopped carbon ﬁber manufactured by fused ﬁlament fabrication (FFF). The results showed that inﬁll density and inﬁll patterns affect the mechanical behav- ior of the sample of chopped carbon ﬁber and the triangular pattern has a better tensile strength [15]. Gavali et al. [16] in their research found that an increase in the chopped carbon ﬁber in PLA composite led to an increase in ﬂexural strength. After reinforcing with 10 wt% of carbon ﬁber, the strength increased to 67 MPa compared with 66 MPa of neat PLA. An increase in the percentage of carbon ﬁber of 15% led to a ﬂexural strength of about 78 MPa [2]. In addition, the increase in the bending strength by adding different percentages of carbon ﬁber in the polyamide (PA) matrix was also found in the research Materials 2022, 15, 3706 3 of 20 3 of 20 carried out in the works [1,6,17,18]. Porter et al. [19] studied the effect of the inﬁll orien- tation and density on the ﬂexural properties of the PLA samples manufactured by FDM. The results showed that the percentage of 10 to 20% is the optimum inﬁll for maximizing the stiffness-to-mass, but it is recommended to use a higher inﬁll to avoid the drop off in ﬂexural rigidity. Mishra et al. [20] researched the effect of different combinations of inﬁll densities and inﬁll patterns on the absorbed energy of the PLA 3D-printed samples. The results concluded that an 85% inﬁll density for each inﬁll pattern demonstrated the highest energy absorption. In [21], Tanveer et al. investigated the impact strength of the PLA samples with 50%, 70%, and 100% inﬁll densities. The authors concluded that impact strength is direct proportional to inﬁll density percentage. Aloyaydi et al. [22] investigated the inﬂuence of inﬁll density on the microstructure and ﬂexural behavior of PLA 3D printed using FDM. 2. Materials and Methods through FFF. The thermal behavi etry (DSC) and Thermogravimetri Parameter Value Unit Filament diameter 2.85 [mm] Layer height 0.2 [mm] Infill density 25; 50; 75; 100 [%] Print speed 50 [mm/sec] Extrusion temperature 260 [°C] Building plate temperature 90 [°C] Nozzle diameter 0.6 [mm] Number of lower and upper layers 8 Number of shell contours 4 Infill pattern Triangle (0°/60° with respect to load direction) Table 2. Dogbone-shaped samples dimensions subjected to tensile tests. Lengt h。。 Over- all。。L0 [mm] Distance。。 between Grips 。。D [mm] Length of Narrow。。Section。。Ln [mm] Radius of Fillet。。R [mm] Width。。W0 [mm] Width of Narrow Section。。 W [mm] Thickness 。。c [mm] 165 115 57 76 19 13 4 Table 3. Samples dimensions subjected to flexural tests. Length。。L [mm] Width。。a [mm] Thickness。。b [mm] 200 20 4 Length L [mm] Width a [mm] Thickness b [mm] 200 20 4 Table 1. Manufacturing parameters for the PAHT CF15 composite samples. pp g , pp g and the speed set to 10 mm/min. y yp g p p ength are presented in Table 2 and Table 3, respectively. All tensile tests were carried t at a 5 mm/min speed The three point flexural test was conducted using a distanc Table 1. Manufacturing parameters for the PAHT CF15 composite samples. Parameter Value Unit Filament diameter 2.85 [mm] Layer height 0.2 [mm] Inﬁll density 25; 50; 75; 100 [%] Print speed 50 [mm/s] Extrusion temperature 260 [◦C] Building plate temperature 90 [◦C] Nozzle diameter 0.6 [mm] Number of lower and upper layers 8 Number of shell contours 4 Inﬁll pattern Triangle (0◦/60◦with respect to load direction) pp g , pp g m and the speed set to 10 mm/min. le 1. Manufacturing parameters for the PAHT CF15 composite samples. Parameter Value Unit Filament diameter 2.85 [mm] Layer height 0.2 [mm] Infill density 25; 50; 75; 100 [%] Print speed 50 [mm/sec] Extrusion temperature 260 [°C] Building plate temperature 90 [°C] Nozzle diameter 0.6 [mm] Number of lower and upper layers 8 Number of shell contours 4 Infill pattern Triangle (0°/60° with respect to l d di ti ) y yp g p p trength are presented in Table 2 and Table 3, respectively. All tensile tests were carried ut at a 5 mm/min speed. The three-point flexural test was conducted using a distance etween the two supporting rollers of 95 mm, with the supporting rollers diameter of 30 mm and the speed set to 10 mm/min. able 1. 2. Materials and Methods through FFF. The thermal behavi etry (DSC) and Thermogravimetri The mechanical and physical properties of the Ultrafuse PAHT CF15 filament supplied by the manufacturer (BASF 3D Length L [mm] Width a [mm] Thickness b [mm] 200 20 4 The carbon ﬁber-reinforced polyamide composites, Ultrafuse PAHT 15, was used for printing the samples. The PAHT CF15 is a high-temperature polyamide-based ﬁlament ﬁlled with 15% chopped carbon ﬁbers for Fused Filament Fabrication (FFF) [32]. PAHT CF15 is a high-performance 3D printing ﬁlament that allows the manufacture of parts that can be subjected to high thermal and mechanical loads [33]. The mechanical and physical properties of the Ultrafuse PAHT CF15 ﬁlament supplied by the manufacturer (BASF 3D Printing Solutions BV, Emmen, The Netherlands) for the ﬂat XY-direction print in Table 4 are shown [33]. The ﬁlament was used in the printing process in the state delivered Table 2. Dogbone-shaped samples dimensions subjected to tensile tests. le 2. Dogbone-shaped samples dimensions subjected to tensile tests. Infill pattern Triangle (0°/60° with respect to load direction) Table 2. Dogbone-shaped samples dimensions subjected to tensile tests. Table 2. Dogbone-shaped samples dimensions subjected to tensile tests. Lengt h。。 Over- all。。L0 [mm] Distance。。 between Grips 。。D [mm] Length of Narrow。。Section。。Ln [mm] Radius of Fillet。。R [mm] Width。。W0 [mm] Width of Narrow Section。。 W [mm] Thickness 。。c [mm] 165 115 57 76 19 13 4 Table 3. Samples dimensions subjected to flexural tests. Length。。L [mm] Width。。a [mm] Thickness。。b [mm] 200 20 4 h b f b f d l d Ul f A d f Length Overall L0 [mm] Distance between Grips D [mm] Length of Narrow Section Ln [mm] Radius of Fillet R [mm] Width W0 [mm] Width of Narrow Section W [mm] Thickness c [mm] 165 115 57 76 19 13 4 Table 3. Samples dimensions subjected to ﬂexural tests. Infill pattern Triangle (0°/60° with respect to load direction) Table 2. Dogbone-shaped samples dimensions subjected to tensile tests. Lengt h。。 Over- all。。L0 [mm] Distance。。 between Grips 。。D [mm] Length of Narrow。。Section。。Ln [mm] Radius of Fillet。。R [mm] Width。。W0 [mm] Width of Narrow Section。。 W [mm] Thickness 。。c [mm] 165 115 57 76 19 13 4 Table 3. Samples dimensions subjected to flexural tests. Length。。L [mm] Width。。a [mm] Thickness。。b [mm] 200 20 4 Length L [mm] Width a [mm] Thickness b [mm] 200 20 4 nting the samples. 1. Introduction Although the properties of various composite materials reinforced with various types of material have been studied, some fundamental properties of commercially available materials and uses in large-scale printing and in various ﬁelds have not yet been studied. The motivation of this paper resulted from the need to analyze the capacity of both FFF technology and commercially available ﬁber-reinforced composites to manufacture components (e.g., unmanned aerial vehicle components) subject to different types of stress [19,29]. Therefore, the purpose of this paper is the investigation of the inﬁll density (ID) inﬂuence on the thermal and mechanical properties of commercially available PAHT CF15 composite, reinforced with carbon ﬁber (CF), used in the Fused Filament Fabrication (FFF) process. The inﬂuence of inﬁll density (25%, 50%, 75%, 100%) on the mechanical performance of the samples was performed by a tensile test and three- point bending test. Microscopic analyses were performed to highlight the distribution and orientation of the carbon ﬁbers in the polymer matrix, the defects in the microstructure of the ﬁlament, and those that occurred during the manufacturing process through FFF. The thermal behavior was characterized by Differential Scanning Calorimetry (DSC) and Thermogravimetric analysis (TGA) to obtain information about material transition, melting, and degradation. Materials 2022, 15, 3706 , , 4 of 20 2. Materials and Methods through FFF. The thermal behavi etry (DSC) and Thermogravimetri All samples were printed with the BCN3D Epsilon W50 printer (BCN3D Technologies, Barcelona, Spain) with the parameters shown in Table 1. Preparation of the samples for manufacturing was carried out in the Cura—BCN3D software, 2.1.2 version (BCN3D Technologies, Barcelona, Spain). The 3D models of the samples for tensile and ﬂexural tests were designed according to ASTM D638 [30] and ASTM D790 [31] standards, respectively. The dimensions of the Type I dogbone-shaped samples for tensile tests and ﬂexural strength are presented in Tables 2 and 3, respectively. All tensile tests were carried out at a 5 mm/min speed. The three-point ﬂexural test was conducted using a distance between the two supporting rollers of 95 mm, with the supporting rollers diameter of 30 mm and the speed set to 10 mm/min. y ( ) g y ( ) transition, melting, and degradation. Materials and Methods All samples were printed with the BCN3D Epsilon W50 printer (BCN3D Technolo- , Barcelona, Spain) with the parameters shown in Table 1. Preparation of the samples manufacturing was carried out in the Cura—BCN3D software, 2.1.2 version ( BCN3D hnologies, Barcelona, Spain). The 3D models of the samples for tensile and flexural s were designed according to ASTM D638 [30] and ASTM D790 [31] standards, respec- ly. The dimensions of the Type I dogbone-shaped samples for tensile tests and flexural ngth are presented in Table 2 and Table 3, respectively. All tensile tests were carried at a 5 mm/min speed. The three-point flexural test was conducted using a distance he microstructure of the filament, and those that occurred during the manufacturing pro- ess through FFF. The thermal behavior was characterized by Differential Scanning Calo- metry (DSC) and Thermogravimetric analysis (TGA) to obtain information about mate- al transition, melting, and degradation. Materials and Methods All samples were printed with the BCN3D Epsilon W50 printer (BCN3D Technolo- ies, Barcelona, Spain) with the parameters shown in Table 1. Preparation of the samples or manufacturing was carried out in the Cura—BCN3D software, 2.1.2 version ( BCN3D echnologies, Barcelona, Spain). The 3D models of the samples for tensile and flexural ests were designed according to ASTM D638 [30] and ASTM D790 [31] standards, respec- l h d f h I d b h d l f l d fl l Table 1. Manufacturing parameters for the PAHT CF15 composite samples. 2. Materials and Methods through FFF. The thermal behavi etry (DSC) and Thermogravimetri Parameter Value Unit Filament diameter 2.85 [mm] Layer height 0.2 [mm] Inﬁll density 25; 50; 75; 100 [%] Print speed 50 [mm/s] Extrusion temperature 260 [◦C] Building plate temperature 90 [◦C] Nozzle diameter 0.6 [mm] Number of lower and upper layers 8 Number of shell contours 4 Inﬁll pattern Triangle (0◦/60◦with respect to load direction) Table 2. Dogbone-shaped samples dimensions subjected to tensile tests. between the two supporting rollers of 95 mm, with the supporting rollers diameter of 30 mm and the speed set to 10 mm/min. Table 1. Manufacturing parameters for the PAHT CF15 composite samples. Parameter Value Unit Filament diameter 2.85 [mm] Layer height 0.2 [mm] Infill density 25; 50; 75; 100 [%] Print speed 50 [mm/sec] Extrusion temperature 260 [°C] Building plate temperature 90 [°C] Nozzle diameter 0.6 [mm] Number of lower and upper layers 8 Number of shell contours 4 Infill pattern Triangle (0°/60° with respect to load direction) Table 2. Dogbone-shaped samples dimensions subjected to tensile tests. Lengt h。。 Over- all。。L0 [mm] Distance。。 between Grips 。。D [mm] Length of Narrow。。Section。。Ln [mm] Radius of Fillet。。R [mm] Width。。W0 [mm] Width of Narrow Section。。 W [mm] Thickness 。。c [mm] 165 115 57 76 19 13 4 Table 3. Samples dimensions subjected to flexural tests. Length。。L [mm] Width。。a [mm] Thickness。。b [mm] 200 20 4 The carbon fiber-reinforced polyamide composites Ultrafuse PAHT 15 was used for Length Overall L0 [mm] Distance between Grips D [mm] Length of Narrow Section Ln [mm] Radius of Fillet R [mm] Width W0 [mm] Width of Narrow Section W [mm] Thickness c [mm] 165 115 57 76 19 13 4 Table 3. Samples dimensions subjected to ﬂexural tests. tively. The dimensions of the Type I dogbone shaped samples for tensile tests and flexural strength are presented in Table 2 and Table 3, respectively. All tensile tests were carried out at a 5 mm/min speed. The three-point flexural test was conducted using a distance between the two supporting rollers of 95 mm, with the supporting rollers diameter of 30 mm and the speed set to 10 mm/min. Table 1. Manufacturing parameters for the PAHT CF15 composite samples. 2. Materials and Methods through FFF. The thermal behavi etry (DSC) and Thermogravimetri Manufacturing parameters for the PAHT CF15 composite samples. Parameter Value Unit Filament diameter 2.85 [mm] Layer height 0.2 [mm] Infill density 25; 50; 75; 100 [%] Print speed 50 [mm/sec] Extrusion temperature 260 [°C] Building plate temperature 90 [°C] Nozzle diameter 0.6 [mm] Number of lower and upper layers 8 Number of shell contours 4 Table 2. Dogbone-shaped samples dimensions subjected to tensile tests. Table 2. Dogbone-shaped samples dimensions subjected to tensile tests. Lengt h。。 Over- all。。L0 [mm] Distance。。 between Grips 。。D [mm] Length of Narrow。。Section。。Ln [mm] Radius of Fillet。。R [mm] Width。。W0 [mm] Width of Narrow Section。。 W [mm] Thickness 。。c [mm] 165 115 57 76 19 13 4 Table 3. Samples dimensions subjected to flexural tests. Length。。L [mm] Width。。a [mm] Thickness。。b [mm] 200 20 4 The carbon fiber-reinforced polyamide composites, Ultrafuse PAHT 15, was used for printing the samples. The PAHT CF15 is a high-temperature polyamide-based filament filled with 15% chopped carbon fibers for Fused Filament Fabrication (FFF) [32]. PAHT CF15 is a high-performance 3D printing filament that allows the manufacture of parts that can be subjected to high thermal and mechanical loads [33]. The mechanical and physical properties of the Ultrafuse PAHT CF15 filament supplied by the manufacturer (BASF 3D Length Overall L0 [mm] Distance between Grips D [mm] Length of Narrow Section Ln [mm] Radius of Fillet R [mm] Width W0 [mm] Width of Narrow Section W [mm] Thickness c [mm] 165 115 57 76 19 13 4 Table 3. Samples dimensions subjected to ﬂexural tests. Infill pattern Triangle (0°/60° with respect to load direction) Table 2. Dogbone-shaped samples dimensions subjected to tensile tests. Lengt h。。 Over- all。。L0 [mm] Distance。。 between Grips 。。D [mm] Length of Narrow。。Section。。Ln [mm] Radius of Fillet。。R [mm] Width。。W0 [mm] Width of Narrow Section。。 W [mm] Thickness 。。c [mm] 165 115 57 76 19 13 4 Table 3. Samples dimensions subjected to flexural tests. Length。。L [mm] Width。。a [mm] Thickness。。b [mm] 200 20 4 The carbon fiber-reinforced polyamide composites, Ultrafuse PAHT 15, was used for printing the samples. The PAHT CF15 is a high-temperature polyamide-based filament filled with 15% chopped carbon fibers for Fused Filament Fabrication (FFF) [32]. PAHT CF15 is a high-performance 3D printing filament that allows the manufacture of parts that can be subjected to high thermal and mechanical loads [33]. 2. Materials and Methods through FFF. The thermal behavi etry (DSC) and Thermogravimetri The PAHT CF15 is a high-temperature polyamide-based filament d with 15% chopped carbon fibers for Fused Filament Fabrication (FFF) [32]. PAHT 5 is a high-performance 3D printing filament that allows the manufacture of parts that be subjected to high thermal and mechanical loads [33]. The mechanical and physical perties of the Ultrafuse PAHT CF15 filament supplied by the manufacturer (BASF 3D The carbon fiber-reinforced polyamide composites, Ultrafuse PAHT 15, was used for rinting the samples. The PAHT CF15 is a high-temperature polyamide-based filament lled with 15% chopped carbon fibers for Fused Filament Fabrication (FFF) [32]. PAHT CF15 is a high-performance 3D printing filament that allows the manufacture of parts that an be subjected to high thermal and mechanical loads [33]. The mechanical and physical roperties of the Ultrafuse PAHT CF15 filament supplied by the manufacturer (BASF 3D The carbon ﬁber-reinforced polyamide composites, Ultrafuse PAHT 15, was used for printing the samples. The PAHT CF15 is a high-temperature polyamide-based ﬁlament ﬁlled with 15% chopped carbon ﬁbers for Fused Filament Fabrication (FFF) [32]. PAHT CF15 is a high-performance 3D printing ﬁlament that allows the manufacture of parts that can be subjected to high thermal and mechanical loads [33]. The mechanical and physical properties of the Ultrafuse PAHT CF15 ﬁlament supplied by the manufacturer (BASF 3D Printing Solutions BV, Emmen, The Netherlands) for the ﬂat XY-direction print in Table 4 are shown [33]. The ﬁlament was used in the printing process in the state delivered by the manufacturer, without being subjected to thermal procedures, being stored in its original packaging. Materials 2022, 15, 3706 5 of 20 Table 4. Mechanical and physical properties of the Ultrafuse PAHT CF15 ﬁlament [33]. Table 4. Mechanical and physical properties of the Ultrafuse PAHT CF15 ﬁlament [33]. Mechanical and Physical Properties Unit Ultrafuse PAHT CF15 Testing Method Dry Specimen Conditioned Specimen Tensile strength [MPa] 103.2 62.9 ISO 527 Elongation at Break [%] 1.8 2.9 ISO 527 Young’s Modulus [MPa] 8386 5052 ISO 527 Flexural Strength [MPa] 160.7 125.1 ISO 178 Flexural Modulus [MPa] 8258 6063 ISO 178 Flexural Strain at Break [%] 2.4 No break ISO 178 Melting temperature [◦C] 234 ISO 11357-3 Glass Transition [◦C] 70 ISO 11357-2 A total of 5 specimens were printed for each type of inﬁll (25%, 50%, 75%, 100%) using a triangle inﬁll pattern, resulting in 20 specimens for the tensile tests and 20 for the ﬂexural ones, respectively. 2. Materials and Methods through FFF. The thermal behavi etry (DSC) and Thermogravimetri To evaluate the tensile properties of the samples, a WDW- 150 S universal testing machine was used at a constant crosshead speed of 5 mm/min. For ﬂexural properties in the three-point loading, the same testing machine was used. The crosshead speed and span length were 3 mm/min and 115 mm, respectively. The microstructure, the distribution and orientation of carbon ﬁbers, and the distribution of voids both in the ﬁlament and the samples were investigated by optical microscopy. The samples and ﬁlament were cross-sectioned, embedded into resin, and polished using a 1 µm grit and 0.5 µm Al2O3 suspension. Using an Omnimet–Buehler microscope (Nikon, Tokyo, Japan), micrographs were taken on the sections perpendicular to the XY plane (cross-section) and the Z building direction (longitudinal section) with magniﬁcations of 50× and 200×. Differential Scanning Calorimetry (DSC) was conducted under the nitrogen atmosphere using a NETZSCH DSC 200 F3 Maia (NETZSCH-Gerätebau GmbH, Selb, Germany) to highlight the glass transition, melting, and crystallization of the samples. DSC tests were carried out on the as-received ﬁlament and extruded material in accordance with ASTM D3418 [34] at a rate of 10 ◦C/min, cooling to −150 ◦C, and then heating to 500 ◦C. Thermogravimetric analysis (TGA) was performed on the NETZSCH TG STA 449F3 Jupiter (NETZSCH-Gerätebau GmbH, Selb, Germany) to identify the degradation temperature and weight loss. TGA was carried out from 20 ◦C to 500 ◦C at a heating rate of 10 ◦C/min according to the ASTM Standard E1131 [35]. TG analyses were performed in the temperature ranges of 20–100 ◦C, 100–300 ◦C, and 300–500 ◦C. NETZSCH Proteus Thermal Analysis software was used to process the data. It should be mentioned that the microstructural analyses were performed on the shell area of the samples, and for the DSC and TGA analyses, the mass of material was taken from the core area of the samples. 3.1.1. Tensile Strength of Carbon Fiber PAHT Composite From Figure 1a, it can be seen that the increase in the inﬁll density determines the increase in the load and inﬂuences the displacement. For the samples with a 100% inﬁll, the highest load (4.9 kN) and displacement (2.54 mm) are registered, and those with a 25% inﬁll have the lowest values (2.9 kN and 1.74 mm, respectively), which conﬁrms the inﬂuence of the inﬁll density on tensile strength, results that are consistent with those published in [13,14]. This is justiﬁed by the fact as the inﬁll density increases, the content of polymeric material increases (which determines the air gaps in the material to decrease) and implicitly the cross-sectional area. Therefore, the parts manufactured by FFF can support a higher load as the inﬁll percentage increases. increases, the tensile strength also increases. These results are in line with those published in [2,13,14,19,21]. Therefore, samples with a 25% infill have a lower strength, of 56.4 MPa, and an elasticity modulus of 5.8 GPa, and those with a 100% infill have a strength that increases to 90.8 MPa and a modulus that increases to 8 GPa. The relationship between the tensile strength and the degree of filling is indicated by an order 2 polynomial, as shown in Figure 1b. Figure 1b shows that for samples with 25% and 50% infills, the differences between the values of maximum tensile strength are small (54.6 MPa and 61.6 MPa, respectively, a difference of 7 MPa) and the tensile modulus is the same, 5.8 GPa. Taking these results Samples with a 75% inﬁll have a smaller maximum displacement (2.22 mm) than those with 50% (2.28 mm) even if the maximum sustained load (4.03 kN) is higher than that at a 50% inﬁll (3.56 kN). It has been demonstrated [21] that the outer layers (shell), which are denser, provide a resistance to crack propagation. Under action load, during failure of the sample, the cracks propagate from shell to core (inner layers) [21,36]. The presence of manufacturing defects both within the shell and core, such as voids and gaps, as shown in micrographs of the samples with 75% inﬁll density, leads to the rapid propagation of cracks and the decrease in interlayer cohesion, as reported in [24,36], which determine breaking of the samples with a 75% inﬁll after a lower displacement than 50% ID. 3.1.1. Tensile Strength of Carbon Fiber PAHT Composite 3.1.1. Tensile Strength of Carbon Fiber PAHT Composite In order to establish the inﬂuence of the inﬁll density on the tensile strength of the samples manufactured by FFF, a comparative study was performed between the four types of inﬁll. The data obtained during the tensile tests were processed, and the load– displacement curves as an average of the values for the ﬁve samples tested with inﬁlls of 25%, 50%, 75%, and 100% are shown in Figure 1a. Materials 2022, 15, 3706 6 of 20 tly the er load Figure 1. Results after tensile tests; (a) load–displacement curves; (b) tensile strength, modulus, and relationship between tensile strength and infill density. Figure 1. Results after tensile tests; (a) load–displacement curves; (b) tensile strength, modulus, and relationship between tensile strength and inﬁll density. Figure 1. Results after tensile tests; (a) load–displacement curves; (b) tensile strength, modulus, and relationship between tensile strength and infill density. Figure 1. Results after tensile tests; (a) load–displacement curves; (b) tensile strength, modulus, and relationship between tensile strength and inﬁll density. Samples with a 75% infill have a smaller maximum displacement (2.22 mm) than those with 50% (2.28 mm) even if the maximum sustained load (4.03 kN) is higher than that at a 50% infill (3.56 kN). It has been demonstrated [21] that the outer layers (shell), which are denser, provide a resistance to crack propagation. Under action load, during failure of the sample, the cracks propagate from shell to core (inner layers) [21,36]. The presence of manufacturing defects both within the shell and core, such as voids and gaps, as shown in micrographs of the samples with 75% infill density, leads to the rapid propa- gation of cracks and the decrease in interlayer cohesion, as reported in [24,36], which de- termine breaking of the samples with a 75% infill after a lower displacement than 50% ID. In order to determine the tensile strength, an average of the values of the maximum stresses and of the modulus of elasticity of the five samples from each type of infill (25%, 50%, 75%, 100%) was calculated. The plot in Figure 1b shows that as the infill density For each inﬁll density, a linear behavior can be ascertained between the load and displacement up to the point of yield strength (plastic deformation of the material begins) and up to the point where the load is maximum and failure and breaking occur. 3.1.1. Tensile Strength of Carbon Fiber PAHT Composite Therefore, the ﬂexural strength will be determined by the strength of the intact areas/layers. In the case of tensile tests, all layers (material) are subjected to the same stress and, therefore, breakage/failure will be initiated when the layer/material on the layer reaches the tensile stress limit. The higher displacement of the samples with an inﬁll of 25% (maximum displacement of 25.8 mm) under the action of lower loads could be inﬂuenced by the existence in the microstructure at the top, bottom, and lateral surfaces of the samples of a greater number of voids and the occurrence of microcracks as well. the polynomial equation of order 2 was chosen (Figure 2b) as it describes with the greatest precision the relation between them (R2 is 0.9985, in contrast with 0.929 in the case of a linear relation) [25,37]. As in the case of tensile tests, it is found that there are small differ- ences between the values of flexural strength for samples with infill densities of 25% and 50%, respectively. Therefore, to reduce manufacturing/printing time and material and en- ergy consumption, an infill with a density of 25% can be used instead of 50%. 3.1.3. Microstructural Characteristics of the Carbon Fiber PAHT Composite Microstructural analyses, both for the filament and for printed samples, were per- formed to highlight aspects specific to 3D printing, namely the distribution and orienta- tion of fibers in the polymer matrix, the presence of voids and deposition defects, the ap- pearance of microcracks, and the influence of the infill percentage on the microstructure. Figure 3 shows the longitudinal (Figure 3a) and cross-sectional (Figure 3b) micrographs of the PAHT CF15 filament Figure 3 shows that the distribution of carbon fibers in the g The average values of the ﬂexural strength as well as the values of the modulus corresponding to the four inﬁll densities are shown in Figure 2b. In Figure 2b, it can be seen that the samples with a 100% inﬁll have the highest ﬂexural strength (114 MPa) and the highest ﬂexural modulus (3.7 GPa). Between samples with a 25% inﬁll density and those with 100%, it is found that there is an increase in bending strength of 83.3%, which means that inﬁll density has a major contribution, as found in previous studies [2,6,13,19,21]. In contrast, 50% inﬁll density samples have a strength of only 8.7% higher than those with a 25% inﬁll. 3.1.1. Tensile Strength of Carbon Fiber PAHT Composite In contrast, compared to other samples, they have the smallest displacement, 18.6 mm. The analysis of the curves shows that the samples with 25% and 50% inﬁlls (Figure 2a) withstand very close loads, with the differences between the values being very small, that is, 0.13 kN for 50% and 0.12 kN for 25% inﬁll densities. fore, the flexural strength will be determined by the strength of the intact areas/layers. In the case of tensile tests, all layers (material) are subjected to the same stress and, therefore, breakage/failure will be initiated when the layer/material on the layer reaches the tensile stress limit. The higher displacement of the samples with an infill of 25% (maximum dis- placement of 25.8 mm) under the action of lower loads could be influenced by the exist- ence in the microstructure at the top, bottom, and lateral surfaces of the samples of a greater number of voids and the occurrence of microcracks as well. Figure 2. Results after three−points flexural tests: (a) flexural test load–displacement curves; (b) flex- ural strength, modulus, and relationship between flexural strength and infill density. Figure 2. Results after three−points ﬂexural tests: (a) ﬂexural test load–displacement curves; (b) ﬂexural strength, modulus, and relationship between ﬂexural strength and inﬁll density. Figure 2. Results after three−points flexural tests: (a) flexural test load–displacement curves; (b) flex- ural strength, modulus, and relationship between flexural strength and infill density. Figure 2. Results after three−points ﬂexural tests: (a) ﬂexural test load–displacement curves; (b) ﬂexural strength, modulus, and relationship between ﬂexural strength and inﬁll density. The average values of the flexural strength as well as the values of the modulus cor- responding to the four infill densities are shown in Figure 2b. In Figure 2b, it can be seen that the samples with a 100% infill have the highest flexural strength (114 MPa) and the highest flexural modulus (3.7 GPa). Between samples with a 25% infill density and those with 100%, it is found that there is an increase in bending strength of 83.3%, which means that infill density has a major contribution, as found in previous studies [2,6,13,19,21]. In contrast, 50% infill density samples have a strength of only 8.7% higher than those with a 25% infill. In order to express the relationship between flexural strength and infill density, It is known that in ﬂexural tests, the exterior surfaces are most subject to stress. 3.1.1. Tensile Strength of Carbon Fiber PAHT Composite ) , g into account, it can be stated that the 25% infill can be used instead of the 50% infill, if the operating conditions of the components allow, because the manufacturing cost is lower using less material and a shorter print time. 3.1.2. Flexural Strength of the Carbon Fiber PAHT Composite In order to determine the tensile strength, an average of the values of the maximum stresses and of the modulus of elasticity of the ﬁve samples from each type of inﬁll (25%, 50%, 75%, 100%) was calculated. The plot in Figure 1b shows that as the inﬁll density increases, the tensile strength also increases. These results are in line with those published in [2,13,14,19,21]. Therefore, samples with a 25% inﬁll have a lower strength, of 56.4 MPa, and an elasticity modulus of 5.8 GPa, and those with a 100% inﬁll have a strength that increases to 90.8 MPa and a modulus that increases to 8 GPa. The relationship between the tensile strength and the degree of ﬁlling is indicated by an order 2 polynomial, as shown in Figure 1b. Figure 1b shows that for samples with 25% and 50% inﬁlls, the differences between the values of maximum tensile strength are small (54.6 MPa and 61.6 MPa, respectively, a difference of 7 MPa) and the tensile modulus is the same, 5.8 GPa. Taking these results into account, it can be stated that the 25% inﬁll can be used instead of the 50% inﬁll, if the operating conditions of the components allow, because the manufacturing cost is lower using less material and a shorter print time. Materials 2022, 15, 3706 7 of 20 a e differ- 7 of 20 a e differ- 3.1.2. Flexural Strength of the Carbon Fiber PAHT Composite It is known that in flexural tests, the exterior surfaces are m f th fl l t th ill b d t i d b th t th f 3.1.2. Flexural Strength of the Carbon Fiber PAHT Composite It is known that in flexural tests, the exterior surfaces are m f h fl l h ill b d i d b h h The ﬂexural behavior of the samples with the four types of inﬁll percentages in Figure 2a is shown. Load–displacement curves show that samples with a 100% inﬁll have the highest strength-to-maximum load of 0.23 kN. 3.1.1. Tensile Strength of Carbon Fiber PAHT Composite In both sections, it is found that the ﬁlament contains, in addition to carbon ﬁber, voids (manufacturing defects of the ﬁlament). The voids, in turn, can inﬂuence the quality of the microstructure of the deposited material and subsequently the mechanical properties of the samples. is found that they are not entirely along the filament but rather have a random orie on, as can be seen in Figure 3a. In both sections, it is found that the filament contain ddition to carbon fiber, voids (manufacturing defects of the filament). The voids, in t an influence the quality of the microstructure of the deposited material and subseque he mechanical properties of the samples. Figure 3. Micrographs of the PAHT CF15 filament. (a) Longitudinal section/view (200×); (b) c section (200×). Figure 3. Micrographs of the PAHT CF15 ﬁlament. (a) Longitudinal section/view (200×); (b) cross-section (200×). gure 3. Micrographs of the PAHT CF15 filament. (a) Longitudinal section/view (200×); (b) c ction (200×). Figure 3. Micrographs of the PAHT CF15 ﬁlament. (a) Longitudinal section/view (200×); (b) cross-section (200×). Figure 4 shows the microstructure of the samples manufactured by FFF with an i ensity of 25%. Figure 4a shows the microstructure, in the longitudinal section, on xterior (lateral) wall of the sample. It can be seen that, after printing, the porosity eases toward the outside of the specimen (blue arrow) but also toward the inside w e hole related to the infill is found (green arrow). It is also found that at the bound etween the successive beads (bead–bead interface), there are linear areas with a hig orosity (voids) than those of the deposited layer (Figure 4a, areas marked with A). henomenon was also noticed in the published results of previous studies [1,6,7]. An ease in porosity is also seen in the printing direction (Z direction) from bottom (Fig c) to top (Figure 4d) [38]. A factor that could lead to an increase in the porosity (void e decrease in the pressure/weight of the layers on those already deposited as the d tion of the extreme layers approaches, and, consequently, on the extreme layers (top teral layers), only the atmospheric pressure from the printing chamber acts. In addi t t di t ib ti i th d it d l i th i fl i f t Figure 4 shows the microstructure of the samples manufactured by FFF with an inﬁll density of 25%. 3.1.1. Tensile Strength of Carbon Fiber PAHT Composite In order to express the relationship between ﬂexural strength and inﬁll density, the polynomial equation of order 2 was chosen (Figure 2b) as it describes with the greatest precision the relation between them (R2 is 0.9985, in contrast with 0.929 in the case of a linear relation) [25,37]. As in the case of tensile tests, it is found that there are small differences between the values of ﬂexural strength for samples with inﬁll densities of 25% and 50%, respectively. Therefore, to reduce manufacturing/printing time and material and energy consumption, an inﬁll with a density of 25% can be used instead of 50%. polymer matrix is approximately uniform. Regarding the orientation of the c 3.1.3. Microstructural Characteristics of the Carbon Fiber PAHT Composite Microstructural analyses, both for the ﬁlament and for printed samples, were per- formed to highlight aspects speciﬁc to 3D printing, namely the distribution and orientation of ﬁbers in the polymer matrix, the presence of voids and deposition defects, the appearance of microcracks, and the inﬂuence of the inﬁll percentage on the microstructure. Figure 3 shows the longitudinal (Figure 3a) and cross-sectional (Figure 3b) micrographs of the PAHT CF15 ﬁlament. Figure 3 shows that the distribution of carbon ﬁbers in the polymer matrix Materials 2022, 15, 3706 rials 2022, 15, x FOR PEE 8 of 208 8 of 208 is approximately uniform. Regarding the orientation of the carbon ﬁbers, it is found that they are not entirely along the ﬁlament but rather have a random orientation, as can be seen in Figure 3a. In both sections, it is found that the ﬁlament contains, in addition to carbon ﬁber, voids (manufacturing defects of the ﬁlament). The voids, in turn, can inﬂuence the quality of the microstructure of the deposited material and subsequently the mechanical properties of the samples. is found that they are not entirely along the filament but rather have a random orie on, as can be seen in Figure 3a. In both sections, it is found that the filament contain ddition to carbon fiber, voids (manufacturing defects of the filament). The voids, in t an influence the quality of the microstructure of the deposited material and subseque he mechanical properties of the samples. is approximately uniform. Regarding the orientation of the carbon ﬁbers, it is found that they are not entirely along the ﬁlament but rather have a random orientation, as can be seen in Figure 3a. 3.1.1. Tensile Strength of Carbon Fiber PAHT Composite Figure 4a shows the microstructure, in the longitudinal section, on the exterior (lateral) wall of the sample. It can be seen that, after printing, the porosity increases toward the outside of the specimen (blue arrow) but also toward the inside where the hole related to the inﬁll is found (green arrow). It is also found that at the boundary between the successive beads (bead–bead interface), there are linear areas with a higher porosity (voids) than those of the deposited layer (Figure 4a, areas marked with A). This phenomenon was also noticed in the published results of previous studies [1,6,7]. An increase in porosity is also seen in the printing direction (Z direction) from bottom (Figure 4c) to top (Figure 4d) [38]. A factor that could lead to an increase in the porosity (voids) is the decrease in the pressure/weight of the layers on those already deposited as the deposition of the extreme layers approaches, and, consequently, on the extreme layers (top and lateral layers), only the atmospheric pressure from the printing chamber acts. In addition, the temperature distribution in the deposited layers is another inﬂuencing factor, as mentioned in the studies presented in [39,40]. he temperature distribution in the deposited layers is another influencing factor, as m oned in the studies presented in [39,40]. Figure 4b shows a detail from Figure 4a where it can be seen that, after melting the ﬁlament and passing the material through the nozzle, a reorientation of the carbon ﬁbers takes place, most of them being oriented along the deposition direction (along the deposited beads), as also presented in the papers [1,18,41]. The direction of deposition, both for the samples tested at tensile and the ﬂexural strengths, was along the samples (in the XY plane). The microstructure of the samples manufactured with an inﬁll density of 50% is presented in Figure 5. In the longitudinal section made in the lateral part of the sample (Figure 5a, areas marked with B), it is found that the area of porosity/defects (voids) between the successive beads in the XY plane is almost the same as for 25% samples but is much more pronounced than for 75% inﬁll density ones (Figure 6a). As in the case of samples manufactured with a 25% inﬁll, on the cross-section (Figure 5c,d), there is a higher porosity in the upper part of the samples. 3.1.1. Tensile Strength of Carbon Fiber PAHT Composite he temperature distribution in the deposited layers is another influencing factor, as m oned in the studies presented in [39,40]. Figure 4b shows a detail from Figure 4a where it can be seen that, after melting the ﬁlament and passing the material through the nozzle, a reorientation of the carbon ﬁbers takes place, most of them being oriented along the deposition direction (along the deposited beads), as also presented in the papers [1,18,41]. The direction of deposition, both for the samples tested at tensile and the ﬂexural strengths, was along the samples (in the XY plane). p g g p p The microstructure of the samples manufactured with an inﬁll density of 50% is presented in Figure 5. In the longitudinal section made in the lateral part of the sample (Figure 5a, areas marked with B), it is found that the area of porosity/defects (voids) between the successive beads in the XY plane is almost the same as for 25% samples but is much more pronounced than for 75% inﬁll density ones (Figure 6a). As in the case of samples manufactured with a 25% inﬁll, on the cross-section (Figure 5c,d), there is a higher porosity in the upper part of the samples. Materials 2022, 15, 3706 erials 2022, 15, x FOR P 9 of 20 Figure 4. Micrographs of the samples with 25% infill density. (a,b) Longitudinal se cross-section top side and (d) bottom side (50×). Figure 4. Micrographs of the samples with 25% inﬁll density. (a,b) Longitudinal section (200×); (c) cross-section top side and (d) bottom side (50×). VIEW 10 of 22 Figure 4. Micrographs of the samples with 25% infill density. (a,b) Longitudinal se cross-section top side and (d) bottom side (50×). Figure 4. Micrographs of the samples with 25% inﬁll density. (a,b) Longitudinal section (200×); (c) cross-section top side and (d) bottom side (50×). IEW 10 of 22 Figure 4a where it can be seen that, a hrough the nozzle, a reorientation of t nted along the deposition direction (a papers [1,18,41]. The direction of dep e flexural strengths, was along the sam ples manufactured with an infill densi nal section made in the lateral part of ound that the area of porosity/defects Figure 4b shows a detail from filament and passing the material th takes place, most of them being orie ited beads), as also presented in the the samples tested at tensile and the plane). 3.1.1. Tensile Strength of Carbon Fiber PAHT Composite (a,b) Longitudinal section (200×); (c,d) bottom and top side of the cross-section (200×). Figure 6. Micrographs of the samples with 75% inﬁll density. (a,b) Longitudinal section (200×); (c,d) bottom and top side of the cross-section (200×). Figure 7a,b show the microstructure inside (core) the samples manufactured by FFF with an infill density parameter of 100%. The different orientations of the carbon fibers are found (Figure 7a,b, purple arrow), depending on the deposition direction of the mol- ten material bead. The microstructure on the lateral side of the sample (Figures 7c,d), com- pared to those of the samples with 25%, 50% and 75% infills, contains fewer voids between beads (Figure 7c, D areas) and the carbon fibers are oriented (Figure 7d) along the direc- i f h i d b d I h i f Fi 7 f i i f d h h id In Figure 5b, carbon ﬁbers are mostly oriented in the direction of longitudinal depo- sition, while in the cross-section (Figure 5d), it can be seen that there are extremely few carbon ﬁbers, which have a different orientation. As previously mentioned, the presence of defects (voids, pores) at the level of the microstructure inﬂuences the mechanical properties. Consequently, the mechanical performances of samples with a 50% inﬁll close to those with an inﬁll density of 25% is justiﬁed. tion of the printed beads. In the cross-section from Figures 7e,f, it is found that the voids in the microstructure have a uniform distribution, that is, they respect to a certain extent a pattern and they are on the same line. Both in the bottom side of the section (Figure 7e) and in the top one (Figure 7f), interlayer cracks (interlaminar interface) were identified and the porosity increased from the bottom to the top side of the sample. Compared with the microstructure of the samples manufactured with 25% and 50% inﬁll densities, respectively, the 75% inﬁll density samples show a microstructure with fewer voids at the boundary between the successive material deposition (bead–bead interface) (Figure 6a), which means that there is a better adhesion between them. In addition, the reduction in defects can be seen in the micrographs of the cross-section (Figure 6c,d), but there remains a more pronounced porosity in the top part (Figure 6c) of the samples compared to the bottom one (Figure 6c). 3.1.1. Tensile Strength of Carbon Fiber PAHT Composite The microstructure of the samp sented in Figure 5. In the longitudin ure 5a, areas marked with B), it is fo Figure 4b shows a detail from Figure 4a where it can be seen that, aft filament and passing the material through the nozzle, a reorientation of th takes place, most of them being oriented along the deposition direction (alo ited beads), as also presented in the papers [1,18,41]. The direction of depo the samples tested at tensile and the flexural strengths, was along the sam plane). The microstructure of the samples manufactured with an infill density sented in Figure 5. In the longitudinal section made in the lateral part of th ure 5a, areas marked with B), it is found that the area of porosity/defects (v the successive beads in the XY plane is almost the same as for 25% sampl more pronounced than for 75% infill density ones (Figure 6a). As in the c manufactured with a 25% infill, on the cross-section (Figures 5c,d), there rosity in the upper part of the samples. Figure 5. Micrographs of the samples with 50% infill density. (a,b) Longitudinal section (200×); (c,d) top side of the cross-section at 50× and 200×. Figure 5. Micrographs of the samples with 50% inﬁll density. (a,b) Longitudinal section (200×); (c,d) top side of the cross-section at 50× and 200×. udin is fo udi is f the successive beads in the XY plan more pronounced than for 75% infi manufactured with a 25% infill, on rosity in the upper part of the sampl e is almost the same as for 25% sam ll density ones (Figure 6a). As in the the cross-section (Figures 5c,d), ther les. the mor Figure 5. Micrographs of the samples with 50% infill density. (a,b) Longitudinal section (200×); (c,d) top side of the cross-section at 50× and 200×. Figure 5. Micrographs of the samples with 50% inﬁll density. (a,b) Longitudinal section (200×); (c,d) top side of the cross-section at 50× and 200×. Materials 2022, 15, 3706 Materials 2022, 15, x FOR 10 of 20 11 of 22 Figure 6. Micrographs of the samples with 75% infill density. (a,b) Longitudinal section (200×); (c,d) bottom and top side of the cross-section (200×). Figure 6. Micrographs of the samples with 75% inﬁll density. (a,b) Longitudinal section (200×); (c,d) bottom and top side of the cross-section (200×). Figure 6. Micrographs of the samples with 75% infill density. 3.1.1. Tensile Strength of Carbon Fiber PAHT Composite As in the case of the 25% and 75% inﬁll density samples, the reorientation of the carbon ﬁbers along the direction of the deposition beads is found (Figure 6b), after the ﬁlament melts and the molten material passes through the extruder. Figure 7a,b show the microstructure inside (core) the samples manufactured by FFF with an inﬁll density parameter of 100%. The different orientations of the carbon ﬁbers are found (Figure 7a,b, purple arrow), depending on the deposition direction of the molten material bead. The microstructure on the lateral side of the sample (Figure 7c,d), compared to those of the samples with 25%, 50% and 75% inﬁlls, contains fewer voids between beads (Figure 7c, D areas) and the carbon ﬁbers are oriented (Figure 7d) along the direction of the printed beads. In the cross-section from Figure 7e,f, it is found that the voids in the microstructure have a uniform distribution, that is, they respect to a certain extent a pattern and they are on the same line. Both in the bottom side of the section (Figure 7e) and in the top one (Figure 7f), interlayer cracks (interlaminar interface) were identiﬁed and the porosity increased from the bottom to the top side of the sample. 11 of 20 gure 7e) entified Materials 2022, 15, 3706 erials 2022, 15, x FOR PEER REVIEW 12 of Figure 7. Micrographs of the samples with 100% infill density. (a,b) Longitudinal section from th interior of the sample (50×, 200×); (c,d) longitudinal section from the lateral side of the sample (50 200×); (e,f) bottom and top side of the cross-section (50×). Figure 7. Micrographs of the samples with 100% inﬁll density. (a,b) Longitudinal section from th interior of the sample (50×, 200×); (c,d) longitudinal section from the lateral side of the sample (50 200×); (e,f) bottom and top side of the cross-section (50×). 12 o rials 2022, 15, x FOR PEER REVIEW Figure 7. Micrographs of the samples with 100% infill density. (a,b) Longitudinal section from the interior of the sample (50×, 200×); (c,d) longitudinal section from the lateral side of the sample (50×, 200×); (e,f) bottom and top side of the cross-section (50×). Figure 7. Micrographs of the samples with 100% inﬁll density. (a,b) Longitudinal section from the interior of the sample (50×, 200×); (c,d) longitudinal section from the lateral side of the sample (50×, 200×); (e,f) bottom and top side of the cross-section (50×). 3.1.4. Thermal Behavior of Carbon Fiber PAHT Composite 3.1.4. Thermal Behavior of Carbon Fiber PAHT Composite Differential Scanning Calorimetry (DSC) was carried out to determine the effect of the processing conditions on the ﬁlament material as well as on the material deposited by the FFF process. Figures 8–12 show the DSC curves obtained from the analyses both of the PAHT CF15 ﬁlament (Figure 8) and for the samples manufactured by FFF with the four inﬁll densities (25%, 50%, 75%, and 100%, respectively) (Figures 9–12). A comparison of the DSC curves highlighted the differences between their thermal characteristics. On the heating curves (red curves), all samples exhibited signiﬁcant endothermic peaks due to melting (Tm). Melting temperature (Tm) is an important indicator in setting the printing temperature, and the values obtained (Tables 5 and 6) are close to those mentioned by the ﬁlament manufacturer (Table 4), the maximum difference being 2.5 ◦C in the case of samples with an inﬁll density of 100%. In addition, melting peaks are characteristic of semi-crystalline materials and indicate that the structure of the samples is crystalline (melting of the crystalline structure). ( g y ) The glass transition temperature (Tg) is the temperature at which the material turns from a glassy, rigid material to a soft, rubbery material due to the molecular motion of amorphous chains [42]. From the DSC curves (Figures 8–12) and the data presented in Table 5, it is found that the temperature Tg decreases as the inﬁll density increases; the lowest glass transition temperature is recorded for the specimens with an inﬁll density of 100% (Figure 12, Table 5). It is well known that carbon ﬁber has a higher thermal conductivity, which inﬂuences the thermal stability of the matrix. The increase in the thermal mobility of the polymer chain due to the chopped carbon ﬁbers leads to the decrease in the glass transition temperature; thus, the transformation from the glassy phase to the rubbery one takes place at a lower temperature. The decrease in glass transition temperature (Tg) as the carbon ﬁber content increases was also mentioned in [7,28,43]. Therefore, the increase in inﬁll density also involves, in addition to a larger volume of polymeric matrix, the increase in the amount of carbon ﬁber that causes the decrease in Tg as the inﬁll density increases. It can be stated that inﬁll density, as a 3D printing parameter, inﬂuences the thermal properties of the material. 3.1.1. Tensile Strength of Carbon Fiber PAHT Composite A common printing defect in FFF of fiber-reinforced composites is the formation of voids [1,6,7]. However, the defects of the voids type contained in the filament are trans- formed into voids in the printed beads. Previous studies [1,6,7] have demonstrated that voids (triangular or different in shape) formed during FFF manufacturing will act as stress concentrators that will reduce the mechanical performance of the parts. Therefore, a thor- ough understanding of the structure and mechanics of FFF of carbon fiber composites is required Based on the microstructure results for the four infill densities used in the man A common printing defect in FFF of ﬁber-reinforced composites is the formation of voids [1,6,7]. However, the defects of the voids type contained in the ﬁlament are transformed into voids in the printed beads. Previous studies [1,6,7] have demonstrated that voids (triangular or different in shape) formed during FFF manufacturing will act as stress concentrators that will reduce the mechanical performance of the parts. Therefore, a thorough understanding of the structure and mechanics of FFF of carbon ﬁber composites Materials 2022, 15, 3706 12 of 20 is required. Based on the microstructure results for the four inﬁll densities used in the manufacture of PAHT CF15 specimens by FFF technology, it can be mentioned that an increase in inﬁll density leads to a decrease in the voids at the bead–bead interface. However, in specimens with a 100% inﬁll density, the increase in interlayer defects (unbonding of layers or the appearance of cracks) is found. In the future, the authors intend to continue research in order to reduce the formation of interbeads and interlayer voids by decreasing the thickness of the deposited layer because a thinner layer could increase the adhesion between beads and layers, as well as mechanical properties, as mentioned in the studies presented in [7,12]. 3.1.4. Thermal Behavior of Carbon Fiber PAHT Composite Exothermic peaks after the glass transition temperature (Tg) represent the cold crystallization temperature (Tc) (Figures 8, 9, 11 and 12). The cold crystallization is an incomplete crystallization because of the fact that during the fast cooling from the molten state, the ﬁlament and the printed samples do not have enough time to crystallize. Upon reheating above Tg, the thermal energy provides sufﬁcient molecular mobility to allow the polyamide to crystallize. For samples with a 50% inﬁll density (Figure 10), the cold crystallization peak is very small; therefore, it has the lowest imperfect crystallization. Small peaks identiﬁed before the melting temperature for printed samples (Figures 9–12) refer to the recrystallization melting temperature. This temperature can be caused by morphological changes such as the completion of crystallization and the reﬁnement of crystals, as stated in the paper [42] too. From the curves in Figures 8–12, it is found that exceeding the melting temperature (Tm) has the effect of starting the degradation of the material, and the endothermic peaks highlight the complete degradation of the material (degradation temperature, Td). Table 5 shows the values of the most important peaks (Tg, Tc, Tm, Td) on the DSC heating curves in Figures 12–17. 13 of 20 line ma crystal- line ma- crystal- 13 of 20 line ma crystal- line ma- crystal- Materials 2022, 15, 3706 Figure 8. DSC thermogram for PAHT CF15 filament. Figure 9. Results of DSC analysis for sample with 25% infill density. Figure 8. DSC thermogram for PAHT CF15 ﬁlament. line structure). Figure 8. DSC thermogram for PAHT CF15 filament. Figure 9. Results of DSC analysis for sample with 25% infill density. Figure 9. Results of DSC analysis for sample with 25% inﬁll density. EVIEW 14 of 2 Figure 8. DSC thermogram for PAHT CF15 filament. Figure 8. DSC thermogram for PAHT CF15 ﬁlament. Figure 8. DSC thermogram for PAHT CF15 filament. Figure 8 DSC thermogram for PAHT CF15 filament Fi 8 DSC th f PAHT CF15 ﬁl t Figure 8. DSC thermogram for PAHT CF15 filament. Figure 9. Results of DSC analysis for sample with 25% infill density. Figure 8. DSC thermogram for PAHT CF15 ﬁlament. Figure 8. DSC thermogram for PAHT CF15 filament. Figure 9. Results of DSC analysis for sample with 25% infill density. Figure 9. Results of DSC analysis for sample with 25% inﬁll density. EVIEW 14 o Figure 10. DSC analysis for sample with 50% infill density. 3.1.4. Thermal Behavior of Carbon Fiber PAHT Composite Figure 10. DSC analysis for sample with 50% inﬁll density. Figure 8. DSC thermogram for PAHT CF15 filament. Figure 8. DSC thermogram for PAHT CF15 ﬁlament. Figure 8. DSC thermogram for PAHT CF15 filament. Figure 9. Results of DSC analysis for sample with 25% infill density. Figure 9 Re ult of DSC a aly i fo a ple ith 25% i fill de ity Figure 9 Results of DSC analysis for sample with 25% inﬁll density VIEW 14 Figure 9. Results of DSC analysis for sample with 25% infill density. Figure 9. Results of DSC analysis for sample with 25% infill density. Figure 9. Results of DSC analysis for sample with 25% inﬁll density. Figure 10. DSC analysis for sample with 50% infill density. Figure 10. DSC analysis for sample with 50% inﬁll density. Figure 10. DSC analysis for sample with 50% infill density. Figure 10. DSC analysis for sample with 50% inﬁll density. Materials 2022, 15, 3706 14 of 20 Figure 11. DSC thermogram for sample with 75% infill density. Figure 12. DSC curves related to sample with 100% infill density. Figure 11. DSC thermogram for sample with 75% inﬁll density. Figure 10. DSC analysis for sample with 50% infill density. Figure 11. DSC thermogram for sample with 75% infill density. Figure 12. DSC curves related to sample with 100% infill density. Figure 12. DSC curves related to sample with 100% inﬁll density. Table 5. Differential Scanning Calorimetry (DSC) results. Figure 11. DSC thermogram for sample with 75% infill density. Figure 11. DSC thermogram for sample with 75% inﬁll density. Figure 10. DSC analysis for sample with 50% infill density. Figure 11. DSC thermogram for sample with 75% infill density. Figure 11. DSC thermogram for sample with 75% infill density. Figure 11. DSC thermogram for sample with 75% inﬁll density. Figure 10. DSC analysis for sample with 50% infill density. Figure 11 DSC thermogram for sample with 75% infill density Figure 11. DSC thermogram for sample with 75% infill density. Figure 11. DSC thermogram for sample with 75% inﬁll density. Figure 11. DSC thermogram for sample with 75% infill density. Figure 12. DSC curves related to sample with 100% infill density. Figure 12. DSC curves related to sample with 100% infill density. Figure 12. DSC curves related to sample with 100% inﬁll density. Figure 12. DSC curves related to sample with 100% infill density. Figure 12. 3.1.4. Thermal Behavior of Carbon Fiber PAHT Composite DSC curves related to sample with 100% infill density. Figure 12 DSC curves related to sample with 100% inﬁll density Figure 12. DSC curves related to sample with 100% infill density. Figure 12. DSC curves related to sample with 100% infill density. Figure 12. DSC curves related to sample with 100% inﬁll density. Table 5. Differential Scanning Calorimetry (DSC) results. Table 5. Differential Scanning Calorimetry (DSC) results. Table 5. Differential Scanning Calorimetry (DSC) results. Samples Glass Transition Temperature Tg [◦C] Crystallization Temperature Tc [◦C] Melting Temperature Tm [◦C] Full Degradation Temperature Td [◦C] Filament PAHT CF15 38.5 77.7 232.1 406.9 Sample 25% ID 49.6 74.7 231.7 403.9 Sample 50% ID 34.6 62.1 232.0 405.1 Sample 75% ID 33.6 77.0 232.0 406.4 Sample 100% ID 32.9 67.5 231.5 405.2 Table 6. Thermogravimetric analysis results for PAHT CF15 ﬁlament and printed samples. Samples Softening Temperature Ts Melting Temperature Tm Burning Temperature Tb Full Degradation Temperature Td Mass Change 20–100 ◦C Mass Change 100–300 ◦C Mass Change 300–500 ◦C [◦C] [◦C] [◦C] [◦C] [%] [%] [%] Filament PAHT CF15 . . . 233.5 376.2 409.2 +0.20 −1.01 −67.52 Table 6. Thermogravimetric analysis results for PAHT CF15 ﬁlament and printed samples. 15 of 20 ansition that the Materials 2022, 15, 3706 Table 6. Cont. Samples Softening Temperature Ts Melting Temperature Tm Burning Temperature Tb Full Degradation Temperature Td Mass Change 20–100 ◦C Mass Change 100–300 ◦C Mass Change 300–500 ◦C [◦C] [◦C] [◦C] [◦C] [%] [%] [%] Sample 25% ID 172.2 234.1 378.2 403.0 −0.12 −1.31 −65.95 Sample 50% ID 178.6 236.5 377.5 405.4 −0.45 −1.29 −65.99 Sample 75% ID 168.9 236.4 372.7 394.5 +0.02 −1.38 −65.12 Sample 100% ID 220.0 232.0 374.4 399.2 −1.59 −2.47 −64.82 p g y g ture on the glass transition temperature (Tg) of the material tested is an objective of futu papers. In the temperature range of 100–300 °C, the mass loss is 1.01% because of wat evaporation, melting (at 233.5 °C, endothermic peak, Tm), and also the beginning of th matrix decomposition (after exceeding the melting temperature). The analysis of th PAHT CF15 filament’s thermogram (Figure 13) shows that another significant mass lo effect begins at over 350 °C, in the temperature range of 300–500 °C. 3.1.4. Thermal Behavior of Carbon Fiber PAHT Composite Above this temper ture, the process of accentuated damage of the matrix begins with burning of the materi (at 376.2 °C, exothermic peak, Tb) until the complete destruction (at 409.2 °C, endotherm peak, Td), where the mass loss is 67.52% ( Figure 16; Figure 17, Table 6). It can be state that the limit of thermal stability in given conditions is about 346 °C (more exactly, 345. °C). Figure 13. TGA curves for PAHT CF15 filament. Figure 13. TGA curves for PAHT CF15 ﬁlament. Materials 2022, 15, x FOR PEER REVIEW 17 of 22 Table 6. Cont. ng ture Melting Temperature Tm Burning Temperature Tb Full Degradation Temperature Td Mass Change 20–100 ◦C Mass Change 100–300 ◦C Mass Change 300–500 ◦C [◦C] [◦C] [◦C] [%] [%] [%] 234.1 378.2 403.0 −0.12 −1.31 −65.95 236.5 377.5 405.4 −0.45 −1.29 −65.99 236.4 372.7 394.5 +0.02 −1.38 −65.12 232.0 374.4 399.2 −1.59 −2.47 −64.82 ture on the glass transition temperature (Tg) of the material tested is an objective of papers. In the temperature range of 100–300 °C, the mass loss is 1.01% because of evaporation, melting (at 233.5 °C, endothermic peak, Tm), and also the beginning matrix decomposition (after exceeding the melting temperature). The analysis PAHT CF15 filament’s thermogram (Figure 13) shows that another significant ma effect begins at over 350 °C, in the temperature range of 300–500 °C. Above this tem ture, the process of accentuated damage of the matrix begins with burning of the m (at 376.2 °C, exothermic peak, Tb) until the complete destruction (at 409.2 °C, endoth peak, Td), where the mass loss is 67.52% ( Figure 16; Figure 17, Table 6). It can be that the limit of thermal stability in given conditions is about 346 °C (more exactly, °C). Figure 13. TGA curves for PAHT CF15 filament. Figure 13. TGA curves for PAHT CF15 ﬁlament. EVIEW 17 of Figure 14. TGA results for sample with 25% infill density. Figure 14. TGA results for sample with 25% inﬁll density. Table 6. Cont. g papers. In th EVIEW 17 of Figure 13. TGA curves for PAHT CF15 filamen Figure 13. TGA curves for PAHT CF15 ﬁlament. Figure 14. TGA results for sample with 25% infill density. Figure 14. TGA results for sample with 25% inﬁll density. Materials 2022, 15, 3706 16 of 20 Figure 14. TGA results for sample with 25% infill density. Figure 15. 3.1.4. Thermal Behavior of Carbon Fiber PAHT Composite It is known that polyamide (PA) [32,44] tends to absorb water readily and, therefore, weight gain (Figure 13) is a consequence of water absorption. The water content inﬂuences the glass transition in the sense that as the water content increases, the glass transition will be at a lower temperature, as also mentioned in the paper [44]. This means that the ﬁlament should be dried before it can be used in 3D printing. Studying the inﬂuence of moisture on the glass transition temperature (Tg) of the material tested is an objective of future papers. In the temperature range of 100–300 ◦C, the mass loss is 1.01% because of water evaporation, melting (at 233.5 ◦C, endothermic peak, Tm), and also the beginning of the matrix decomposition (after exceeding the melting temperature). The analysis of the PAHT CF15 ﬁlament’s thermogram (Figure 13) shows that another signiﬁcant mass loss effect begins at over 350 ◦C, in the temperature range of 300–500 ◦C. Above this temperature, the process of accentuated damage of the matrix begins with burning of the material (at 376.2 ◦C, exothermic peak, Tb) until the complete destruction (at 409.2 ◦C, endothermic peak, Td), where the mass loss is 67.52% (Figures 16 and 17, Table 6). It can be stated that the limit of thermal stability in given conditions is about 346 ◦C (more exactly, 345.75 ◦C). g ( y ) Regarding the TG analyses related to the samples manufactured by FFF, it is found that up to 100 ◦C, all specimens have low mass loss due to water evaporation, except for the one with a 75% inﬁll density, which registers an extremely small increase of 0.02%. The 0.02% increase in mass may be due to the absorption of water, which also leads to a decrease in the glass transition temperature. For the 100% inﬁll density sample, the lowest glass transition temperature, of 32.9 ◦C, is found (Figure 12, Table 5). The specimens with an inﬁll density of 100% have the highest mass loss, of 1.59% (Figure 17), and those with the lowest loss are those with an inﬁll of 25% (0.12%). It can be concluded that at a 100% inﬁll, which involves a greater volume of polyamide (matrix) and a greater volume of carbon ﬁber, the mass losses will be higher as well. 3.1.4. Thermal Behavior of Carbon Fiber PAHT Composite Thermogravimetric analysis curves for sample with 50% infill density. Figure 16. TGA results for sample with 75% infill density. Figure 15. Thermogravimetric analysis curves for sample with 50% inﬁll density. Figure 15. Thermogravimetric analysis curves for sample with 50% infill density. Figure 16. TGA results for sample with 75% infill density. Figure 16. TGA results for sample with 75% inﬁll density. EVIEW 18 of Figure 15. Thermogravimetric analysis curves for sample with 50% infill density. Figure 15. Thermogravimetric analysis curves for sample with 50% inﬁll density. Figure 15. Thermogravimetric analysis curves for sample with 50% infill density. Figure 15. Thermogravimetric analysis curves for sample with 50% infill density. Figure 15. Thermogravimetric analysis curves for sample with 50% inﬁll density. Figure 15. Thermogravimetric analysis curves for sample with 50% infill density. Figure 15. Thermogravimetric analysis curves for sample with 50% infill density. Figure 16. TGA results for sample with 75% infill density. Figure 15. Thermogravimetric analysis curves for sample with 50% inﬁll density. Figure 15. Thermogravimetric analysis curves for sample with 50% infill density. Figure 16. TGA results for sample with 75% infill density. Figure 16. TGA results for sample with 75% inﬁll density. VIEW 18 Figure 17. Thermogravimetric analysis results for sample with 100% infill density. Regarding the TG analyses related to the samples manufactured by FFF, it is fou th t t 100 °C ll i h l l d t t ti t Figure 17. Thermogravimetric analysis results for sample with 100% inﬁll density. Figure 15. Thermogravimetric analysis curves for sample with 50% infill density. Figure 15. Thermogravimetric analysis curves for sample with 50% inﬁll density. Figure 15. Thermogravimetric analysis curves for sample with 50% infill density. VIEW 1 Figure 16. TGA results for sample with 75% infill density. Figure 16. TGA results for sample with 75% infill density. Figure 16. TGA results for sample with 75% inﬁll density. Figure 17. Thermogravimetric analysis results for sample with 100% infill density. Figure 17. Thermogravimetric analysis results for sample with 100% inﬁll density. 17 of 20 Materials 2022, 15, 3706 17 of 20 The results of thermogravimetry analyses (TGA) are shown in Figures 13–17 and Table 6. Figure 13 displays the TGA curve of the PAHT CF 15 ﬁlament and the results show a weight gain of 0.20% up to the temperature of 100 ◦C. 3.1.4. Thermal Behavior of Carbon Fiber PAHT Composite In the temperature range of 100–300 ◦C, there are mass losses of over 1% for all four inﬁll densities, and the samples with an inﬁll of 100% have the highest loss, 2.47% (Figure 17). However, the lowest mass loss is related to the 50% inﬁll density sample (1.29%) (Figure 15, Table 6). The thermograms in Figures 14–17 show that in the temperature range of 300–500 ◦C, there are higher mass losses, more precisely above the temperature of about 345 ◦C (344.75 ◦C). As in the case of the TG analysis of the PAHT CF15 ﬁlament (Figure 13), exceeding the temperature of 345 ◦C determines the beginning of the accentuated degradation of the material for all the four inﬁll densities. The highest burning temperature is recorded for the 25% inﬁll density sample (378.2 ◦C) (Figure 14, Table 6) and the lowest for the 75% inﬁll density one (372.7 ◦C) (Figure 16). The complete destruction of the matrix is at a maximum temperature of 405.4 ◦C recorded for the sample with a 50% inﬁll density (Figure 15), which also has the highest mass loss, of 65.99%, and the lowest temperature of complete degradation is related to the specimen with a 75% inﬁll density (394.5 ◦C). Compared to the 25% and 50% inﬁll density (ID) samples, the 75% and 100% inﬁll density ones have the lower melting temperatures, burning, complete degradation, and also lower mass losses (Table 6, Figures 14–17). One explanation could be that in the samples with IDs of 75% and 100%, at the microstructure level, from the areas the material was taken for analysis (DSC, TGA), the volume of the voids and/or the volume of the carbon ﬁber distributed in the polymer matrix are higher than those at 25 and 50%, respectively, which would mean a smaller volume of polyamide, which, in turn, requires a lower amount of energy (temperature) for both melting and decomposition and, therefore, after degradation, a lower mass loss is obtained. 4. Conclusions This paper presents a study on the thermal and mechanical behavior of fabricated samples of polyamide reinforced with 15% carbon ﬁber, PAHT CF15, commercially avail- able, using the additive manufacturing process Fused Filament Fabrication (FFF). The Materials 2022, 15, 3706 18 of 20 18 of 20 samples were manufactured with 25%, 50%, 75%, and 100% inﬁll densities (IDs) and sub- jected to tensile and three-point ﬂexural tests, DSC and TGA thermal analyses, as well as microstructural analyses. • The results of three-point ﬂexural and tensile tests showed that the inﬁll density percentage has a major inﬂuence on mechanical properties. As the inﬁll density increases so does the tensile and ﬂexural strength • The results of three-point ﬂexural and tensile tests showed that the inﬁll density percentage has a major inﬂuence on mechanical properties. As the inﬁll density increases, so does the tensile and ﬂexural strength. • It was found that for the samples with 25% and 50% inﬁll densities, the differences between the maximum tensile and ﬂexural strengths are small, 7 MPa and 5.4 MPa, respectively. It is well known that reducing inﬁll densities results in lower printing costs and times. Therefore, if the conditions of use of the components made of PAHT CF15 allow, using a 25% inﬁll density in the FFF process could be seen as an option. g y p p • The microstructure analysis showed that after the printing process, the carbon ﬁbers in all the PAHT CF15 samples are preferentially aligned along the printing direction, and it does not eliminate the voids present in the ﬁlament material. It was also found that the content of voids between successive deposition beads is higher the lower the inﬁll density. The presence of a high volume of voids (defects) in the structure of the material leads to a decrease in the mechanical properties of the components manufactured by FFF. Therefore, it is necessary to reduce or eliminate voids by adopting in situ post-processing techniques or changing the printing parameters. g g g g • DSC analyses indicated that both the PACH CF15 ﬁlament and the printed samples show glass transition, cold crystallization, and melting and degradation peaks. It was found that the inﬁll density percentage inﬂuences the glass transition temperature (Tg); therefore, the increase in ID determines the decrease in the transition temperature from the glassy to the rubbery phase (Figures 8–12, Table 5). 4. Conclusions 19 of 20 19 of 20 Materials 2022, 15, 3706 References 1. Zhang, X.; Fan, W.; Liu, T. Fused deposition modeling 3D printing of polyamide-based composites and its applications. Compos. Commun. 2020, 21, 100413. [CrossRef] , , [ ] 2. Shanmugam, V.; Rajendran, D.J.J.; Babu, K.; Rajendran, S.; Veerasimman, A.; Marimuthu, U.; Singh, S.; Das, O.; Neisiany, R.E.; Hedenqvist, M.S.; et al. The mechanical testing and performance analysis of polymer-ﬁbre composites prepared through the additive manufacturing. Polym. Test. 2021, 93, 106925. [CrossRef] g y 3. Fidan, I.; Imeri, A.; Gupta, A.; Hasanov, S.; Nasirov, A.; Elliott, A.; Alifui-Segbaya, F.; Nanami, N. The trends and challenges of ﬁber reinforced additive manufacturing. Int. J. Adv. 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Conclusions The highest value of Tg, 49.6 ◦C, was recorded for samples with an ID of 25%, while for those with an ID of 100%, Tg is 32.9 ◦C. g • The results of the TGA analysis of the PAHT CF5 ﬁlament showed that exceeding the temperature of 345.75 ◦C (thermal stability limit) determines the beginning of the accentuated degradation process of the material with signiﬁcant mass loss (Table 6, Figures 13–17). In the case of samples manufactured with the four inﬁll densities, the thermal stability limit is close to the ﬁlament, namely at 344.75 ◦C (in the case of specimens with IDs of 75% and 100%). It was found that the increase in the percentage of ID causes a decrease in the thermal degradation temperature (onset temperature). Samples with IDs of 75% and 100% recorded a temperature of 344.75 ◦C and those with IDs of 25 and 50%, 348.5 ◦C and 349.6 ◦C, respectively (Figures 13–17). Author Contributions: Conceptualization, L.-A.C., M.A.P. and S.-M.Z.; investigation, L.-A.C., M.A.P. and S.-M.Z.; methodology, L.-A.C., M.A.P., S.-M.Z., C.L. and I.S.P.; project administration, S.-M.Z.; software, C.L., V.-M.S. and G.R.B.; validation, G.R.B., I.S.P. and V.-M.S.; writing—original draft, L.-A.C., M.A.P. and S.-M.Z.; writing—review and editing, L.-A.C., M.A.P. and S.-M.Z. All authors have read and agreed to the published version of the manuscript. Author Contributions: Conceptualization, L.-A.C., M.A.P. and S.-M.Z.; investigation, L.-A.C., M.A.P. and S.-M.Z.; methodology, L.-A.C., M.A.P., S.-M.Z., C.L. and I.S.P.; project administration, S.-M.Z.; software, C.L., V.-M.S. and G.R.B.; validation, G.R.B., I.S.P. and V.-M.S.; writing—original draft, L.-A.C., M.A.P. and S.-M.Z.; writing—review and editing, L.-A.C., M.A.P. and S.-M.Z. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by a grant of the Ministry of Research, Innovation and Digitiza- tion, CNCS/CCCDI—UEFISCDI, project number PN-III-P2-2.1-PED-2019-0739, within PNCDI III. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: We also acknowledge PRO-DD Structural Founds Project (POS-CCE, O.2.2.1., ID 123, SMIS 2637, ctr. no 11/2009) for providing the infrastructure used in this work. Acknowledgments: We also acknowledge PRO-DD Structural Founds Project (POS-CCE, O.2.2.1., ID 123, SMIS 2637, ctr. no 11/2009) for providing the infrastructure used in this work. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. 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https://openalex.org/W2015093284	http://diposit.ub.edu/dspace/bitstream/2445/165801/1/648569.pdf	English	null	Vacancy patterning and patterning vacancies: controlled self-assembly of fullerenes on metal surfaces	Nanoscale	2,014	public-domain	9,968	Probst §* e-mails: Michael.Probst@uibk.ac.at; francesc.illas@ub.edu Vacancy Patterning and Patterning Vacancies: Controlled Self-Assembly of Fullerenes on Metal Surfaces Alexander Kaiser §$, Francesc Viñes$, Francesc Illas$, Marcel Ritter %, Frank Hagelberg#, Michael §Institut für Ionenphysik und Angewandte Physik, University of Innsbruck, Technikerstrasse 25, A-6020 Innsbruck, Austria $Departament de Química Física & Institut de Química Teòrica i Computacional (IQTCUB), Universitat de Barcelona, c/Martí i Franquès 1, E-08028 Barcelona, Spain %Institute for Basic Sciences in Engineering Science, University of Innsbruck, 6020, Technikerstraße 13, Austria #Department of Physics and Astronomy, East Tennessee State University, Johnson City, TN 37614, USA *e-mails: Michael Probst@uibk ac at; francesc illas@ub edu Introduction Introduction The increasing research interest on Self-Assembled Monolayers (SAMs), patterned arrangements typically consisting of organic molecules, is related to their promising applications in biosensors, 1 optoelectronics, 2 and tribology. 3 Typically, such arrangements are studied on metal surfaces, although recent research endeavors have been applied on SAM formation on graphene layers, an appealing field for graphene bandgap engineering, 4 but also in order to get, for instance, ordered magnetic organic layers. On the other hand defined vacancy patterns on surfaces were created, on a nanometer scale, as early as in 2002 within the millipede project. 5 It involved a two-dimensional (2D) array of Atomic Force Microscopy (AFM) tips which resembled nm-scale millipedes which imprinted user-defined patterns of vacancies on a given thin polymer surface. Comentario [1]: Add Ref. Nature Physics 2013, 9, 368 [This is now correct]. Surface vacancies are particularly attractive adsorption sites for subsequently deposited molecules, given the low-coordination of vacancy neighboring surface atoms, and their concomitant improved chemical activity. When the adsorption of a molecule turns out to be thus strongly enhanced by the presence of a vacancy, as compared to the clean surface, individual population of vacancies can become preferred to molecular clustering, i.e. SAM formation. Thus, a vacancy-determined pattern of adsorbate molecules can be constructed. We call this process Controlled Self-Assembly (CSA), and we shall investigate below its feasibility taking fullerene C60 adsorption on gold, aluminum, and beryllium surfaces as cases and key examples. Another related process is a type of SAM formation which, on the contrary, induces the creation of surface vacancies. Conceptually, the fullerenes, first adsorbed on the clean surface, would induce vacancy formation beneath them and reside in their self-made ‘nests’. Vacancy formation triggered by the adsorption of a molecule or a cluster on a surface is not rare, and has been observed, for instance, on Pt clusters on CeO2 substrates. 6 As far as fullerenes are considered, vacancy incitement in the course of C60 monolayer formation on various surfaces has been investigated by many experimental and theoretical studies. 7 On one hand, these structures remain stable at Room Temperature (RT),7d and C60 adsorption is found to induce dimple formation on the Au(111) substrate, with well-separated individual C60 nucleation centers for cluster formation upon continued deposition. Vacancies were found to compete with step edges for adsorbates, underlying the importance of under-coordination in the increased adsorbent capacity. Abstract A density functional theory study accounting for van der Waals interactions reveals the potential of metal surface vacancies as anchor points for the construction of user-defined 2D patterns of adsorbate molecules via a controlled self-assembly process. Vice versa, energetic criteria indicate the formation of regular adsorbate-induced vacancies after adsorbate self-assembly on clean surfaces. These processes are exemplified by adsorbing C60 fullerene on Al(111), Au(111), and Be(0001) surfaces with and without single, triple, and septuple atom pits. An analysis of vacancy-adatom formation energetics precedes the study of the adsorption processes. Keywords: Adsorption · Self-Assembly · Fullerene · Vacancy Formation · Nanopatterns · Metal Surfaces Keywords: Adsorption · Self-Assembly · Fullerene · Vacancy Formation · Nanopatterns · Metal 1 1 Fransis! 12/4/2014 12:05 Comentario [1]: Add Ref. Nature Physics 2013, 9, 368 [This is now correct]. Introduction On the other hand Tzeng et al. observed that annealing to 750 K desorbs all C60 molecules of Au(111) while preserving the structure of C60.8 However, vacancy formation was not discussed in their work. 8 Comentario [2]: We agreed on (i) put a single number per reference and (ii) avoid overcitation, i.e., for giving a feed-back that there is much research, one can cite a few cases only. 2 The adsorption of C60 on metal surfaces is indeed a topic that has driven much attention over the last years, and the nature of the C60substrate chemical bonding is still a matter of debate. The topic has spanned over a variety of transition metals (Au, 7a-f, 9 Ag, 7f, 7l, 9e, 9f, 10 Ni, 10b, 11 Pd, 12 Pt, 7k, 10b, 13 and Cu 7f, 9h, 11, 14) and even few p-group metals (Al 7g-j, 14f and Si 15), combining experiments and theoretical calculations. The latter mostly rely on Density Functional Theory (DFT), although for such systems the inclusion of a proper description of van der Waals (vdW) dispersive forcers may be determinant. Two recent studies of C60 adsorbed on smooth Au(111) and Ag(111) surfaces with and without van-der-Waals (vdW) correction revealed a strong covalent, partly ionic interaction of ~1-3 eV strength, where the metalC60 charge transfer plays a key role. 9b, 9e However, pristine metal surfaces are found to reconstruct when exposed to fullerenes: Note that a clean Au(111) surface tends to adopt a herringbone-structure, 16 which, as revealed by X-Ray Diffraction (XRD) measurements and structural analysis, undergoes at 160 °C a surface transformation accompanied by removal of the herringbone structure and introduction of surface vacancies. Fullerenes were found to reside atop of a regular (2√3×2√3)R30° pattern of vacancies with C-Au distances of 2.49 Å.7b These surface reconstructions, such as vacancy or crater formation, were experimentally observed and confirmed by DFT theory for C60 adsorbed on Pt(111),7k Ag(111),7l Al(111),7i, 7j and Au(110).7e Here we consider representatives and structurally similar surfaces of a noble metal, Au(111), a p-group metal, Al(111), and an alkaline earth metal, Be(001). These metals were selected for several different reasons; most of the related literature deals with C60 adsorption on Au, which therefore is presented as a good reference system and test field. Introduction Secondly, Al is used as electrode material in organic photovoltaics, where it is in contact with the functionalized fullerene Phenyl-C61-Butyric acid Methyl ester (PCBM).17 Lastly, Be is a metal candidate for walls of future fusion reactors,18 and studies of the interaction of carbonaceous materials with beryllium are, to the best of our knowledge, very rare. Experiments with Be need special precautions due to its high toxicity. Therefore it is interesting to now how elements adjacent to it in the periodic table could serve as proxies for Be. Comparing the energetics of Be and Al in this work also serves this purpose. At variance with most of the large amount of work already done in the field, we considered well-separated C60 molecules in large unit cells to specifically assess the interaction of C60 with the surface, contrary to previous studies dealing with close-packed C60 monolayers. 3 Furthermore, our approach explicitly includes vdW forces, which has only been accounted for in one publication of C60 adsorption on Au(111) where, however, vacancy formation was not considered.9b We also analyze vacancy adatom formation energetics by comparing adsorption energies, charge transfer, and bond nature for C60 adsorption on clean surfaces, as well as in single, triple, and septuple atom pits. Energetic criteria for vacancy formation inducement and controlled self-assembly are discussed. Computational Details Table 1: Lattice constants (a, c in Å), cohesive energies (CE in eV), work functions (WF in eV), and surface energies (SE in J/m -2) compared with literature data. Table 1: Lattice constants (a, c in Å), cohesive energies (CE in eV), work functions (WF in eV), and surface energies (SE in J/m -2) compared with literature data. a/c lit. CE lit. WF lit. SE lit. Al 4.013 4.0422 3.73 3.23-4.2123 4.13 4.24-4.5424 1.1 0.94-1.2724b, 25 Au 4.082 4.07922 3.74 3.73-3.9726 5.06 4.83-6.0124b, 27 1.4 1.25-1.6124b, 28 Be 2.236/ 3.54 2.28229/ 3.57529 3.85 3.32-4.030 5.27 4.98-5.62 24b 2.1 2.1-2.724b Computational Details All DFT calculations and structural optimizations were carried out using the VASP code.19 A basis set of Plane-Waves (PW) was used with a PW kinetic energy cutoff of 415 eV. Valence electrons were described using the Perdew-Burke-Ernzerhof (PBE) exchange- correlation functional, whereas core electrons were described by using the Projector Augmented Wave (PAW) method. The vdW forces are treated by the long-range correction as proposed by Grimme.19a, 20 Default vdW parameter values were used for C, Al, and Be atoms, whereas for Au atoms we used the values supplied by Amft et al.21 A sufficiently accurate k-point mesh was chosen for all calculations, which happened to be the Γ point for large cells A (5×5) unit cell was chosen for the six atomic layer slab model representing the Al(111) and Au(111) surfaces with two frozen and four flexible layers —i.e. (2+4) approximation— and a vacuum thickness of 14 Å, similar to that used previously in related works.9e A larger (8×8) unit cell had to be used for the Be(0001) surface, in order to achieve a large enough separation of C60 from its periodic images. The distance between the carbon atoms of C60 and their periodic images is in all simulations larger than 7 Å, giving results close to vacuum conditions. In the direction perpendicular to the surface, the distance between the topmost carbon atoms and the bottom metal layer of the periodic slab image is always larger than 6.5 Å. To construct input geometries, C60 was manually located above the surface or the center of a vacancy with one of its carbon hexagons pointing downwards at a substantially larger distance than that found in the optimized geometry. During the optimization C60 is allowed to fully relax, as well as the top four surface metal layers. We first validated our computational settings by optimizing metal lattice constants (a, in case of Be also c) and calculating cohesive energies (CE), work functions (WF) and surface energies (SE). As can be seen in Table 1 we found good agreement with literature (lit.) values. 4 Our resulting optimized lattice constants were adopted throughout this work. Last but not least, molecules were visualized in the Vish visualization shell 31. An estimated number of 44 single core wall clock years on Intel Xeon E5 and similar processors was spent in total for the optimizations. Vacancy Formation (3) (3) In other words, Ead is the energy required to remove ν atoms from the bulk and add them collectively on top of the surface, whereas Evac is the energy gained by removing ν atoms from the clean surface and adding them to the bulk reservoir. 33 The adatom binding energy Ebad and the vacancy binding energy Ebvac are calculated using the atomic vacuum energy, Tat, as reference energy instead of the bulk value; Ebad(ν) = T(0) + ν Tat – T(ν ), (4) Ebvac(ν) = T(-ν) + ν Tat – T(0). (5) (4) (5) Thus the energy Ebad/Ebvac is that needed to remove one by one the ad/vacancy atoms from the surface to the vacuum. The adiabatic removal of an atom from the surface can be seen as a two-step process, where vacancy-adatom formation is followed by removal of the adatom. Note that the overall five defined energies are not independent from each other: Eva = Ead + Evac , (6) Eva + Ebad = Ebvac . (7) (6) (7) The energetic results for each surface and vacancy size are summarized in Table 2 together with the structural deformation def. The deformation def is measured as the distance of an atom to its initially ideal crystal position in percent of the nearest neighbor distance and corrected for periodic boundary conditions, and Table 2 shows the maximal values. From our findings, Eva is the most important value for the prediction of possible surface reconstruction, since it is the minimum energy that has to be compensated by the interaction between the adsorbate species (C60 in our case) and the surface to allow for vacancy formation. In particular, the dissociation energy D for removing C60 from the surface vacancy - also called the adsorption energy - needs to be even larger than the sum of Eva and D for the case of a clean surface so as to make the reconstruction energetically favorable. From the Eva values of Table 2 we observe that, for a single atom vacancy, Be (2.09 eV) is more stable than Al (1.6 eV), which is in turn more stable than Au (1.11 eV). The enhanced stability of Be could be explained by its higher melting point (1551 K) compared to Al (933 K), and Au (1338 K). Vacancy Formation y First we dealt with the formation of surface vacancies, since they, through surface reconstruction, can enhance the binding strength of adsorbed C60 and even lower the total energy of the C60/surface system. Often these vacancies are termed pits or craters. Deep pits with 3 or 7 atom vacancies have shown to increase adsorption energies of the C60/Au(111) system according to DFT calculations without dispersion corrections. 7d Single-atom and regular multi-atom vacancies have been optimized starting from pristine ideal surfaces, from which 1, 3, or 7 (ν) atoms have been removed. Three-atom vacancies form a triangular pit, whereas seven-atom vacancies form a hexagonal pit. These structures are similar to those depicted by Tang et al., yet residing in a larger unit cell. 7d The vacancy-adatom formation energy is defined as 32 The vacancy-adatom formation energy is defined as 32 Eva(ν) = T(-v) + T(v) – 2 T(0), (1) (1) where T(±ν) is the total energy of the clean surface, T(0), with ν additional or -ν removed atoms, i.e. T(-3) is the total energy of a surface with a three-atom vacancy, whereas T(7) is the total energy of a surface with seven adatoms. Eva is defined as the energetic cost of building a vacancy from a clean surface, where the removed atoms attach, as a cluster, to the surface at an infinite distance from the vacancy. Thus the vacancy-adatom formation energy Eva can be represented as sum of the adatom formation energy, Ead, and the vacancy formation energy, Evac. Both are defined with reference to the total energy of a bulk atom, Tbulk: where T(±ν) is the total energy of the clean surface, T(0), with ν additional or -ν removed atoms, i.e. T(-3) is the total energy of a surface with a three-atom vacancy, whereas T(7) is the total energy of a surface with seven adatoms. Eva is defined as the energetic cost of building a vacancy from a clean surface, where the removed atoms attach, as a cluster, to the surface at an infinite distance from the vacancy. Thus the vacancy-adatom formation energy Eva can be represented as sum of the adatom formation energy, Ead, and the vacancy formation energy, Evac. Both are defined with reference to the total energy of a bulk atom, Tbulk: 5 Ead(ν) = T(ν) – T(0) – ν Tbulk , (2) Evac(ν) = T(-ν) + v Tbulk – T(0). Vacancy Formation As far as deformation is concerned, Al deforms rather dramatically whereas Au and Be vary much less. Overall the deformation increases from the fixed bottom layers to the top layer and towards the vacancies or adatoms. Comentario [4]: AK: the bulk lattice constant is relaxed, but a bulk vacancy would lead also to relaxation, which is not included in the cohesive energy. The values are very similar however if this relaxation is also taken into account. I think it is too long and boring for the reader to write this here in more detail? The computed vacancy and adatom formation energies appear to be in accord to previous computed values in the literature. In particular, the Eva formation energy of 1.72 eV reported by Stumpf and Scheffler for Al(111) 33 is composed of Evac = 0.67 eV and Ead = 1.05 eV. Less agreement is found with DFT calculations within the Local Density Approximation (LDA) which yielded an Evac energy of 0.36 eV for Al(111), 35 and 0.83 eV for Au(111). 7l The value for Al(111) seems especially low since another study within the Generalized Gradient Approximation (GGA) and LDA by Kiejna et al. 36 yielded values of 0.61 and 0.66 eV, respectively, again in perfect agreement with our value of 0.69 eV. Previous studies reported Evac for Au to be in between 0.77 to 0.82 eV, 37 in accordance with Li et al., 7l but slightly larger than our value of 0.58 eV. Other theoretical and experimental values for Au showed values between 0.82 and 0.93 eV, thus again slightly higher. 38 We examined whether this discrepancy of Evac for a single atom was due to our vdW correction, yet the estimate without Grimme’s correction yielded a similarly low value of 0.55 eV. Comentario [5]: FV: Yes, maybe too long and boring. Let us drop it, and only put it in case a referee asks for it. Vacancy Formation 34 However, by this line of reasoning, Au should be more stable than Al, which is not reflected by our results. In contrast, they suggest that surface reconstruction should proceed more easily in gold than in aluminum or beryllium. Eva is, as expected, not directly proportional to ν, as fewer bonds-to-neighbors have to 6 be broken for the removal of more and more atoms. An increase by a factor of 1.8-2.2 is observed when going from one- to three-atom vacancies, and of 2.3-3.2 from one- to seven-atom vacancies. Thus, after the first three atoms removed, removing any additional atom comes at a significantly reduced energy expense (circa 147, 258, and 278 meV atom -1 for Al, Au, and Be, respectively). It should be noted that in the case of septuple vacancy pits the 5×5×(6+Lz) the unit- cell might be too small and interactions between neighboring pits could influence our results, since only two rows of atoms separate the craters. In these cases results should be taken as mere indications rather than absolute values. Table 2: Energies of vacancy-, adatom-, and vacancy-adatom formation as well as of structural deformation for single, triple, and septuple atom pits. ν Eva/ eV Ead/ eV Evac/ eV Ebad/ eV Ebvac/ eV def /% Al 1 1.60 0.91 0.69 2.83 4.42 18.7 3 3.11 1.59 1.52 9.61 12.72 52.3 7 3.70 2.23 1.48 23.91 27.61 70.5 Au 1 1.11 0.53 0.58 3.20 4.31 3.0 3 2.49 1.30 1.20 9.91 12.40 9.6 7 3.52 1.76 1.76 24.39 27.91 7.5 Be 1 2.09 0.84 1.25 3.01 5.11 7.1 3 3.77 1.40 2.36 10.16 13.92 8.0 7 4.88 2.13 2.74 24.84 29.72 12.8 A deep inspection of the formation energy values encompassed in Table 2 unveils that the two contributions to Eva (Ead and Evac) carry almost equal weights for Au. In the case of Al the adatom formation energy dominates, whereas in case of Be it is the vacancy formation energy. 7 Regardless of the case, Ebad and Ebvac scale with ν. Single-atom vacancy binding energies Ebvac are larger than cohesive energies for the three metals. This means that the removal of a single atom from the surface takes more energy than removing an atom from the perfect bulk. Thus, indirectly, Ebvac carries information about the stability of a material against sputtering, in a better fashion than the cohesive energy. C60 Adsorption In view of the high electron affinity of C60, a metalC60 charge transfer is expected, which leads to ionic binding in addition to possible covalent bonds. The abovementioned dissociation energy D is defined as: D = TC60 + T(-ν) – T(-ν,C60), (8) (8) where TC60 is the total energy of C60 in vacuum (cell size dimensions of 20 Å) and T(-ν,C60) the total energy of the surface with ν vacancies and C60 adsorbed upon. Results for D are collected in in Table 3. For all considered materials and surface structures we obtain a high value of D around or larger than 2 eV. The winner is gold with a particularly high adsorption energy whereas Al and Be share the second rank. In a previous study Tang et al. obtained, by DFT but without applying any dispersion correction, adsorption energies of 2.07, 2.33, and 2.56 eV for single, triple, and septuple atom vacancies, respectively, as compared to a value of 1.2 eV for a dense monolayer of C60 at clean Au(111). 7d Note that in this work a denser fullerene monolayer was used, and, consequently, the reduced adsorption energies may well be the result of steric 8 repulsions in between fullerenes. Other DFT works report adsorption strengths of the order of 1.9-2.2 eV for fullerene adsorbed on clean gold, 8-9 and classical force field models depicted binding strengths of 1.26 and 3.12 eV for C60 on surface reconstructed grooves and dimples, respectively. 9g In any case vdW dispersion seems to play a major role in the attachment of fullerenes to metal surfaces, as had also been found for similar systems such as graphene. 39 For example, C60 was found to adsorb on Au(111) with a hexagon parallel to the surface over an hcp three-fold hollow site, 9e with a primarily covalent bond of 1.27 eV strength, and a charge transfer of 0.2 e, according to DFT calculations without vdW description. A subsequent DFT study including vdW 9b showed that adsorption is dominated by dispersive forces, yet bonding remained covalent in nature. The optimized structure, close to the presently obtained one, implies a binding to the fcc three-fold hollow site with binding strengths in the range of 1.71-2.97 eV depending on the method for dispersion correction. As a general trend vacancies strongly enhance the adsorption energy. C60 Adsorption It first increases with the vacancy number ν, but it may well happen that at some point the extension of the pit exceeds that of C60. Consequently, D decreases again from ν = 3 to ν = 7 for Al and Be. Note that the vdW interactions play a major role to enhance D; optimization without the Grimme correction resulted in rather small adsorption energies of 0.16 eV for clean Al and 0.07 eV for clean Be with C60 locating far away from the surface. A previous DFT study of C60/Au(111) without accounting for dispersion obtained a similarly low binding energy of 0.18 eV. 9d However, the C60 desorption temperature of both Au(111) and Al(111) lies around 700 K, suggesting a strong covalent bonding with the surface. 7i,8 Table 3: Dissociation energies D (equivalent to the adsorption energy of C60 at the substrate), Bader charge transfer qCT, and number of covalent bonds nCB. Al(111) Au(111) Be(001) ν 0 1 3 7 0 1 3 7 0 1 3 7 D /eV 1.99 3.97 4.28 3.44 3.39 4.21 4.94 5.20 2.45 3.69 4.21 3.58 qCT /e -2.3 -4.2 -4.1 -5.2 -0.08 -0.2 -0.18 -0.11 -4.8 -4.3 -5.2 -6.1 nCB 6 6 4 6 0 0 0 0 6 6 9 9 A Bader electron density analysis reveals large charge transfer from Al and Be to C60. 40 It increases dramatically with ν up to 6 electrons for ν = 7, that are transferred from Be to C60, Table 3: Dissociation energies D (equivalent to the adsorption energy of C60 at the substrate), Bader charge transfer qCT, and number of covalent bonds nCB. Al(111) Au(111) Be(001) ν 0 1 3 7 0 1 3 7 0 1 3 7 D /eV 1.99 3.97 4.28 3.44 3.39 4.21 4.94 5.20 2.45 3.69 4.21 3.58 qCT /e -2.3 -4.2 -4.1 -5.2 -0.08 -0.2 -0.18 -0.11 -4.8 -4.3 -5.2 -6.1 nCB 6 6 4 6 0 0 0 0 6 6 9 9 A Bader electron density analysis reveals large charge transfer from Al and Be to C60 40 It Table 3: Dissociation energies D (equivalent to the adsorption energy of C60 at the substrate), Bader charge transfer qCT, and number of covalent bonds nCB. A Bader electron density analysis reveals large charge transfer from Al and Be to C60. C60 Adsorption 40 It increases dramatically with ν up to 6 electrons for ν = 7, that are transferred from Be to C60, A Bader electron density analysis reveals large charge transfer from Al and Be to C60. 40 It increases dramatically with ν up to 6 electrons for ν = 7, that are transferred from Be to C60, A Bader electron density analysis reveals large charge transfer from Al and Be to C60. 40 It increases dramatically with ν up to 6 electrons for ν = 7, that are transferred from Be to C60, 9 although the electron affinity of isolated C60 is only positive up to C60 2-. 41 A completely different behavior is found for Au which retains the electrons, so the charge transfer is limited to 0.1-0.2 e. Previous ultraviolet photoelectron spectroscopy experiments estimated a charge transfer from gold to C60 of 1±0.2 e. This exceeds the value predicted by our as well as other DFT calculations by a large margin and remains unexplained so far. 7f,9e The true locations of charge accumulation and depletion are resolved in Charge Density Difference (CDD) maps, see Figure 1. Again the bonding mechanism at the Au surface is completely different than the bonding at Al and Be. For Au depletion regions that resemble well- localized p-orbitals (blue lobes) are found, extending far inside the Au surface. The charge is transferred to the red-green clouds that might be interpreted as covalent bonds, as also found by Hamada et al. and Hinterstein et al. by DFT-based CDD plots showing the directional character of C-Au bonds. 7e,9b Interestingly, the CDD plots do not show all these features without taking vdW into account. 9e We also looked at the Electron Localization Functions (ELF) and found no apparent covalent bonds for C60 on Au in contrast to C60 on either Al or Be, where clear signatures of covalent bonding appear at the places of charge accumulation between C60 and the respective surface. In the case of Be, two C atoms are seen to share bonds to one surface atom. For Al, in contrast, every C atom is clearly bonded to one specific surface atom. This results in different optimized geometries because the bottom hexagon of C60 resides on top of an Al atom but in the center of three Be atoms. C60 Adsorption The number of covalent bonds nCB observed from ELF is given in Table 3 as well. The deeper the vacancies get, the deeper the fullerene dives into the surface, and different atoms become available for covalent bonding. The ELF covalent regions of the clean surface are much smaller than the ones in the presence of vacancies, mirroring the corresponding difference in D. 10 10 Figure 1: CDD volume rendering of regions with charge accumulation (greenred = lowhigh) and depletion (bluelight blue = lowhigh) due to adsorption of C60 for clean surfaces. For the sake of clarity very small charge differences are omitted. Comentario [6]: Perspektive zu stark, Be kleiner als Al, kleiner als Au. FV: If possible, lighter background. Make a single image with first row being the three top-views, and second row being the ones here showing CDD. Figure 1: CDD volume rendering of regions with charge accumulation (greenred = lowhigh) and depletion (bluelight blue = lowhigh) due to adsorption of C60 for clean surfaces. For the sake of clarity very small charge differences are omitted. The total Density Of States (DOS) for the C60 adsorbed on Al system is projected on the Al atoms in Figure 2a and compared with the DOS of a pristine Al surface. Note that the amplitudes for Al+C60 without and with the seven-atom vacancy were resized to meet the amplitudes of the pristine Al DOS at low energy. Small changes appear both below and above the Fermi level (EF) upon absorption of C60, where the discrete states of C60 are superposed with the quasi-continuum of the surface. The DOS is smeared out due to the seven-atom vacancy and further by the presence of C60. The same comparison for Au yields a similar picture with a much lower degree of smearing, while for Be the DOS is relatively unaffected by adsorption of C60 and vacancy formation. These observations are in accordance with the fact that Al mostly deforms in response to vacancy formation and C60 adsorption. Figure 2: DOS of Aluminum in C60+Al(ν) compared to the clean Al surface (a) and DOS of carbon in C60+Al(ν) compared to C60 in vacuum (b). Figure 2: DOS of Aluminum in C60+Al(ν) compared to the clean Al surface (a) and DOS of carbon in C60+Al(ν) compared to C60 in vacuum (b). C60 Adsorption 11 The scaled DOS projected on carbon atoms is shown in Figure 2b; compared to C60 in vacuum the C60 states when adsorbed on Al are shifted to lower energies by ~1 eV. Furthermore, in the vicinity of EF the sharp peaks of the molecule are tremendously smeared out, yielding a metallic behavior, and, even so, a small part of the LUMO is shifted below EF, in accordance with previous DFT calculations. 9e This effect is stronger when C60 is adsorbed to a vacancy than on the clean surface. Largely similar observations are made for the cases of Au and Be. The findings are consistent with previous Scanning Tunneling Spectroscopy (STS) experiments on Au(110), which revealed that a significant fraction of C60 LUMO broadens and shifts downwards towards the Fermi level. 7a Discussion We first discuss fullerene adsorption induced vacancy formation. Upon adsorption, D is released, and this can give rise to vacancy formation, since D(ν) > Eva(ν) for all structures considered here except for septuple-atom vacancies for Al and Be. However, other adsorption energy dissipation channels exist. A better suited criterion to determine whether a vacancy formation is thermodynamically allowed is that the D energy on a vacancy must surpass that on the clean surface plus the vacancy-adatom formation energy: D(ν) > D(0) + Eva(ν). This ensures the adsorption-on-a-vacancy state to be the ground state, being of higher stability than the adsorption on the pristine surface. The latter more strict criterion is only fulfilled for Al(111) single-atom vacancies, where a total energy gain of 0.38 eV is recorded. However, the missing energy for the other structures is not very high (0.29 eV for Au and 0.85 eV for Be in the case of the single-atom vacancy) and therefore could easily be supplied, for instance, by addition of the adatom nearby C60, or by forming surface dimers, or even larger clusters. In addition, adsorbate location at step edges could gain extra energy, 7g, 33 given the enhanced adsorption-ability of surface under-coordinated metal atoms located at edges between terraces. 42 Besides, the small energy cost could be thermally supplied. Overall, adsorption of C60 reduces the cost of vacancy formation compared to pristine surfaces. This clearly points for the formation of surface metal vacancies that follow the pattern of C60 adsorbates. The concept is backed up by STM experiments which showed bright and dim C60 on Au(111), where dim molecules were identified to reside on top of multiatom vacancies. 7c This is an example of how the adsorption of a C60 monolayer yielded a periodic ordering of adsorption-induced vacancies on the surface. The conditions for C60 CSA are more difficult to assess. One can propose a Gedankenexperiment involving a metal surface with already well-separated vacancies and a temperature large enough to allow for C60 diffusion over vacancy-free surface areas. Thus, the first deposited C60 can move freely on the clean surface until it finds a vacancy. A recent study 12 estimated an activation energy of 13 meV for single fullerene diffusion on Au(111); 9a such a small activation energy would allow C60 to easily find vacancies, supporting the CSA feasibility. Discussion Since the binding energy on the vacancy is by 1-2 eV larger than on the clean surface, the C60 will remain anchored there unless the temperature is too high. A second C60 has two possible fates. Either it moves nearby to the first C60 and initializes cluster nucleation, releasing a few hundreds of meV, 43 or it finds an unoccupied vacancy, releasing a much higher margin of energy. From an energetic point of view there would be a temperature window, where nucleation is suppressed because the binding to other C60 is not strong enough, while simultaneously C60 forms stable bonds to vacancies. The above-introduced competing process of induced vacancy formation could, however, complicate this picture. We conclude that with accurate temperature control, the self- assembly on pre-structured template surfaces —containing surface vacancies— could be controlled, leading to user-defined patterns of adsorbate molecules, given that adsorption- induced vacancy formation is energetically unfavorable, such as on Au(111) and Be(001). Previous STM experiments of C60/Au(111) at RT assisted by DFT calculations also revealed vacancies as preferred bonding sites. 7d At 46 K C60 molecules seem to attach to the elbow sites of the Au(111) herringbone reconstruction. When annealing to RT these sites are vacated, and nucleation at step edges occurs without vacancy formation. When starting directly at RT, Tang et al. found individual molecules at elbow sites and further cluster nucleation in between them upon further deposition, whereas at low temperature clusters only nucleated around single molecules adsorbed at elbow sites. This controversy led the authors to argue that vacancies form below C60 to stabilize its adsorption at RT. The hypothesis was supported by a C60 height of 6.2 Å at 46 K while at RT C60 lies 2.2 Å deeper. This ‘sinking’ is in agreement with the adsorption over seven-atom pits, and demonstrates that C60 can be safely stored over vacancies at RT, supporting the idea of the CSA process that will be necessary for envisaged nanodevices. XRD studies also showed surface reconstruction with pit formation to accommodate C60 on Au(110), 7e and on herringbone Au(111). 7b The latter displayed C-Au distances of 2.49 Å with a C60 hexagon facing the single-atom vacancy, close to our value of 2.33 Å, indicating a mixed covalent/ionic bonding. As far as aluminum is concerned, the Al(111) surface also reconstructs under a compressed C60 overlayer. 7g-j Stengel et al. Discussion proposed that two Al adatoms from single-atom vacancies under two out of three adsorbed C60 form a dimer that stays in the interstitial region between C60 molecules, further stabilizing the system. 7g The structure naturally explains why one out of three C60 protrudes from the surface. The authors gave adsorption energies ranging 0.98- 1.38 eV, yet not accounting for vdW forces, and estimated a total energy gain due to the reconstruction of 0.8/3 = 0.27 eV per C60 molecule. In another related study 7l LDA DFT calculations yielded adsorption energies on pristine surfaces of 1.37, 1.25, and 1.52 eV for Al, 13 Au, and Ag, respectively. On vacancies the adsorption energies increased to 2.34, 2.69, and 2.61 eV, respectively. The authors also contemplated structures where vacancy atoms occupy the free space in between C60. Using the unreconstructed surface as energy reference, the adsorption energies are 1.40, 1.35, and 1.59 eV. Here the energy needed to create a vacancy is accounted for, and still these adsorption energies are by 0.10, 0.03, and 0.07 eV larger than the adsorption energies found on clean surfaces. This study suggests C60-induced vacancy formation on all considered surfaces, and is fully compatible with our results for aluminum. Au, and Ag, respectively. On vacancies the adsorption energies increased to 2.34, 2.69, and 2.61 eV, respectively. The authors also contemplated structures where vacancy atoms occupy the free space in between C60. Using the unreconstructed surface as energy reference, the adsorption energies are 1.40, 1.35, and 1.59 eV. Here the energy needed to create a vacancy is accounted for, and still these adsorption energies are by 0.10, 0.03, and 0.07 eV larger than the adsorption energies found on clean surfaces. This study suggests C60-induced vacancy formation on all considered surfaces, and is fully compatible with our results for aluminum. No literature values were found for beryllium which turned out to be most resistant with respect to vacancy formation among the three metals investigated in this work. The stability of Be surface could be useful for the CSA process. Conclusion We studied the energetic criteria for vacancy formation and fullerene adsorption on gold, aluminum and beryllium. To this end we employed density functional theory calculations taking van der Waals interactions into account. We found that the cost for vacancy formation increases in the order Au < Al < Be. A strong adsorption of C60 is found for all metals, surpassing dissociation energies of 2 eV. Adsorption on surface vacancies is enhanced compared to pristine surfaces, so that C60 molecules might occupy user-defined patterns of vacancies (vacancy patterning). 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Phys. 2011, 135 (11), 114702; (d) Tang, L.; Zhang, X.; Guo, Q.; Wu, Y.-N.; Wang, L.-L.; Cheng, H.-P., Two bonding configurations for individually adsorbed C60 molecules on Au(111). Phys. Rev. Acknowledgements Part of this work was supported by the Austrian Ministry of Science BMWF as part of the UniInfrastrukturprogramm of the Research Platform Scientific Computing at the University of Innsbruck and funded by the Austrian Science Fund (FWF) DK+ project Computational Interdisciplinary Modeling, W1227-N16. This work was also supported by the Spanish MICINN grant CTQ2012-30751, and, in part, by the Generalitat de Catalunya (grants 2009SGR1041 and XRQTC). F.V. thanks the MINECO for the postdoctoral Juan de la Cierva and Ramón y Cajal 14 grants (JCI-2010-06372, RYC-2012-10129), F.I. acknowledges additional funding through the 2009 ICREA Academia award for excellence in university research. F.H. acknowledges support from the Tennessee NSF-EPSCoR Grant TN-SCORE (NSF EPS 1004083). 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https://openalex.org/W3023554475	https://escholarship.org/content/qt1d43k4p9/qt1d43k4p9.pdf?t=lnqbo9	English	null	Cost of power interruptions to electricity consumers in the United States (US)	Energy	2,006	public-domain	7,519	Lawrence Berkeley National Laboratory Lawrence Berkeley National Laboratory Title Cost of Power Interruptions to Electricity Consumers in the United States (U.S.) Permalink https://escholarship.org/uc/item/1d43k4p9 Authors Hamachi LaCommare, Kristina Eto, Joseph H. Publication Date 2006-02-16 Peer reviewed Lawrence Berkeley National Laboratory Lawrence Berkeley National Laboratory Title Cost of Power Interruptions to Electricity Consumers in the United States (U.S.) Permalink https://escholarship.org/uc/item/1d43k4p9 Authors Hamachi LaCommare, Kristina Eto, Joseph H. Publication Date 2006-02-16 Peer reviewed Lawrence Berkeley National Lab Lawrence Berkeley National Laborato Title Cost of Power Interruptions to Electricity Consumers Permalink https://escholarship.org/uc/item/1d43k4p9 Authors Hamachi LaCommare, Kristina Eto, Joseph H. Publication Date 2006-02-16 Peer reviewed Lawrence Berkeley National Laboratory Lawrence Berkeley National Laboratory Title Cost of Power Interruptions to Electricity Consumers in the United States (U.S.) Permalink https://escholarship.org/uc/item/1d43k4p9 Authors Hamachi LaCommare, Kristina Eto, Joseph H. Publication Date 2006-02-16 Peer reviewed Powered by the California Digital Library University of California eScholarship.org LBNL-58 Cost of Power Interruptions to Electricity Consumers in the United States (U.S.) Kristina Hamachi LaCommare and Joseph H. Eto Environmental Energy Technologies Division February 2006 http://eetd.lbl.gov/ea/EMS/EMS_pubs.html To be published in a forthcoming issue of Energy: The International Journal. This work described in this paper was funded by the Assistant Secretary of Energy Efficiency and Renewable Energy, Distributed Energy Program of the U.S. Departme of Energy under Contract No. DE-AC02-05CH11231. ERNEST ORLANDO LAWRENCE BERKELEY NATIONAL LABORATORY LBNL-58164 ERNEST ORLANDO LAWRENCE BERKELEY NATIONAL LABORATORY Environmental Energy Technologies Division February 2006 February 2006 To be published in a forthcoming issue of Energy: The International Journal. This work described in this paper was funded by the Assistant Secretary of Energy Efficiency and Renewable Energy, Distributed Energy Program of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231. Ernest Orlando Lawrence Berkeley National Laboratory is an equal opportunity employer. Kristina Hamachi LaCommare* and Joseph H. Eto Kristina Hamachi LaCommare* and Joseph H. Eto Ernest Orlando Lawrence Berkeley National Laboratory One Cyclotron Road, MS 90R4000 Berkeley CA 94720-8136 Received: 7 April 2005 * Corresponding author, Fax: (510) 486-7976, email address: KSHamachi@lbl.gov Disclaimer This document was prepared as an account of work sponsored by the United States Government. While this document is believed to contain correct information, neither the United States Government nor any agency thereof, nor The Regents of the University of California, nor any of their employees, makes any warranty, express or implied, or assumes any legal responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights. Reference herein to any specific commercial product, process, or service by its trade name, trademark, manufacturer, or otherwise, does not necessarily constitute or imply its endorsement, recommendation, or favoring by the United States Government or any agency thereof, or The Regents of the University of California. The views and opinions of authors expressed herein do not necessarily state or reflect those of the United States Government or any agency thereof, or The Regents of the University of California. 1 Abstract The massive electric power blackout in the northeastern U.S. and Canada on August 14-15, 2003 catalyzed discussions about modernizing the U.S. electricity grid. Industry sources suggested that investments of $50 to $100 billion would be needed. This work seeks to better understand an important piece of information that has been missing from these discussions: what do power interruptions and fluctuations in power quality (power-quality events) cost electricity consumers? We developed a bottom-up approach for assessing the cost to U.S. electricity consumers of power interruptions and power-quality events (referred to collectively as “reliability events”). The approach can be used to help assess the potential benefits of investments in improving the reliability of the grid. We developed a new estimate based on publicly available information, and assessed how uncertainties in these data affect this estimate using sensitivity analysis. 2 1. Introduction The highly publicized power outage in the Northeastern U.S. and Canada on August 14-15, 2003 produced criticism that the U.S. power system was “antiquated”, comparable to that of a “Third- World nation”, and in need of modernization [1, 2]. Qualitative critiques of the power system have been published [3, 4], but these assessments have largely neglected to quantify an important piece of information: how much do power interruptions and fluctuations in power quality (power-quality events) actually cost electricity consumers? Accurately estimating this cost will help assess the potential benefits of investments in improving the reliability of the grid. This work develops a bottom-up approach for estimating this cost in order to better understand the significance of uncertainties inherited in these estimates. There have been several efforts to assess the cost of power interruptions and power quality. In 1992, Keane and Woo [5] applied customer outage costs to derive an optimal reserve margin for generation that minimizes the sum of customer outage and generation capacity costs. The resulting optimal reserve margin is compared with and determined to be less than the one implied from the commonly used “1-day-in-10-years loss-of-load” criterion. Because very few data were available on outage costs, rules of thumb were widely used to derive the estimates in the study. Shortly after, the Electric Power Research Institute (EPRI) reported the first ever power-quality- cost estimate of $26 billion per year for the U.S.1 [6]. However, power quality is only a subset of reliability events that affect customers and does not include losses of power for periods longer 3 than a few seconds. In addition, the estimate does not address the residential and commercial sectors. Finally, the estimate is based on the assumption derived anecdotally from professional judgment that 1.5 to three cents of spending per dollar sales went to power-quality equipment. Later, EPRI extrapolated from this figure and began reporting power-interruption costs of $50 billion per year [7]. During the same period, a U.S. Department of Energy (DOE) study published cost-of-reliability estimates ranging from $150 to $400 billion per year, based on an extrapolation from a value-of- service study of the Duke Power service territory to the entire U.S. [8]. Sources of potential error and bias in this estimate include the extrapolation from a single utility service territory to the entire U.S. and the focus only on costs to the industrial sector [9]. In 2001, Eto et al. 1 Billion refers to 109 U.S. dollars. 1 Billion refers to 109 U.S. dollars. 1. Introduction [10] performed a scoping study of electricity reliability trends in the U.S., which began by updating a comprehensive list of literature on electricity reliability costs. The report also described three distinct end-use approaches for tracking trends in reliability needs and presented an insurance industry perspective on electricity reliability as an example of a financial tool for addressing customers’ reliability needs. Finally and most recently, EPRI estimated the national cost of power interruptions including power-quality events at $119 billion per year [11]. The estimate is based on a limited national survey of selected customer groups asking these customers to estimate costs for several distinct power-outage scenarios. Key assumptions of this study, which are reviewed in LaCommare and 4 Eto [12], are that the costs experienced by the non-surveyed customer groups (excluding residential customers) were 25 to 50 percent of the costs experienced by the surveyed population. Costs to residential customers were not estimated. The outage cost scenarios all asked customers to estimate costs they would experience on summer weekday afternoons, which is the time when customers typically reported the highest costs from outages. These studies have helped improve our understanding of the sources of uncertainty associated with estimating customer outage costs, which is essential for valuing the overall cost of power interruptions to U.S. electricity customers. However, little has been done to analyze systematically the significance of these uncertainties. A number of recent analyses, which seek to improve energy demand forecasts, are suggestive of an approach that may facilitate and improve our ability to understand the cost of power interruptions and power-quality events using a bottom-up approach. In particular, research using end-use approaches has improved our understanding of how energy is used. For example, Turiel et al. [13] estimates the energy intensity of major end uses in the commercial sector using an end-use approach, work that is important to understanding how each end use contributes to the overall demand for electricity in the commercial sector. Bottom-up analyses such as these, allow for more accurate estimates of energy consumption patterns and help improve the modeling accuracy of energy forecasting models. Chateau and Lapillonne [14] stress the importance of end-use models and approaches as an important tool for forecasting energy demand in long term energy planning. 1. Introduction Our work builds upon the analytical rigor and strengths inherent in these 5 5 bottom-up approaches for energy analysis by adapting it to improve our understanding of the cost of outages. In the work described in this article, we developed a bottom-up approach for estimating the cost of power interruptions to U.S. electricity consumers. We illustrated the use of the approach by drawing on existing data from a variety of sources to develop a new (“base case”) estimate of the total economic cost to U.S. electricity consumers (not including power quality events.) We examined uncertainties and gaps in the information used to develop our estimate as one application of our end-use approach in order to define the degree of variability that might be expected in future estimates. Based on the identified uncertainties, we prioritized future data- collection activities whose results can be used to refine estimates of outage costs. 2. A Bottom-Up Approach for Estimating the Economic Cost of Power Interruptions and Power-Quality Events A key aspect of our work is the development of a bottom-up approach for estimating the economic costs of power interruptions and power-quality events to U.S. electricity consumers. The framework relies on a simple mathematical expression that determines the economic cost of power interruptions and power-quality events as follows: Cost of Power Interruptions and Power Quality = k j i m i n j k j i k j i j i p k V C F N , , 1 1 , , , , , 1 × × × ∑∑∑ = = = (1) Cost of Power Interruptions and Power Quality = k j i m i n j k j i k j i j i p k V C F N , , 1 1 , , , , , 1 × × × ∑∑∑ = = = (1) (1) i j k 1 1 1 = = = where, where, N = number of electricity customers in customer class i for region j 6 F = the frequency of reliability events of type k experienced annually by customer class i in region j F = the frequency of reliability events of type k experienced annually by customer class i in region j C = the cost of reliability event type k per customer in customer class i for region j (2002- Consumer-Price-Index-weighted dollars/event) V = the vulnerability of customers to reliability event type k in customer class i for region j (a fraction between 0 and 1) m = the number of customers in each customer class n = the number of regions p = the number of types of reliability event p = the number of types of reliability event i,j,k = indices for customer class, region, and type of reliability event, respectively The simplicity of this formula belies the complexities involved in estimating each of the four parameters in equation (1). Indeed, the data used to estimate the parameters are often difficult to define clearly. The following subsections outline current limitations on availability of the data needed to estimate each parameter, while section 3 describes the process we used to collect these data for our study. 2.1 Customer Data The number of customers considered in estimating the cost of power interruptions or power- quality events will have a significant impact on the magnitude of the estimate. Substantial uncertainty can result from differences in how customers are defined. In the U.S., customers may be defined in any of the following ways: (1) as a single electricity account with one (or more) meter(s), such as a single-family detached residence; (2) as a single site/facility with multiple 7 7 accounts, each possibly consisting of multiple meters, such as an apartment building; or (3) as multiple premises under common ownership, each with one or more accounts/meters, such as a chain of retail establishments. It is important to note that customers do not equal people and that one electricity customer often refers to one household, residence, or commercial or industrial facility or business. accounts, each possibly consisting of multiple meters, such as an apartment building; or (3) as multiple premises under common ownership, each with one or more accounts/meters, such as a chain of retail establishments. It is important to note that customers do not equal people and that one electricity customer often refers to one household, residence, or commercial or industrial facility or business. 2.2 Reliability Event Data Although many U.S. utilities maintain detailed records of customer outage experience, these data are usually reported in summarized form as two reliability-event indices [15]: the System Average Interruption Duration Index (SAIDI) and System Average Interruption Frequency Index (SAIFI). SAIDI and SAIFI describe the duration and frequency, respectively, of sustained interruptions experienced by customers of a utility in one year [16, 17]. According to the Institute of Electrical and Electronics Engineers (IEEE), a “sustained interruption” is any interruption that lasts at least five minutes and is not classified as “momentary”. Consistent with IEEE’s definition of a sustained interruption, a momentary outage is defined as any event lasting less than five minutes. SAIDI represents the average duration of customers’ power interruptions, while SAIFI represents the average number of interruptions per customer per year for a specified electricity supply system. We used SAIDI and SAIFI data to quantify the duration and frequency of sustained electricity interruption events for a typical year. We also used the Momentary Average 8 Interruption Frequency Index (MAIFI) index, which is a useful measure for assessing the frequency of momentary interruptions; however, these data are not as commonly collected. Unfortunately, SAIDI, SAIFI, and MAIFI data do not distinguish among the types of customers experiencing reliability events. Because of the way electricity distribution systems are designed and operated, larger commercial and industrial customers in some areas may experience fewer and shorter power interruptions than smaller commercial and residential customers [18]. Power quality, or the degree to which voltage and current deviate from a perfect 60-cycle sinuosoid, has emerged as an important issue in recent years. Despite the growing importance of power-quality events as a category of reliability events, there is even less information available on these events than on power outages. There has been only one comprehensive study of power quality, and the data collected for it are now more than a decade old [19]. Currently, there are no ongoing public-domain data-collection efforts for power quality [17]. Hence, although the bottom-up approach we designed allows for consideration of power-quality events, the lack of sufficient power-quality data prevented us from considering these events in our base-case estimate. 2.3 Cost per Outage Data Estimating customer outage costs involve several sources of uncertainty, two of which pertain to the accuracy of the cost measurement as well as how representative the estimate is of the area it covers. Typically, outage-cost estimates are based on what customers say they will experience 9 under different hypothetical outage circumstances in surveys [9]. The key source of uncertainty in these estimates is the degree to which the costs customers report for hypothetical circumstances correspond with the actual costs they would experience [20]. No studies have attempted to validate the results obtained from these surveys, yet this is a significant source of uncertainty in the cost estimates that have been prepared to date. Another important source of uncertainty inherent to customer survey data is small sample size; this can lead to significant errors if the sample is insufficient to appropriately represent the population. A common manifestation of this uncertainty arises from extrapolation to a wide geographic area from a smaller subset that is not representative of the wider geography. Assessing actual costs is complicated by the differing impacts of reliability events on various classes of customers – residential, commercial, and industrial. Costs experienced by non- residential customers or firms are, in principle, simpler to estimate than the difficult-to-quantify “hardship” costs experienced by residential customers. Basic accounting categories for commercial customers, such as labor and materials costs and revenue losses, are straightforward (though not necessarily easy to use) to apply in determining the cost of work interruptions caused by power losses. Residential-sector costs, however, include elements such as the cost of consumable goods (e.g. flashlights and candles) and inconvenience costs (e.g. resetting clocks, changing plans, and coping with inconvenience, fear, anxiety, etc.). A subtle issue that is gaining recognition is that business losses are not always directly proportional to the duration of a reliability event [21]. In such cases, the key factor is the length of business or production downtime caused by an outage of any length. For example, a partial 10 loss of voltage or a voltage sag can cause the same amount of downtime as a complete loss of power, if, for example, machines need to be rebooted or production processes need to be restarted. This issue poses a major challenge in estimating the economic cost of power interruptions and power-quality events. 2.3 Cost per Outage Data The economic cost and perceived risk of reliability events has led many customers to invest in a wide variety of technologies and measures to reduce their vulnerability to these events. Back-up or stand-by generators are probably the most well known customer investments; smaller investments like strip-surge protectors should be considered in this same category, however. In between are a host of energy-storage technologies, such as batteries and flywheels, which can reduce a customer’s vulnerability to power interruption and power-quality events. Comprehensive, national data on these investments are lacking. Hence, in the illustrative example presented in the next section, we did not explicitly consider the potential of these measures to reduce customers’ vulnerability to reliability events. That is, we implicitly set this factor to be equal to 1.0. 3.1 Customers by Class and Region We used residential, commercial, and industrial electricity demand sectors to describe customer classes as shown in Table 1. Customer population data were taken directly from the Energy Information Administration (EIA) Electric Sales and Revenue publication [22]. The data are reported for the year 2001 by state and demand sector. We partitioned the U.S. into regions so that we could represent variations in outage costs as a function of differences in the population. The regions correspond to U.S. Census Divisions as mapped by EIA, with a slight variation in the Pacific; we extracted California, where most of the Pacific region population resides, and treated it as a separate region. 3. Deriving a New Estimate of the Economic Cost of Power Interruptions to U.S. Electricity Consumers 3. Deriving a New Estimate of the Economic Cost of Power Interruptions to U.S. Electricity Consumers Having established our framework, we applied it to develop a new estimate of the cost of power interruptions, based on a review of the best data available in the public domain (the “base case”). 11 As described earlier, even the best data available have important limitations and our new estimate ultimately represents a mix of nationally aggregated and regional data components. As described earlier, even the best data available have important limitations and our new estimate ultimately represents a mix of nationally aggregated and regional data components. 2 When we removed the outliers, the SAIDI average decreased by 13 percent, from 122 to 106 minutes, but the SAIFI and MAIFI means changed very little. The standard deviation for both SAIDI and SAIFI were roughly halved when the outlying 10 percent of the data points were removed; the MAIFI standard deviations were reduced by more than 10 percent f. This suggests that trimming the highest and lowest five percent of data points helped to improve significantly the robustness of our means. 3.2 Duration and Frequency of Reliability Events Despite the existence of well-defined indices for a majority of reliability events, reliability event data are not collected systematically or consistently. The National Regulatory Research Institute reports that only 23 of 40 surveyed states require annual reporting of reliability statistics [23]. As a result, deriving reliability event data on a national scale is a challenge. We conducted an Internet search to gather publicly available reliability-event data. To address biases introduced by extreme outlying values, we used “trimmed means”: we removed the 12 highest and lowest five percent of observations and then calculated the means of the reduced observations [24]. Table 2 shows the trimmed means and standard deviations for SAIDI, SAIFI, and MAIFI used for our base case calculation. Trimming the data sets had a noticeable effect on the resulting average duration of outages in the U.S.2 Because our Internet search did not produce adequate data for all regions, national reliability event data were used in the base case with regional considerations presented in our sensitivity analysis. Several other studies have also examined national SAIDI, SAIFI, and MAIFI statistics (see Table 3). Our calculated trimmed means of 106 minutes, 1.2 sustained interruptions, and 4.3 momentary interruptions for SAIDI, SAIFI, and MAIFI, respectively, are very similar to the estimates reported from these studies.Our data thus appear to be reasonable inputs for calculating the cost of power interruptions to U.S. electricity customers. Since we did not find similarly comprehensive data on the frequency of power-quality events, we elected to exclude the costs of these events in our base-case estimate. 3.3 Cost of Reliability Events Limited information is available on the cost of reliability events. The cost data is taken from a recent analysis of the cost of power interruptions to customers [9]. The study is a meta-analysis 13 of 24 independent customer surveys conducted by eight electric utility companies in the U.S. during the past 13 years. The study developed analytic expressions, called “customer damage functions”, using a regression approach that expresses customer outage costs as a function of customer class, region, event duration, and other descriptive variables based on survey responses from more than 2,000 large commercial and industrial (C&I), 5,200 small and medium C&I, and 11,000 residential customers. The resulting cost per outage per customer data we derived using the damage functions were normalized and reported in year-2002 Consumer-Price-Index (CPI)-weighted dollars. 3 The residential population is not included because it is such a large share of the total population, yet has little overall impact on the total cost of power interruptions in the U.S. 3.2 Duration and Frequency of Reliability Events The independent descriptive variables used to derive customer outage costs were obtained from the most recently available EIA Commercial Building Energy Consumption Survey (CBECS) and Manufacturing Energy Consumption Survey (MECS) data. To account for outage cost differences by time of day, week, and season, we weighted the costs for morning, night, weekend, weekday, and summer and winter time periods. The outage cost associated with each time period was then weighted to derive the cost per outage per customer throughout the year. Table 4 shows the costs per outage per customer used to calculate the total cost of power interruptions to U.S. electricity customers in our base case analysis. The table includes costs for momentary and sustained interruptions as well as costs for the typically reported one-hour outage length as a basis of comparison to external estimates. Again, the U.S. outage cost is used in our base case estimate with regional consideration in the sensitivity analysis. 14 3.4 Base-Case Estimate of the Economic Cost of Power Interruptions We find that, based on publicly available data and subject to the limitations discussed in this section, the economic cost of power interruptions to U.S. electricity consumers is $79 billion annually. Figure 1 shows how these costs are distributed among the three customer classes. The commercial sector accounts for more than 72 percent ($57 billion) of the total outage cost. This large share is due to the high outage cost for this sector (shown in Table 4) coupled with the large population of commercial customers. The industrial sector represents nearly 26 percent of the total cost, and the residential sector accounts for less than 2 percent of the total. Although per outage per customer costs were significantly higher for the industrial sector than for the commercial and residential sectors, the industrial sector’s smaller population-- 1.6 million customers, compared to 14.9 million commercial customers--led to a lower total estimated cost than for the commercial sector. Commercial sector outage costs were roughly one order of magnitude lower than those for the industrial sector. The 114.3 million residential customers represent by far the largest population of customers in the three sectors, but their outage costs are two to three orders of magnitude lower than for the commercial sector. Therefore, despite its large customer population, the residential sector’s contribution to the total is minor. 3.2 Duration and Frequency of Reliability Events 15 Figure 2 shows that momentary outages accounted for two-thirds of the overall annual U.S. outage cost; the 4.3 momentary outages that were derived from a trimmed mean of collected Internet data cost the U.S. $52 billion each year; the 1.2 sustained outages (totaling 106 minutes together) also representing a reduced mean of the collected data account for the remaining $26 billion of the $79 billion total. Figure 2 shows that momentary outages accounted for two-thirds of the overall annual U.S. outage cost; the 4.3 momentary outages that were derived from a trimmed mean of collected Internet data cost the U.S. $52 billion each year; the 1.2 sustained outages (totaling 106 minutes together) also representing a reduced mean of the collected data account for the remaining $26 billion of the $79 billion total. ows that momentary outages accounted for two-thirds of the overall annual U.S. Figure 3 shows our base-case estimate by region and customer class. The South Atlantic region bears the largest burden, $14.7 billion, or nearly 19 percent, of the total outage cost for the nation. The Pacific-region cost, with only $2.8 billion (or 3.6 percent of the total) has the lowest share of national outage costs. Figure 3 also compares our regional estimates to the population of commercial and industrial customers in each region to determine how well correlated the regional estimates are to population density.3 The differences in the total costs by region appear to correspond well to these regional population differences. In the South Atlantic region, the higher total cost is clearly linked to the large population. The lowest total costs are seen in the Pacific and New England regions where the customer populations are the smallest. These findings point to the underlying parameters (i.e., customer definitions and populations, outage characteristics, and outage costs) that likely have the greatest influence on our estimate. Next, we explore the sensitivity of each of these parameters to understand better the effects of their inherent uncertainties on the total cost of power interruptions. 16 Estimates To understand the impacts of uncertainty on results from our bottom-up approach, we performed a sensitivity analysis in which we varied the key parameters to quantify their impact on our results. This sensitivity analysis expresses the degree of uncertainty associated with the input parameters as well as the variability in the resulting cost estimate. Figure 4 shows the resulting total cost of power interruption estimates for each of the following sensitivity analyses, in which we varied: Figure 4 shows the resulting total cost of power interruption estimates for each of the following sensitivity analyses, in which we varied: 1. The duration and frequency of reliability events and the cost per outage per customer by region, using the limited region-specific data we collected; 2. The duration and frequency of reliability events by one standard deviation greater and less than the values used in our base-case estimate; 3. The value of outages based on two assumptions of their timing--whether they occur on a summer weekday afternoon or summer weekend night; and 4. The duration and frequency of reliability events experienced by commercial and industrial sectors to reflect the fact that they tend to be disproportionately shorter in duration and of decreased frequency relative to events experienced by the residential sector. We found that the annual cost of power interruptions: We found that the annual cost of power interruptions: 17 • Could be as low as $22 billion or as high as $135 billion when we consider a reasonable range in the annual duration and frequency of power interruptions, which accounts for gaps in the data for certain regions and possible year-to-year variations in reliability; • Could be as high as $119 billion if, as many studies (incorrectly) assume, all reliability events take place during summer weekday afternoons when power usage and costs are high; • Could be as low as $23 billion if the typically fewer and shorter interruptions experienced by larger commercial and industrial customers are accounted for, which is the result of the design of many utility distribution systems; and • Is relatively insensitive to regional variation in reliability event and outage cost data—the $90 billion estimate accounting for this variation is only 14 percent higher than the base-case estimate that used U.S.-averaged values. Each sensitivity exercise helped shed light on key uncertainties associated with calculating the cost of power interruptions. Estimates The modest impact of regional differences suggests that climate and the distribution of customers by class across the country has a limited impact on the overall cost of power interruptions in the U.S. However, we detected significant differences in the other three sensitivity studies. Changing the base-case SAIDI, SAIFI, and MAIFI values by just one standard deviation, changes the overall estimate by one order of magnitude, from $22 billion to $135 billion annually. This illustrates the large effect that outage duration and frequency, which can realistically be expected to vary within this range, have on the overall cost. Assumptions about the timing of events have almost as large an impact, mainly because commercial and industrial customers’ outage costs depend on whether events occur during their operating hours. This demonstrates the importance of using time-representative outage costs when calculating 18 total costs. Finally, the total cost estimate is dramatically lowered when we account for higher levels of electric reliability typically enjoyed by industrial sector customers relative to commercial and residential customers. This variable, too, has a significant influence on overall cost-of-power-interruptions estimates and should be accounted for in future analyses. 5. Conclusion Our analysis establishes a bottom-up approach for systematically comparing and analyzing estimates of the economic cost of power interruptions and power quality to U.S. electricity customers. Using this approach, our base-case estimate of the annual cost for power interruptions to U.S. electricity consumers is $79 billion. Our analysis of the uncertainty in this estimate suggests that the costs could be as high as $135 billion or as low as $22 billion based on the particular sensitivity assumptions we employed. Thus, we present these figures not as the final word on the cost of power interruptions, but rather to illustrate the range of variability in Our analysis establishes a bottom-up approach for systematically comparing and analyzing estimates of the economic cost of power interruptions and power quality to U.S. electricity customers. Using this approach, our base-case estimate of the annual cost for power interruptions to U.S. electricity consumers is $79 billion. Our analysis of the uncertainty in this estimate suggests that the costs could be as high as $135 billion or as low as $22 billion based on the particular sensitivity assumptions we employed. Thus, we present these figures not as the final word on the cost of power interruptions, but rather to illustrate the range of variability in estimates, which is directly related to the assumptions on which the estimates are based and the uncertainty inherent to these assumptions. In view of the large range of plausible estimates and the enormous costs associated with the private and public decisions that may be based on them, we encourage policy makers, regulators, and industry to work jointly to undertake activities needed to improve the information available on reliability events and their costs. The cost of such improved data collection would be modest relative to the potential costs of reliability events in the U.S. 19 Using our bottom-up approach, we find that the majority of outage costs are borne by the commercial and industrial sectors, not the residential sector. Although there are important variations in the composition of customer types within each region of the country, the total cost of reliability events by region tend to correlate roughly with the number of commercial and industrial customers in each region. In addition, costs tend to be driven by the frequency rather than the duration of reliability events. 5. Conclusion Momentary power interruptions, which are more frequent, have a larger impact on the total cost of interruptions than sustained interruptions, which are less frequent. Priorities for future improvements in outage cost data collection fall into two broad categories: better information is needed on customers’ reliability experiences and on the cost of power interruptions to individual customers. Specific improvements include: undertaking a coordinated, nationwide collection of updated information on the cost of reliability events to customers; validating the outage costs obtained from customer surveys with actual market data (e.g., customer ownership of backup generation), establishing consistent definitions and tracking protocols for the frequency, duration, timing, and number and type of customers affected by reliability events, including power-quality events, by customer class; and collecting information on efforts by customers to reduce their vulnerability to reliability events through investments in technology and other measures. Acknowledgements We acknowledge the guidance and direction provided by Imre Gyuk and Sam Baldwin, U.S. We acknowledge the guidance and direction provided by Imre Gyuk and Sam Baldwin, U.S. DOE. The work described in this paper was funded by the Office of Electric Transmission and We acknowledge the guidance and direction provided by Imre Gyuk and Sam Baldwin, U.S. DOE. The work described in this paper was funded by the Office of Electric Transmission and DOE. The work described in this paper was funded by the Office of Electric Transmission and 20 Distribution, Energy Storage Program and by the Assistant Secretary for Energy Efficiency and Renewable Energy, Office of Planning, Budget, and Analysis of the U.S. Department of Energy under Contract No. DE-AC03-76SF00098. References [1] Fialka J. Power industry sets campaign to upgrade grid. Wall Street Journal, New York, A3. 25 August 2003. [2] Schieffer B. Face the Nation with Bob Schieffer. Washington D.C. 17 August 2003. [3] Hirst E., Kirby B. Operating reserves and bulk-power reliability. Energy 1998; 23 (11): 949- 959. [4] Billinton R., Allan R. Basic power system reliability concepts. 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Understanding the cost of power interruptions to U.S. electricity customers. report no. LBNL-55718. Berkeley, California. Lawrence Berkeley National Laboratory. 2004. [13] Turiel I, Craig P, Levine M, McMahon J. Estimation of energy intensity by end-use for commercial buildings. Energy 1987; 12 (6): 435-446. [14] Chateau B, Lapillonne B. Accounting and end-use models. References Energy 1990; 15 (3, 4): 261-278. [15] Kueck, J, Kirby B, Overholt P, Markel L. Measurement practices for reliability and power quality, a toolkit of reliability measurement practices. report no. ORNL/TM-2004/91. Oak Ridge, Tennessee. Oak Ridge National Laboratory. 2004. [16] IEEE. IEEE Standard 1159-1995, recommended practice for monitoring power quality. ISBN no. 1-55937-549-3. New York, New York. IEEE. 1995. ISBN no. 1-55937-549-3. New York, New York. IEEE. 1995. [17] Electric Power Research Institute. Distribution reliability indices tracking within the United States. report no. TR-1008459. Palo Alto, California. EPRI. 2003. States. report no. TR-1008459. Palo Alto, California. EPRI. 2003. [18] IEEE. Draft recommended practice for utility interface of photovoltaic (PV) systems. report no. P929. New York, New York. IEEE. 1999. [19] Electrotek Inc. An assessment of distribution system power quality: volumes 1-3. report no. TR-106294-V1-3. Palo Alto, California. EPRI. 1996. 22 [20] Woo C-K, Pupp RL. Costs of service disruptions to electricity consumers. Energy 1992; 17 (2): 109-126. [20] Woo C-K, Pupp RL. Costs of service disruptions to electricity consumers. Energy 1992; 17 (2): 109-126. [21] Eto, J, Divan D, Brumsickle W. Pilot evaluation of electricity-reliability and power-quality monitoring in California's Silicon Valley with the I-Grid® system. report no. LBNL-52740. Berkeley, California. Lawrence Berkeley National Laboratory. 2004. [22] Energy Information Administration. Electric sales and revenue 2001. Washington, D.C. 2001. [23] National Regulatory Research Institute. The state public service commission reliability survey. 2000. [24] Mosteller F, Tukey JW. Data analysis and regression: a second course in statistics. Reading, Massachusetts: Addison-Wesley Publishing Company, 1977. 23 Figure and Table Captions Figure 1. Base-Case Estimate of the Cost of Power Interruptions by Customer Class1 Figure 2. Base-Case Estimate of the Cost of Power Interruptions by Type of Interruption1 Figure 3. Base-Case Estimate of the Cost of Power Interruptions by Region and Customer Class with C&I Population1 Figure 4. Summary of U.S. Cost of Power Interruption Sensitivity Cases1 Table 1. Number of Customers by Region and Sector in 2001 Table 2. Summary of Trimmed Mean Reliability Event Data Table 3. Summary of U.S. Reliability Event Estimates by External Studies Table 4. Customer Outage Costs for the U.S. in 2001 Figure and Table Captions Figure and Table Captions g p Figure 1. Base-Case Estimate of the Cost of Power Interruptions by Customer Class1 Figure 2. Base-Case Estimate of the Cost of Power Interruptions by Type of Interruption1 Figure 3. Base-Case Estimate of the Cost of Power Interruptions by Region and Customer Class with C&I Population1 Figure 4. Summary of U.S. Cost of Power Interruption Sensitivity Cases1 Table 2. Summary of Trimmed Mean Reliability Event Data y y Table 3. Summary of U.S. Reliability Event Estimates by External Studies Table 4. Customer Outage Costs for the U.S. in 2001 Table 4. Customer Outage Costs for the U.S. in 2001 24 24 Residential 2% Commercial 72% Industrial 26% $1.5 Billion $20.4 Billion $56.8 Billion U.S. Total: $79 Billion 1Costs shown in U.S. 2002 CPI-weighted dollars Figure 1. Base-Case Estimate of the Cost of Power Interruptions by Customer Class1 Residential 2% Commercial 72% Industrial 26% $1.5 Billion $20.4 Billion $56.8 Billion U.S. Total: $79 Billion 1Costs shown in U.S. 2002 CPI-weighted dollars Fi 1 B C E ti t f th C t f P I t Residential 2% U.S. Total: $79 Billion 1Costs shown in U.S. 2002 CPI-weighted dollars g Figure 1. Base-Case Estimate of the Cost of Power Interruptions by Customer Class1 Figure 1. Base-Case Estimate of the Cost of Power Interruptions by Custo 25 25 Momentary Interruptions 67% Sustained Interruptions 33% U.S. Total: $79 Billion $52.3 Billion $26.3 Billion 1Costs shown in U.S. 2002 CPI-weighted dollars Figure 2. Base-Case Estimate of the Cost of Power Interruptions by Type of Interruption1 U.S. Total: $79 Billion Sustained Interruptions 33% Momentary Interruptions 67% 1Costs shown in U.S. 2002 CPI-weighted dollars Base-Case Estimate of the Cost of Power Interruptions by Type of Interruption Figure 2. Base-Case Estimate of the Cost of Power Interruptions by Type of Interruption1 Figure 2. Base-Case Estimate of the Cost of Power Interruptions by Type of In 26 $0 $2 $4 $6 $8 $10 $12 $14 $16 New England Middle Atlantic East North Central West North Central South Atlantic East South Central West South Central Mountain Pacific California Billions-$ 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 C&I Population (Millions) Residential Industrial Commercial C&I Population $3.6 B $8.1 B $2.8 B $6.7 B $10.6 B $5.5 B $14.7 B $6.7 B $10.4 B $9.7 B U.S. Total = $79 Billion 1Costs shown in U.S. 2002 CPI-weighted dollars Figure 3. Figure and Table Captions Base-Case Estimate of the Cost of Power Interruptions by Region and Customer Class with C&I Population1 1Costs shown in U.S. 2002 CPI-weighted dollars Figure 3. Base-Case Estimate of the Cost of Power Interruptions by Region and Customer Class with C&I Population1 Costs shown in U.S. 2002 CPI-weighted dollars Figure 3. Base-Case Estimate of the Cost of Power Interruptions by Region and Customer Class with C&I Population1 27 $0 $20 $40 $60 $80 $100 $120 $140 $160 LBNL Base-Case Estimate Regional Data SAIDI, SAIFI, MAIFI Increased by 1 Standard Deviation SAIDI, SAIFI, MAIFI Decreased by 1 Standard Deviation All Outages Valued Using Costs for a Summer Weekday Afternoon All Outages Valued Using Costs for a Summer Weekend Night C&I Customers Experience Fewer, Shorter Outages Billions-$ Momentary Interruptions Sustained Interruptions $79 B $135 B $22 B $119 B $23 B $26 B $90 B 1Costs shown in U.S. 2002 CPI-weighted dollars Figure 4. Summary of U.S. Cost of Power Interruption Sensitivity Cases1 $0 $20 $40 $60 $80 $100 $120 $140 $160 LBNL Base-Case Estimate Regional Data SAIDI, SAIFI, MAIFI Increased by 1 Standard Deviation SAIDI, SAIFI, MAIFI Decreased by 1 Standard Deviation All Outages Valued Using Costs for a Summer Weekday Afternoon All Outages Valued Using Costs for a Summer Weekend Night C&I Customers Experience Fewer, Shorter Outages Billions-$ Momentary Interruptions Sustained Interruptions $79 B $135 B $22 B $119 B $23 B $26 B $90 B 1Costs shown in U.S. 2002 CPI-weighted dollars Figure 4. Summary of U.S. Cost of Power Interruption Sensitivity Cases1 1Costs shown in U.S. 2002 CPI-weighted dollars Costs shown in U.S. 2002 CPI weighted dollars Figure 4. Summary of U.S. Cost of Power Interruption Sensitivity Cases1 28 Table 1. Number of Customers by Region and Sector in 2001 RESIDENTIAL COMMERCIAL INDUSTRIAL TOTAL U.S. ESTIMATE 114,317,707 14,939,895 1,582,573 130,840,175 BY REGION New England 5,822,935 714,049 62,677 6,599,661 Middle Atlantic 15,045,495 2,127,033 103,713 17,276,241 East North Central 18,705,754 2,110,172 158,780 20,974,706 West North Central 8,287,837 1,139,609 170,937 9,598,383 South Atlantic 22,473,797 2,842,220 270,840 25,586,857 East South Central 7,356,975 1,135,507 78,545 8,571,027 West South Central 12,883,403 1,722,873 292,035 14,898,311 Mountain 7,368,280 1,001,310 212,842 8,582,432 Pacific 3,922,426 494,778 66,699 4,483,903 California 11,841,144 1,559,258 154,261 13,554,663 Source: Energy Information Administration. 2003. Electric Sales and Revenue 2001. EIA/DOE. January. Washington D.C. Table 1. Number of Customers by Region and Sector in 2001 RESIDENTIAL COMMERCIAL INDUSTRIAL Table 1. Figure and Table Captions Number of Customers by Region and Sector in 2001 29 Table 2. Summary of Trimmed Mean Reliability Event Data SAIDI SAIFI MAIFI Trimmed Mean 106 min. 1.2 4.3 (Standard Deviation) (54 min.) (0.5) (3.6) 29 Table 2. Summary of Trimmed Mean Reliability Event Data SAIDI SAIFI MAIFI Trimmed Mean 106 min. 1.2 4.3 (Standard Deviation) (54 min.) (0.5) (3.6) Table 2. Summary of Trimmed Mean Reliability Event Data SAIDI SAIFI MAIFI Trimmed Mean 106 min. 1.2 4.3 (Standard Deviation) (54 min.) (0.5) (3.6) 30 Table 3. Summary of U.S. Reliability Event Estimates by External Studies SAIFI SAIDI MAIFI EPRI Reporta 1.1 107 IEEE 1995 Surveyb 1.3 120 5.5 EEI Annual Reportc 1998 1.2 118 5.4 1999 1.4 101 11.6 aSource: EPRI 2003 Distribution Reliability Indices Tracking Within the U.S. TR-1008459. May. Palo Alto CA. bSource: IEEE 1995 Survey (http://resourceinsight.com/work/naruc_pbr_97.pdf) cSource: Power Sources Manufacturer's Association (http://www.psma.com/HTML/newsletter/Q2_2001/page11.html) Table 3. Summary of U.S. Reliability Event Estimates by External Studies y ( p g p p ) cSource: Power Sources Manufacturer's Association (http://www.psma.com/HTML/newsletter/Q2_2001/page11.html) 31 Table 4. Customer Outage Costs for the U.S. in 2001a Residential Commercial Industrial Momentary Interruption $2.18 $605 $1,893 Sustained Interruption $2.99 $1,067 $4,227 One-hour Interruption $2.70 $886 $3,253 aCosts shown in U.S. 2002 CPI-weighted dollars Table 4. Customer Outage Costs for the U.S. in 2001a
W1972969625.txt	https://bmcmededuc.biomedcentral.com/counter/pdf/10.1186/1472-6920-9-7	en		A case study for teaching information literacy skills	BMC medical education	2,009	cc-by	4,102	BMC Medical Education BioMed Central Open Access Research article A case study for teaching information literacy skills Karla V Kingsley1 and Karl Kingsley2 Address: 1College of Education, Teacher Education Department, University of New Mexico, Albuquerque, NM, USA and 2Department of Biomedical Sciences, School of Dental Medicine, University of Nevada, Las Vegas, Las Vegas, NV, USA Email: Karla V Kingsley - karlak@unm.edu; Karl Kingsley - karl.kingsley@unlv.edu * Corresponding author Published: 29 January 2009 BMC Medical Education 2009, 9:7 doi:10.1186/1472-6920-9-7 Received: 13 August 2008 Accepted: 29 January 2009 This article is available from: http://www.biomedcentral.com/1472-6920/9/7 © 2009 Kingsley and Kingsley; 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. Abstract Background: The Internet has changed contemporary workplace skills, resulting in a need for proficiency with specific digital, online and web-based technologies within the fields of medicine, dentistry and public health. Although younger students, generally under 30 years of age, may appear inherently comfortable with the use of technology-intensive environments and digital or online search methods, competence in information literacy among these students may be lacking. Methods: This project involved the design and assessment of a research-based assignment to help first-year, graduate-level health science students to develop and integrate information literacy skills with clinical relevance. Results: One cohort of dental students (n = 78) was evaluated for this project and the results demonstrate that although all students were able to provide the correct response from the content-specific, or technology-independent, portion of the assignment, more than half (54%) were unable to demonstrate competence with a web-based, technology-dependent section of this assignment. No correlation was found between any demographic variable measured (gender, age, or race). Conclusion: More evidence is emerging that demonstrates the need for developing curricula that integrates new knowledge and current evidence-based practices and technologies, traditionally isolated from graduate and health-care curricula, that can enhance biomedical and clinical training for students. This study provides evidence, critical for the evaluation of new practices, which can promote and facilitate the integration of information literacy into the curriculum. Background Use of the Internet has changed contemporary American life in ways that were unimaginable two decades ago. Proficiency with digital technology and online communications are crucial skill-based methodologies for conducting evidence-based research in all realms, including the fields of medicine, public health, and higher education. On a typical day almost half (49%) of Internet users search for information related to their work, leisure activities, education, and/or health care, according to research conducted by the Pew Internet and American Life Project [1]. In the six-year period between 2002 and 2008, the percentage of users who searched for general information on the Web climbed by 69%; during the same period, the number of Internet users who searched specifically for information about health-related topics surged by 79% [2]. As everyPage 1 of 6 (page number not for citation purposes) BMC Medical Education 2009, 9:7 day life becomes increasingly digitized, Internet users face new challenges as they endeavor to solve information problems. Without a doubt, in-depth knowledge about subject matter, theory, and pedagogy are vital components of contemporary college and university-level teaching. In order to ensure the effective integration of information and communications technologies (ICT) into teaching, faculty also need an adequate understanding of and proficiency with ICT [3,4]; however, this is not always the case. Researchers at the Pew Internet & American Life project noted a substantial generation gap between college professors and their students with regard to Internet usage, interests, and abilities [5]. A measure of reluctance among university faculty regarding the use of web-based technology in the classroom remains, and as a result, researchers continue to underscore the need for professional development for educators at the university level [4,6,7]. University students may appear to be more comfortable in technology-intensive environments than are their professors, but it does not necessarily follow that they have the knowledge and critical thinking skills to effectively locate, filter, and evaluate information found online. A core competency for operating in electronic environments is information literacy; however, until recently information literacy initiatives were primarily the concern of librarians [8]. A national survey conducted by the Pew Internet & American Life project, entitled Information searches that solve problems, found that 63% of those who used the Internet were successful in finding the information they needed, but only 57% of users seeking information specifically about health-related matters were successful [9]. Although support for evidence-based medicine (EBM) has grown in recent years, as means of improving patient outcomes as well as improving the overall quality and effectiveness of healthcare delivery, case studies assessing clinically integrated EBM courses incorporating ICT in the form of digital technologies and online web searches are less abundant [10,11]. Recent studies have found that methods for teaching EBM are not only inconsistent among medical and dental schools, but may also be underdeveloped, suggesting a general lack of consensus regarding which methods represent best educational practice [11,12]. Although a variety of methods exist for both teaching and learning of EBM skills, it is becoming increasingly clear that these methods should incorporate substantial components of ICT, e-learning and must include guidance to acquire the skills for filtering and establishing the quality of current information gathered during this process [10,12]. These data demonstrate the need to integrate information literacy skills (ILS), specifically using web-based technol- http://www.biomedcentral.com/1472-6920/9/7 ogies that students will likely use in clinical practice following graduation. This study describes the development and dissemination of a research-based assignment, integrating web-based technologies to acquire theoretical and applied knowledge and concepts of a dental curriculum, within a specific first-year dental course. In addition, assessment of student performance, as well as recommendations for future modifications, are presented to provide a focused, targeted assignment with the potential to be adapted and implemented in a variety of teaching and learning environments. Methods Course Current dental students (n = 78) enrolled in a first year dental course, DEN7110: Oral Pathogens and Oral Immunology, during Spring 2008 were given an assignment designed to help them develop and integrate information literacy skills with clinical relevance. In brief, this first year (DS1) course is designed to build a foundational knowledge base of oral pathogens and immunity, to help students describe the impact of oral pathogens on the orofacial areas, the concepts of mucosal immune mechanisms and the pathogenic mechanisms of the oral flora. Dr. Kingsley is a lecturer in this course. Assignment Description This assignment was designed with three specific objectives and outcomes in mind: 1. Describe the scientific basis of a caries vaccine and provide an example of its application in patients. (Upon completion of this exercise, the student will be able to discuss biomedical science concepts of caries immunology and caries vaccines in the context of oral health and disease); 2. Compile a bibliography of eight (8) articles that represent the current literature in the area of caries microbiology and virulence factors (3) and caries vaccines (5) in refereed journals (The student will be able to critically evaluate relevant primary scientific literature regarding caries immunology and caries vaccines using and integrating web-based technologies, such as PubMed); 3. From the articles in this bibliography, provide an analysis of the two articles that are considered the "best" evidence and defend the selection of each one. (The student will be able to build and review an updated bibliography of current literature regarding caries vaccines) In brief, students were given a review article of vaccines against caries (dental or tooth cavity formation) from 2001 and were then asked to provide answers related to content (technology-independent) and also to use specific web-based, online technologies to find more recently Page 2 of 6 (page number not for citation purposes) BMC Medical Education 2009, 9:7 http://www.biomedcentral.com/1472-6920/9/7 published peer-reviewed citations (technology-dependent) [see Additional file 1]. ment) where the subjects cannot be identified or linked, directly or through identifiers, to the individual subjects. Assessment and Evaluation The assignment consisted of four questions designed to gauge students' content knowledge in various aspects of caries immunology and caries vaccines. Three questions, which addressed separate aspects of fundamental knowledge, were divided into two parts, A and B. Part A of each question was content specific; obtaining full credit was based solely upon the students' ability to list or define the correct response(s). Part B of each question involved technology or web-based technology to search for citations and building relevant literacy skills; obtaining full credit was based upon the students' ability comprehend the overall task, translate this knowledge into a new context and apply this knowledge in a new, specific situation, evidenced by the citation. Parts A and B were scored separately, as correct or incorrect and responses tallied. Results Human Subjects Exemption Student assessment data for this assignment were retrieved and each record was assigned a numerical, non-duplicated identifier to prevent the disclosure, and ensure the confidentiality, of personally identifiable private information. Gender, age and race were noted separately for each student record, in separate tables, prior to assignment. This protocol was reviewed by the UNLV Biomedical Institutional Research Board (IRB), and was deemed excluded from IRB review (OPRS#0811-2911). Informed Consent was waived pursuant to the exemption to human subjects research under the Basic HHS Policy for Protection of Human Research Subjects, (46.101) Subpart A (b) regarding IRB Exemption for 2) research involving the use of education tests (cognitive, diagnostic, aptitude, achieveTechnology-independent questions Specifically, 100% of students had correct responses to the content-specific or technology-independent portion of questions 1, 2 and 3 (Part A): 1A, 2A and 3A (Fig. 1A). Fewer than half (46%) provided correct responses to the information literacy or technology-dependent portions of question 1 (Part B), although a significantly greater proportion of students had correct responses to question 2 and question 3 (89% and 98%, respectively). Responses were scored as incorrect if the source was not found evidence-based and available through PubMed, as stated explicitly in the instructions (Appendix 1), with most incorrect responses citing websites and not peer-reviewed sources. Half of students providing incorrect responses to 2B also provided an incorrect/incomplete response to question 1B. Student responses to question 4, the analysis and synthesis of information portion, were also separated into technology-dependent (4A) and technology-independent (4B) sections (Figure 2). Nearly all students (97%) were Technology-dependent questions B 100 100 80 80 Percentage of responses Percentage of responses A The DS1 student assignment evaluations were provided in non-identifiable format to the study authors, revealing only the number of responses and percent of correct responses for each of four questions, part A and B (n = 624). Analysis of this assignment revealed that virtually all students (n = 78) had sufficiently demonstrated their knowledge of major ideas, relating to the content-specific or technology-independent portions of questions 1–3 (Part A), however many students demonstrated lack of proficiency with information literacy and the technologydependent application of skills (Part B) (Figure 1). 60 40 20 0 -20 -40 60 40 20 0 -20 -40 -60 -60 1A 2A 3A 1B 2B 3B Figure First year 1 (DS1) student assignment evaluations First year (DS1) student assignment evaluations. A) Student responses to technology-independent questions revealed all students reported correct responses to content-specific sections (1A, 2A and 3A). B) Student responses to technologydependent questions revealed only 54% of students reported incorrect responses to 1B, with fewer incorrect responses to 2B and 2C (11 and 2%, respectively) (blue = correct, yellow = incorrect). Page 3 of 6 (page number not for citation purposes) BMC Medical Education 2009, 9:7 http://www.biomedcentral.com/1472-6920/9/7 cohort average of 19%. Neither of these differences were statistically significant (p > 0.05). Analysis and Synthesis question Discussion 100 Percentage of responses 80 60 40 20 0 -20 -40 -60 4A 4B Figure First year 2 (DS1) responses to analysis and synthesis question First year (DS1) responses to analysis and synthesis question. Nearly all students were able to provide correct responses to the technology-dependent (4A) and technology-independent (4B) sections of this question (97%). able to provide appropriate citations utilizing the webbased interface (4A), with the same proportion (97%) demonstrating their ability to analyze and summarize data obtained from one of these sources (4B). Interestingly, neither of these two students that provided an incorrect response to 4A had missed any other previous technology-dependent question. To determine if other characteristics or demographic factors may have affected student performance, age, gender and race for incorrect responses were compared with the cohort averages (Table 1). Although age did not appear to be a significant factor, it should be noted that the average age was under 30 and did not vary significantly within the cohort (24.9 +/- 2.2 years). A slightly higher proportion of males missed one or more of the technology-dependent questions (Part B) 82%, compared with the cohort average of 76%. A somewhat higher proportion of non-white students also missed one or more of the technologydependent questions (Part B) 25%, compared with the In this study we examined graduate students' ability to utilize web-based technologies as an integral part of the research process. In order to complete the assignment, students made use of several different technology-dependent skills: the ability to locate online library resources, as well as an understanding of how information is organized within the library system, how to access online databases, and how to interpret and evaluate research materials within the context of a specific discipline. The current study adds to the small but rapidly growing corpus of research specifically focused on university students' levels of information literacy. As web-based technologies grow more prevalent in the digital era, so too does the need for students to acquire and fine-tune their 21st-century skills, including their information finding abilities. As previously stated, a national survey conducted by the Pew Internet & American Life project found that 63% of those who used the Internet were successful in finding the information they needed, and only 57% of users seeking information related to health-related information [9]. As the results of Q1 Part B from this case study clearly demonstrated, fewer than half of graduate-level health science students were able to demonstrate competence on the first web-based, technology-dependent assignment. Because no standardized methods yet exist for both teaching and learning of EBM skills, it is imperative that health science and dental curricula, should incorporate substantial components of ICT, e-learning and specific guidance for acquiring the skills for filtering and establishing the quality of current information from the evidence base during this process. It is also interesting to note that analysis of the cohort using demographic variables, limited in this study to age, gender and race, did not provide any evidence that these results were correlated with any particular group – although the group composition was overwhelmingly young, white and male. Some research has suggested that Table 1: Characteristics of respondents Demographic Cohort Incorrect respondents Age Ave = 24.9 years, STD = 2.2 Ave = 24.8 years, STD = 2.7 21.8% (51/234) Gender Male = 76% (59/78); Female = 24% (19/78) Male = 82% (42/51); Female = 18% (19/51) Race White = 81% (63/78); Other = 19% (15/78) White = 75% (38/51); Other = 25% (13/51) Page 4 of 6 (page number not for citation purposes) BMC Medical Education 2009, 9:7 age and income are the factors most responsible for the "digital divide" [13], while more recent studies indicate that race and gender differences are the primary factors that predict information literacy skills and associated academic performance measures [14]. Because the digital divide appears to increase with each ten year age bracket (60+ > 50–59 > 40–49, etc.) it is not surprising that no correlation was found in this study, in part because there were no significant differences between the age of those who responded incorrectly (24.8 y/o) and the average age of all students in the cohort (24.9 y/o), and also because of the relative age of this particular cohort does not approach the ages where significant differences in ICT and ILS were previously found. This study also did not find any significant differences based on race or gender, although a higher proportion of those scoring incorrectly were minorities (25%), compared with their overall representation in the cohort (19%), suggesting that studies including a larger number of students, as well as a greater proportion of females and minorities, may find significant differences. Additional factors, representing potential confounders of the previously mentioned studies [13,14], include the role of income and its association with prior educational experience before entering dental school. Although access to these specific data for students in this cohort was not available for the study authors, summary data exists and has been released for this cohort that may be relevant to the present study. The Office of Admissions released statistics that demonstrate 11.5% (9/78) of this cohort had no four year degree, 8.9% (7/78) attended undergraduate institutions that offered no masters- or doctoral-level programs and 76.9% (60/78) attended public institutions of higher learning – all potential influences of the undergraduate education experience in gaining information retrieval and literacy skills. For example, many capstone or senior-level courses are cross-listed with masters- or doctoral-level courses that tend to expand critical thinking skills, foster student-student teaching and learning, and may reinforce evidence-based learning [15,16]. Because these courses are more likely to take place during the final or senior year and in masters- or doctoral-granting institutions, those students who enter dental school without completing their undergraduate degree, often citing financial reasons, may be more likely to miss these learning opportunities. Finally, although the role of English as a second language (ESL) may represent one additional difficulty facing students and ICT and ILS, only one student was listed as having graduated from a non-US institution, and no further data regarding ESL for this cohort was available. http://www.biomedcentral.com/1472-6920/9/7 Conclusion These results strongly suggest the need for designing and incorporating information literacy and integration of technology-dependent, applied research assignments into graduate-level curricula. Although some evidence exists for guidance on successful evaluation strategies during the process of developing information literacy skills [17,18], relatively few examples of specific courses and specific recommendations can be found [19]. The results of this study suggest that placement of ILS and ICT teaching and learning modules should be integrated and incorporated early in the graduate curriculum. Furthermore these results also demonstrate cause for concern, considering that levels of information literacy can either enhance, or constrain, students' ability to complete technologydependent assignments or conduct research, which are increasingly common skills needed for everyday clinical practice [20]. The nature and extent of technology-enhanced pedagogy and curricula are also directly tied to levels of information literacy on the part of educators. While there is a significant body of literature that discusses technology integration in schools and classrooms at all levels of education, more research is needed that specifically addresses the issue of information literacy, particularly with regard to university-level learners, and even more specifically – integration of technology and web-based applications in dental, medical and health science settings to prepare clinicians for the demands of 21st century practice [21-23]. During the process of analyzing and presenting these data, several areas for future research were identified, which may have significant potential as the subject of future research endeavors and studies. Since the ultimate goal is to provide teaching and learning opportunities related to information retrieval skills in the context of evidencebased practices, two foci have been identified as having higher priority. First is the identification of additional first-year dental courses in other introductory clinical, preclinical and behavioral science courses within the curriculum that can facilitate similar integration of modules and assignments into student coursework. Second is the incorporation of multiple searching strategies and bibliographic databases in order to providing expanding learning opportunities, to provide comparisons and contrasts, as well as facilitating more detailed feedback that could be used to improve the effectiveness of the information retrieval process and subsequent evaluation by students. Competing interests The authors declare that they have no competing interests. Page 5 of 6 (page number not for citation purposes) BMC Medical Education 2009, 9:7 Authors' contributions KK and KVK conceived and coordinated this project and were equal contributors. KK administered the assessment and KVK assisted with the interpretation and analysis of data generated and made significant contributions to the writing and editing of this manuscript. Additional material http://www.biomedcentral.com/1472-6920/9/7 13. 14. 15. 16. 17. Additional file 1 Caries vaccine student assignment. Four part student assignment with technology-dependent and technology-independent questions given for first year dental students. Click here for file [http://www.biomedcentral.com/content/supplementary/14726920-9-7-S1.doc] 18. 19. 20. 21. 22. Acknowledgements Brodie M, Flournoy RE, Altman DE, Blendon RJ, Benson JM, Rosenbaum MD: Health information, the Internet, and the digital divide. Health Aff 2000, 19:255-265. Jackson LA, Zhao Y, Kolenic A 3rd, Fitzgerald HE, Harold R, Von Eye A: Race, gender and information technology use: the new digital divide. Cyberpsychol Behav 2008, 11:437-442. Klem WL, Weiss PM: Evidence-based resources and the role of librarians in developing evidence-based practice curricula. J Prof Nurs 2005, 21:380-387. Brown ST, Kirkpatrick MK, Mangum D, Avery J: A review of narrative pedagogy strategies to transform traditional nursing education. J Nurs Educ 2008, 47:283-286. Wallace MC, Shorten A, Crookes PA: Teaching information literacy skills: an evaluation. Nurse Educ Today 2000, 10:485-489. Shorten A, Wallace MC, Crookes PA: Developing information literacy: a key to evidence-based nursing. Int Nurs Rev 2001, 48:86-92. Levine AE, Bebermeyer RD, Chen JW, Davis D, Harty C: Development of an interdisciplinary course in information resources and evidence-based dentistry. J Dent Educ 2008, 72:1067-1076. Gillette J: Evidence-based dentistry for everyday practice. J Evid Based Dent Pract 2008, 8:144-148. Metros S, Woolsey K: This issue: Digital literacies in the age of sight and sound. Theory into Practice 2008, 47:79-82. Shaker L: In Google we trust: Information integrity in the digital age. First Monday 2006, 11(4): [http://www.firstmonday.org/ issues/issue11_4/shaker/]. Tillman H: Evaluating Quality on the Net 2003 [http://www.hopetill man.com/findqual.html]. KK would like to acknowledge the UNLV Teaching and Learning Center (TLC), Gillian Galbraith and Barbara Shallcross for their assistance with this manuscript. 23. References The pre-publication history for this paper can be accessed here: 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. Madden M, Fox S: Finding answers online in sickness and in health. Washington, DC: Pew Internet & American Life Project; 2006. Fallows D: Search engine use. 2008 [http://www.pewinternet.org/ pdfs/PIP_Search_Aug08.pdf]. Washington, DC: Pew Internet & American Life Project Bouras A, Albe V: Viewpoints of higher education teachers about technologies. International Journal of Technology Design in Education 2008, 18:285-305. Sutherland R, Armstrong V, Barnes S, Breeze N, Gall M, Mattthewman S, Olivero F, Taylor A, Triggs P, Wishart J, John P: Transforming teaching and learning: embedding ICT into everyday classroom practices. Journal of Computer Assisted Learning 2004, 20:413-425. Jones S: The Internet goes to college: how students are living in the future with today's technology. 2008 [http://www.pewin ternet.org/report_display.asp?r=71]. Washington, DC: Pew Internet & American Life Project Girod M, Girod G: Simulation and the need for practice in teacher preparation. Journal of Technology and Teacher Education 2008, 16:307-338. Langer AM, Knefelkamp L: College students' technology arc: A model for understanding progress. Theory into Practice 2008, 47:186-196. Mackey TP, Jacobson TE: Information literacy: A collaborative endeavor. College Teaching 2005, 53(4):140-144. Estabrook L, Witt E, Rainie L: Information searches that solve problems: How people use the Internet, libraries, and government agencies when they need help. 2007 [http:// www.pewinternet.org/PPF/r/231/report_display.asp]. Washington, DC: Pew Internet & American Life Project Coppus SF, Emparanza JI, Hadley J, Kulier R, Weinbrenner S, Arvanitis TN, Burls A, Cabello JB, Decsi T, Horvath AR, Kaczor M, Zanrei G, Pierer K, Stawiarz K, Kunz R, Mol BW, Khan KS: A clinically integrated curriculum in evidence-based medicine for just-intime learning through on-the-job training: the EU-EBM project. BMC Med Educ 2007, 7:46. Das K, Malick S, Khan KS: Tips for teaching evidence-based medicine in a clinical setting: lessons from adult learning theory. Part one. J R Soc Med 2008, 101:493-500. Khan KS, Coomarasamy A: A hierarchy of effective teaching and learning to acquire competence in evidenced-based medicine. BMC Med Educ 2006, 6:59. Pre-publication history http://www.biomedcentral.com/1472-6920/9/7/prepub Publish with Bio Med 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 researc h 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 BioMedcentral Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp Page 6 of 6 (page number not for citation purposes)
https://openalex.org/W2135468776	https://europepmc.org/articles/pmc4934508?pdf=render	English	null	Scalp In-Transit Metastatic Melanoma Treated with Interleukin-2 and Pulsed Dye Laser	Healthcare	2,013	cc-by	1,491	1. Introduction No particular regimen is considered standard therapy for widespread metastatic melanoma, and effectiveness of single-agent interventions has been questioned [1]. Here, we report a new approach with intralesional interleukin-2 (IL-2) and V-beam pulsed dye laser for the treatment of scalp melanoma metastases. Michael Z. Wang and Jerry D. Brewer * Department of Dermatology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA; E-Mail: wang08380@gmail.com Department of Dermatology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA; E-Mail: wang08380@gmail.com * Author to whom correspondence should be addressed; E-Mail: brewer.jerry@mayo.edu; Tel.: +1-507-284-3579; Fax: +1-507-284-2072. Received: 20 August 2013; in revised form: 16 October 2013 / Accepted: 21 October 2013 / Published: 25 October 2013 Abstract: No particular regimen is considered standard therapy for widespread metastatic melanoma, although surgery is the primary choice for regional nodal metastases. Systemic interleukin-2 (IL-2) is an effective immunotherapy for melanoma, but standard doses are associated with severe toxicity. We report a patient who was treated with intralesional low-dose IL-2 and V-beam pulsed dye laser for the treatment of scalp melanoma metastases. This treatment resulted in rapid regression of metastatic tumors with limited adverse effects. Keywords: interleukin-2; melanoma; pulsed dye laser therapy Healthcare 2013, 1, 96-99; doi:10.3390/healthcare1010096 healthcare ISSN 2227-9032 www.mdpi.com/journal/healthcare Case Report Scalp In-Transit Metastatic Melanoma Treated with Interleukin-2 and Pulsed Dye Laser Michael Z. Wang and Jerry D. Brewer * Department of Dermatology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA; E-Mail: wang08380@gmail.com * Author to whom correspondence should be addressed; E-Mail: brewer.jerry@mayo.edu; Tel.: +1-507-284-3579; Fax: +1-507-284-2072. Received: 20 August 2013; in revised form: 16 October 2013 / Accepted: 21 October 2013 / Published: 25 October 2013 Abstract: No particular regimen is considered standard therapy for widespread metastatic melanoma, although surgery is the primary choice for regional nodal metastases. Systemic interleukin-2 (IL-2) is an effective immunotherapy for melanoma, but standard doses are associated with severe toxicity. We report a patient who was treated with intralesional low-dose IL-2 and V-beam pulsed dye laser for the treatment of scalp melanoma metastases. This treatment resulted in rapid regression of metastatic tumors with limited adverse effects. K d i t l ki 2 l l d d l th OPEN ACCESS Healthcare 2013, 1, 96-99; doi:10.3390/healthcare1010096 Case Report Scalp In-Transit Metastatic Melanoma Treated with Interleukin-2 and Pulsed Dye Laser Michael Z. Wang and Jerry D. Brewer * 2. Case Report The patient was a 61-year-old man. Twenty-two years ago, a primary melanoma was removed from his neck. Eleven years later, a second primary melanoma developed on his scalp, and was surgically excised. Seven years later, a third melanoma (superficial spreading type) developed on the scalp (Clark level III, Breslow thickness 0.9 mm, and mitotic rate 2/mm2), and was treated with wide local excision Healthcare 2013, 1 97 and sentinel lymph node biopsy. Two lymph nodes harvested were negative. Half a year later, multiple in-transit metastases appeared on his scalp without signs of distant metastases. The lesions were surgically excised. However, multiple in-transit metastases continued to develop in the region rapidly. Thereafter, surgical excision and topical imiquimod 5% were initiated resulting in complete resolution of one lesion, whereas two others persisted. The patient was referred to our institution (Figure 1A). The melanoma was genetically wild-type. Treatment began with systemic granulocyte-macrophage colony-stimulating factor (GMCSF) (250 mcg/day; alternating between 2 weeks of treatments and cessations), topical imiquimod 5% (twice/day), V-beam pulsed dye laser (every 2–4 weeks; 595 nm; 10-mm spot size), and periodic punch excisions from June through November 2010. Despite improvement, new lesions continued to develop. Intralesional IL-2 injections (6 million IUs, 2–3 times/week) were then added to the therapeutic regimen. Subsequently, within 1 month, all the tumors vanished (Figure 1A) with pathological confirmation. No severe toxicity was observed, and patient only had flu-like symptoms. Treatment was discontinued in December 2010. The patient remained clinically melanoma-free until July 2011, when a new solitary lesion appeared on vertex scalp. The 0.5 × 0.5-cm lesion was a freely mobile, subcutaneous nodule and was confirmed to be metastatic melanoma. IL-2 injections resumed with the previous protocol for a longer period, and complete response was achieved. By September 2011, biopsy showed no residual cancer in the area. Figure 1. (A1) In-transit metastasis of melanoma at presentation (referral to our institution); (A2) After initial treatment with pulsed dye laser; (A3) At the initiation of interleukin-2 injections, in conjunction with continued pulsed dye laser therapy; (A4) Four and a half weeks after the addition of interlesional interleukin-2; (B) Possible mechanisms of the synergetic effects of intralesional interleukin-2 (IL-2) and local laser therapy. 98 Healthcare 2013, 1 Figure 1. Cont. 3. Results and Discussion 3. Results and Discussion The management of recurrent melanoma remains challenging. This patient with prolonged but localized in-transit melanoma showed remarkably improved clinical outcome when intralesional IL-2 was added to the interventions. The synergistic effect of IL-2 with V-beam laser is supported by animal models that show enhanced antitumor immunity with judicious combination of immunologic stimulation and laser devitalization of tumor nodules [2]. The benefits of systemic IL-2 are offset by its toxicities, limiting its usage to selected patients with advanced metastatic melanoma. We demonstrated that intralesional administration of IL-2 in combination with V-beam laser achieved rapid regression of in-transit metastasis with limited adverse effects. IL-2 induced vascular leakage is advantageous in localized treatment because local edema may initiate tumor necrosis that stimulates the immune response. Furthermore, intralesional injection of IL-2 achieves much higher local concentration and limits systemic adverse effects. Therefore a greater therapeutic effect could be expected. Indeed, studies indicated that intralesional IL-2 therapy was more effective than systemic, perilesional, and distant site IL-2 treatments [3]. One report showed that a complete local response was achieved in 69% of metastasized melanoma patients (33/48) by intratumorally injection of IL-2 [4]. Preferential absorption of laser radiance by hemoglobin and the subsequent conversion of light energy into thermal energy may cause blood vessel coagulation [5], which may enhance the local edematous and ischemic effect of IL-2. In addition, cancer cells are more vulnerable than normal cells to hyperthermia-induced cellular damage [6]. Considerable improvement in clinical outcome has been reported after hyperthermic therapy in tumors of many types, including melanoma [6]. Figure 1B summarizes the possible synergetic effect of intralesional IL-2 and V-beam therapy. Healthcare 2013, 1 Healthcare 2013, 1 99 4. Conclusions Our results indicate that this combined therapy is a promising strategy for the treatment of in-transit metastatic melanoma. Further study in a larger patient population is warranted. Conflicts of Interest The authors declare no conflict of interest. © 2013 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 license (http://creativecommons.org/licenses/by/3.0/). der Zee, J. Heating the patient: A promising approach? Ann. Oncol. 2002, 13, 1173–118 References 1. Garbe, C.; Eigentler, T.K.; Keilholz, U.; Hauschild, A.; Kirkwood, J.M. Systematic review of medical treatment in melanoma: Current status and future prospects. Oncologist 2011, 16, 5–24. 1. Garbe, C.; Eigentler, T.K.; Keilholz, U.; Hauschild, A.; Kirkwood, J.M. Systematic review of medical treatment in melanoma: Current status and future prospects. Oncologist 2011, 16, 5–24. 2. Katz, K.A.; Jonasch, E.; Hodi, F.S.; Soiffer, R.; Kwitkiwski, K.; Sober, A.J.; Haluska, F.G. Melanoma of unknown primary: Experience at Massachusetts General Hospital and Dana-Farber Cancer Institute. Melanoma Res. 2005, 15, 77–82. 2. Katz, K.A.; Jonasch, E.; Hodi, F.S.; Soiffer, R.; Kwitkiwski, K.; Sober, A.J.; Haluska, F.G. Melanoma of unknown primary: Experience at Massachusetts General Hospital and Dana-Farber Cancer Institute. Melanoma Res. 2005, 15, 77–82. 3. Jacobs, J.J.; Sparendam, D.; den Otter, W. Local interleukin 2 therapy is most effective against cancer when injected intratumourally. Cancer Immunol. Immunother. 2005, 54, 647–654. 3. Jacobs, J.J.; Sparendam, D.; den Otter, W. Local interleukin 2 therapy is most effective against cancer when injected intratumourally. Cancer Immunol. Immunother. 2005, 54, 647–654. 4. Weide, B.; Derhovanessian, E.; Pflugfelder, A.; Eigentler, T.K.; Radny, P.; Zelba, H.; Pföhler, C.; Pawelec, G.; Garbe, C. High response rate after intratumoral treatment with interleukin-2: Results from a phase 2 study in 51 patients with metastasized melanoma. Cancer 2010, 116, 4139–4146. 5. Babilas, P.; Shafirstein, G.; Baumler, W.; Baier, J.; Landthaler, M.; Szeimies, R.M.; Abels, C. Selective photothermolysis of blood vessels following flashlamp-pumped pulsed dye laser irradiation: In vivo results and mathematical modelling are in agreement. J. Invest. Dermatol. 2005, 125, 343–352. 5. Babilas, P.; Shafirstein, G.; Baumler, W.; Baier, J.; Landthaler, M.; Szeimies, R.M.; Abels, C. Selective photothermolysis of blood vessels following flashlamp-pumped pulsed dye laser irradiation: In vivo results and mathematical modelling are in agreement. J. Invest. Dermatol. 2005, 125, 343–352. 6. Van der Zee, J. Heating the patient: A promising approach? Ann. Oncol. 2002, 13, 1173–1184. 6. Van der Zee, J. Heating the patient: A promising approach? Ann. Oncol. 2002, 13, 1173–1184. © 2013 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 license (http://creativecommons.org/licenses/by/3.0/).

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